bims-proteo Biomed News
on Proteostasis
Issue of 2025–03–30
37 papers selected by
Eric Chevet, INSERM
  1. UbiREAD deciphers proteasomal degradation code of homotypic and branched K48 and K63 ubiquitin chains.
  2. Combinatorial ubiquitin code degrades deubiquitylation-protected substrates.
  3. Crosslinking Profiling of Molecular Glue Degrader-Induced E3 Ligase Interactome to Expand Target Space.
  4. Membrane-Bounded Intracellular E3 Ubiquitin Ligase-Targeting Chimeras (MembTACs) for Targeted Membrane Protein Degradation.
  5. Leucine aminopeptidase LyLAP enables lysosomal degradation of membrane proteins.
  6. Structure-guided design of a truncated heterobivalent chemical probe degrader of IRE1α.
  7. Serine mistranslation induces the integrated stress response through the P stalk.
  8. The ER-phagy receptor FAM134B is targeted by Salmonella Typhimurium to promote infection.
  9. Mechanisms of COPII coat assembly and cargo recognition in the secretory pathway.
  10. Proteostasis and lysosomal repair deficits in transdifferentiated neurons of Alzheimer's disease.
  11. Mycobacterium tuberculosis phagosome Ca2+ leakage triggers multimembrane ATG8/LC3 lipidation to restrict damage in human macrophages.
  12. Mimicry of molecular glues using dual covalent chimeras.
  13. SOD2 is a regulator of proteasomal degradation promoting an adaptive cellular starvation response.
  14. Induced proximity at the cell surface.
  15. Ceramide mediates cell-to-cell ER stress transmission by modulating membrane fluidity.
  16. Ltc1 localization by EMC regulates cell membrane fluidity to facilitate membrane protein biogenesis.
  17. Rapid ER remodeling induced by a peptide-lipid complex in dying tumor cells.
  18. Evidence supporting a catalytic pentad mechanism for the proteasome and other N-terminal nucleophile enzymes.
  19. Challenges in predicting PROTAC-mediated protein-protein interfaces with AlphaFold reveal a general limitation on small interfaces.
  20. ANXA11 biomolecular condensates facilitate protein-lipid phase coupling on lysosomal membranes.
  21. The USP43/RNF2 axis negatively regulates antiviral innate immunity by promoting TBK1 ubiquitination and degradation.
  22. DBeQ derivative targets vacuolar protein sorting 4 functions in cancer cells and suppresses tumor growth in mice.
  23. The impact of Coronavirus Nsp1 on host mRNA degradation is independent of its role in translation inhibition.
  24. Drosophila and human Headcase define a new family of ribonucleotide granule proteins required for stress response.
  25. S-palmitoylation modulates ATG2-dependent non-vesicular lipid transport during starvation-induced autophagy.
  26. Praja2 controls P-body assembly and translation in glioblastoma by non-proteolytic ubiquitylation of DDX6.
  27. Melatonin-mediated cGAS-STING signal in senescent macrophages promote TNBC chemotherapy resistance and drive the SASP.
  28. SEC24C deficiency causes trafficking and glycosylation abnormalities in an epileptic encephalopathy with cataracts and dyserythropoeisis.
  29. Plasticity of the mammalian integrated stress response.
  30. Capturing the Conformational Heterogeneity of HSPB1 Chaperone Oligomers at Atomic Resolution.
  31. irCLIP-RNP and Re-CLIP reveal patterns of dynamic protein assemblies on RNA.
  32. Induction of a transcriptional adaptation response by RNA destabilization events.
  33. Deubiquitinase-Targeting Chimeras (DUBTACs) as a Potential Paradigm-Shifting Drug Discovery Approach.
  34. RHOD mediates ATG9A trafficking to promote autophagosome formation during autophagy in cancer.
  35. A large-scale sORF screen identifies putative microproteins involved in cancer cell fitness.
  36. STING agonist-based ER-targeting molecules boost antigen cross-presentation.
  37. SUMOylation facilitates the stability of BCR-ABL to promote chronic myeloid leukemia progression.
  1. Mol Cell. 2025 Mar 20. pii: S1097-2765(25)00152-2. [Epub ahead of print]
    UbiREAD deciphers proteasomal degradation code of homotypic and branched K48 and K63 ubiquitin chains.
    Leo Kiss, Leo C James, Brenda A Schulman.
      Ubiquitin chains define the fates of their modified proteins, often mediating proteasomal degradation in eukaryotes. Yet heterogeneity of intracellular ubiquitination has precluded systematically comparing the degradation capacities of different ubiquitin chains. We developed ubiquitinated reporter evaluation after intracellular delivery (UbiREAD), a technology that monitors cellular degradation and deubiquitination at high temporal resolution after bespoke ubiquitinated proteins are delivered into human cells. Comparing the degradation of a model substrate modified with various K48, K63, or K48/K63-branched ubiquitin chains revealed fundamental differences in their intracellular degradation capacities. K48 chains with three or more ubiquitins triggered degradation within minutes. K63-ubiquitinated substrate was rapidly deubiquitinated rather than degraded. Surprisingly, in K48/K63-branched chains, substrate-anchored chain identity determined the degradation and deubiquitination behavior, establishing that branched chains are not the sum of their parts. UbiREAD reveals a degradation code for ubiquitin chains varying by linkage, length, and topology and a functional hierarchy within branched ubiquitin chains.
    Keywords:  K48; K63; branched ubiquitin chains; deubiquitination; electroporation; proteasome; protein degradation; proteostasis; ubiquitin; ubiquitin code
    DOI:  https://doi.org/10.1016/j.molcel.2025.02.021
  2. Nat Commun. 2025 Mar 24. 16(1): 2496
    Combinatorial ubiquitin code degrades deubiquitylation-protected substrates.
    Mai Morita, Miyu Takao, Honoka Tokuhisa, Ryotaro Chiba, Shota Tomomatsu, Yoshino Akizuki, Takuya Tomita, Akinori Endo, Yasushi Saeki, Yusuke Sato, Fumiaki Ohtake.
      Protein ubiquitylation is maintained by a dynamic balance of the conjugation and deconjugation of ubiquitin. It remains unclear how deubiquitylation-stabilized substrates are directed for degradation. Branched ubiquitin chains promote substrate degradation through the proteasome. TRIP12 and UBR5 are HECT-type E3 ubiquitin ligases, which are specific for lysine 29 (K29) and lysine 48 (K48) linkages, respectively. Here, we show that the deubiquitylase (DUB) OTUD5 is cooperatively modified by TRIP12 and UBR5, resulting in conjugation of K29/K48 branched ubiquitin chains and accelerated proteasomal degradation. TRIP12-OTUD5 antagonism regulates TNF-α-induced NF-κB signaling. Mechanistically, OTUD5 readily cleaves K48 linkages, but does not affect K29 linkages. Consequently, K29 linkages overcome OTUD5 DUB activity to facilitate UBR5-dependent K48-linked chain branching. This mechanism is applicable to other OTUD5-associated TRIP12 substrates. Thus, the combination of DUB-resistant and proteasome-targeting ubiquitin linkages promotes the degradation of deubiquitylation-protected substrates, underscoring the role of branched ubiquitin chains in shifting the ubiquitin conjugation/deconjugation equilibrium.
    DOI:  https://doi.org/10.1038/s41467-025-57873-9
  3. Angew Chem Int Ed Engl. 2025 Mar 25. e202505053
    Crosslinking Profiling of Molecular Glue Degrader-Induced E3 Ligase Interactome to Expand Target Space.
    Yali Xu, Wensi Zhao, Hui-Jun Nie, Jiamin Wang, Jingjing Fu, Hao Hu, Zihao Liu, Shengna Tao, Mingya Zhang, Yubo Zhou, Jia Li, Minjia Tan, Xiao-Hua Chen.
      Molecular glue (MG) degraders, small molecules with significant therapeutic potential for targeting undruggable proteins, are emerging as new modality in drug discovery. Profiling the E3 ligase interactome induced by MG degraders provides insights into their mechanism of action and identifies clinically relevant neo-substrates for degradation, thereby offering new therapeutic opportunities. However, established methods face significant challenges in comprehensive and accurate profiling of MG degrader-induced E3 ligase interactome. Herein, we introduce the concept of globally crosslinking profiling of the MG degrader-induced E3 ligase interactome in living cells, achieved by integrating genetic code expansion technology with mass spectrometry-based proteomics. Our approach presents an efficient and robust strategy for identifying neo-substrates recruited to cereblon E3 ligase by the known degraders CC-885 and DKY709, offering valuable insights for clinical evaluation and significantly expanding their target space. Moreover, we developed two novel MG degraders with potent anti-proliferative effects on cancer cells, and application of our method identified neo-substrates, revealing a previously unrecognized target landscape and advancing our understanding of E3 ligase-neo-substrate interactions. Overall, our study provides a powerful tool for neo-substrate identification and expanding target space of E3 ligase, opening new opportunities for developing next-generation MG degraders to address the clinical challenge of undruggable targets.
    Keywords:  Chemoproteomics; Genetic code expansion; Molecular glue degrader; Targeted protein degradation; photo-crosslinking
    DOI:  https://doi.org/10.1002/anie.202505053
  4. Angew Chem Int Ed Engl. 2025 Mar 27. e202501857
    Membrane-Bounded Intracellular E3 Ubiquitin Ligase-Targeting Chimeras (MembTACs) for Targeted Membrane Protein Degradation.
    Zhi Zhu, Mengwu Mo, Feiyu Wang, Huiming Zhang, Ying Zhang, Chaoyong Yang, Jinbiao Shang.
      Targeted protein degradation (TPD) represents a potent therapeutic strategy aimed at dismantling disease-associated target proteins. PROTAC is the most widely developed technique for intracellular protein degradation, while its degradation ability on membrane proteins has been hindered by the need for complex synthetic processes and limited permeability. In this study, we developed the membrane-bounded intracellular E3 ubiquitin ligase-targeting chimeras (MembTACs) that simultaneously recruit intracellular E3 ubiquitin ligase and bind to the desired membrane proteins for targeted degradation of membrane proteins. We demonstrate that the MembTACs can effectively utilize intracellular E3 ubiquitin ligase to degrade the therapeutically relevant membrane proteins of EpCAM and Met via the proteasome pathway. We anticipate that the new platform will expand the range of PROTAC applications and provide a new dimension for targeted membrane protein degradation.
    Keywords:  Intracellular E3 Ubiquitin Ligase; MembTACs; PROTACs; membrane protein degradation; pHLIP
    DOI:  https://doi.org/10.1002/anie.202501857
  5. Science. 2025 Mar 28. 387(6741): eadq8331
    Leucine aminopeptidase LyLAP enables lysosomal degradation of membrane proteins.
    Aakriti Jain, Isaac Heremans, Gilles Rademaker, Tyler C Detomasi, Peter Rohweder, Dashiell Anderson, Justin Zhang, Grace A Hernandez, Suprit Gupta, Teresa von Linde, Mike Lange, Martina Spacci, Jiayi Luo, Y Rose Citron, James A Olzmann, David W Dawson, Charles S Craik, Guido Bommer, Rushika M Perera, Roberto Zoncu.
      Breakdown of every transmembrane protein trafficked to lysosomes requires proteolysis of their hydrophobic helical transmembrane domains. Combining lysosomal proteomics with functional genomic datasets, we identified lysosomal leucine aminopeptidase (LyLAP; formerly phospholipase B domain-containing 1) as the hydrolase most tightly associated with elevated endocytosis. Untargeted metabolomics and biochemical reconstitution demonstrated that LyLAP is a processive monoaminopeptidase with preference for amino-terminal leucine. This activity was necessary and sufficient for the breakdown of hydrophobic transmembrane domains. LyLAP was up-regulated in pancreatic ductal adenocarcinoma (PDA), which relies on macropinocytosis for nutrient uptake. In PDA cells, LyLAP ablation led to the buildup of undigested hydrophobic peptides, lysosomal membrane damage, and growth inhibition. Thus, LyLAP enables lysosomal degradation of membrane proteins and protects lysosomal integrity in highly endocytic cancer cells.
    DOI:  https://doi.org/10.1126/science.adq8331
  6. RSC Med Chem. 2025 Mar 18.
    Structure-guided design of a truncated heterobivalent chemical probe degrader of IRE1α.
    Breanna L Zerfas, Yingpeng Liu, Jianwei Che, Katherine A Donovan, John M Hatcher, Fidel Huerta, Rebecca J Metivier, Radosław P Nowak, Leah Ragosta, Tiffany Tsang, Eric S Fischer, Lyn H Jones.
      IRE1α is an ER protein involved in the unfolded protein response (UPR) and dysregulation of the ER stress pathway has been implicated in several diseases. Inhibitors of the cytoplasmic endonuclease or kinase domains of the enzyme have limited utility and targeted degradation would address additional scaffolding functions of the protein. Here, we describe the design and development of IRE1α proteolysis targeting chimeras (PROTACs) based on a lysine-reactive salicylaldehyde RNase inhibitor, and present the structure-activity relationships (SARs) that delivered the first highly selective degraders of a native ER-membrane associated protein. Medicinal chemistry optimization exploited ternary complex computational modelling to inform design, HiBiT-SpyTag IRE1α degradation and NanoBRET cereblon occupancy cell-based assays to generate SARs, and mass spectrometry-based proteomics to assess broad selectivity in an unbiased manner. Merging IRE1α and CRBN ligand chemotypes provided the truncated chimera CPD-2828 with physicochemical properties more akin to an oral molecular glue degrader than a traditional PROTAC.
    DOI:  https://doi.org/10.1039/d5md00028a
  7. J Biol Chem. 2025 Mar 25. pii: S0021-9258(25)00296-0. [Epub ahead of print] 108447
    Serine mistranslation induces the integrated stress response through the P stalk.
    Hong Zhang, Jiqiang Ling.
      Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes that support robust and accurate protein synthesis. A rapidly expanding number of studies show that mutations in aaRSs lead to multiple human diseases, including neurological disorders and cancer. How aaRS mutations impact human health is not fully understood. In particular, our knowledge of how aminoacylation errors affect stress responses and fitness in eukaryotic cells remains limited. The integrated stress response (ISR) is an adaptive mechanism in response to multiple stresses. However, chronic activation of the ISR contributes to the development of multiple diseases such as neuropathies. In this study, we show that Ser misincorporation into Ala and Thr codons, resulting from either aaRS editing defects or mutations in tRNAs, actives the ISR. We further demonstrate that activation of the ISR by Ser mistranslation does not depend on the accumulation of uncharged tRNAs, but rather requires the P stalk associated with the ribosome, implying that ribosome stalling and collision are involved. Our work highlights that certain types of aminoacylation errors can lead to chronic activation of the ISR, potentially affecting fitness and disease progression.
    Keywords:  AlaRS; ThrRS; Translational fidelity; stress response; tRNA misacylation
    DOI:  https://doi.org/10.1016/j.jbc.2025.108447
  8. Nat Commun. 2025 Mar 25. 16(1): 2923
    The ER-phagy receptor FAM134B is targeted by Salmonella Typhimurium to promote infection.
    Damián Gatica, Reham M Alsaadi, Rayan El Hamra, Boran Li, Rudolf Mueller, Makoto Miyazaki, Qiming Sun, Subash Sad, Ryan C Russell.
      Macroautophagy/autophagy is a key catabolic-recycling pathway that can selectively target damaged organelles or invading pathogens for degradation. The selective autophagic degradation of the endoplasmic reticulum (hereafter referred to as ER-phagy) is a homeostatic mechanism, controlling ER size, the removal of misfolded protein aggregates, and organelle damage. ER-phagy can also be stimulated by pathogen infection. However, the link between ER-phagy and bacterial infection remains poorly understood, as are the mechanisms evolved by pathogens to escape the effects of ER-phagy. Here, we show that Salmonella enterica serovar Typhimurium inhibits ER-phagy by targeting the ER-phagy receptor FAM134B, leading to a pronounced increase in Salmonella burden after invasion. Salmonella prevents FAM134B oligomerization, which is required for efficient ER-phagy. FAM134B knock-out raises intracellular Salmonella number, while FAM134B activation reduces Salmonella burden. Additionally, we found that Salmonella targets FAM134B through the bacterial effector SopF to enhance intracellular survival through ER-phagy inhibition. Furthermore, FAM134B knock-out mice infected with Salmonella presented severe intestinal damage and increased bacterial burden. These results provide mechanistic insight into the interplay between ER-phagy and bacterial infection, highlighting a key role for FAM134B in innate immunity.
    DOI:  https://doi.org/10.1038/s41467-025-58035-7
  9. Nat Rev Mol Cell Biol. 2025 Mar 25.
    Mechanisms of COPII coat assembly and cargo recognition in the secretory pathway.
    Katie W Downes, Giulia Zanetti.
      One third of all proteins in eukaryotes transit between the endoplasmic reticulum (ER) and the Golgi to reach their functional destination inside or outside of the cell. During export, secretory proteins concentrate at transitional zones of the ER known as ER exit sites, where they are packaged into transport carriers formed by the highly conserved coat protein complex II (COPII). Despite long-standing knowledge of many of the fundamental pathways that govern traffic in the early secretory pathway, we still lack a complete mechanistic model to explain how the various steps of COPII-mediated ER exit are regulated to efficiently transport diverse cargoes. In this Review, we discuss the current understanding of the mechanisms underlying COPII-mediated vesicular transport, highlighting outstanding knowledge gaps. We focus on how coat assembly and disassembly dictate carrier morphogenesis, how COPII selectively recruits a vast number of cargo and cargo adaptors, and finally discuss how COPII mechanisms in mammals might have adapted to enable transport of large proteins.
    DOI:  https://doi.org/10.1038/s41580-025-00839-y
  10. Nat Cell Biol. 2025 Mar 26.
    Proteostasis and lysosomal repair deficits in transdifferentiated neurons of Alzheimer's disease.
    Ching-Chieh Chou, Ryan Vest, Miguel A Prado, Joshua Wilson-Grady, Joao A Paulo, Yohei Shibuya, Patricia Moran-Losada, Ting-Ting Lee, Jian Luo, Steven P Gygi, Jeffery W Kelly, Daniel Finley, Marius Wernig, Tony Wyss-Coray, Judith Frydman.
      Ageing is the most prominent risk factor for Alzheimer's disease (AD). However, the cellular mechanisms linking neuronal proteostasis decline to the characteristic aberrant protein deposits in the brains of patients with AD remain elusive. Here we develop transdifferentiated neurons (tNeurons) from human dermal fibroblasts as a neuronal model that retains ageing hallmarks and exhibits AD-linked vulnerabilities. Remarkably, AD tNeurons accumulate proteotoxic deposits, including phospho-tau and amyloid β, resembling those in APP mouse brains and the brains of patients with AD. Quantitative tNeuron proteomics identify ageing- and AD-linked deficits in proteostasis and organelle homeostasis, most notably in endosome-lysosomal components. Lysosomal deficits in aged tNeurons, including constitutive lysosomal damage and ESCRT-mediated lysosomal repair defects, are exacerbated in AD tNeurons and linked to inflammatory cytokine secretion and cell death. Providing support for the centrality of lysosomal deficits in AD, compounds ameliorating lysosomal function reduce amyloid β deposits and cytokine secretion. Thus, the tNeuron model system reveals impaired lysosomal homeostasis as an early event of ageing and AD.
    DOI:  https://doi.org/10.1038/s41556-025-01623-y
  11. Sci Adv. 2025 Mar 28. 11(13): eadt3311
    Mycobacterium tuberculosis phagosome Ca2+ leakage triggers multimembrane ATG8/LC3 lipidation to restrict damage in human macrophages.
    Di Chen, Antony Fearns, Maximiliano G Gutierrez.
      The role of canonical autophagy in controlling Mycobacterium tuberculosis (Mtb), referred to as xenophagy, is understood to involve targeting Mtb to autophagosomes, which subsequently fuse with lysosomes for degradation. Here, we found that Ca2+ leakage after Mtb phagosome damage in human macrophages is the signal that triggers autophagy-related protein 8/microtubule-associated proteins 1A/1B light chain 3 (ATG8/LC3) lipidation. Unexpectedly, ATG8/LC3 lipidation did not target Mtb to lysosomes, excluding the canonical xenophagy. Upon Mtb phagosome damage, the Ca2+ leakage-dependent ATG8/LC3 lipidation occurred on multiple membranes instead of single or double membranes excluding the noncanonical autophagy pathways. Mechanistically, Ca2+ leakage from the phagosome triggered the recruitment of the V-ATPase-ATG16L1 complex independently of FIP200, ATG13, and proton gradient disruption. Furthermore, the Ca2+ leakage-dependent ATG8/LC3 lipidation limited Mtb phagosome damage and restricted Mtb replication. Together, we uncovered Ca2+ leakage as the key signal that triggers ATG8/LC3 lipidation on multiple membranes to mitigate Mtb phagosome damage.
    DOI:  https://doi.org/10.1126/sciadv.adt3311
  12. Nat Commun. 2025 Mar 24. 16(1): 2855
    Mimicry of molecular glues using dual covalent chimeras.
    Eden Kapcan, Karolina Krygier, Maya da Luz, Nickolas J Serniuck, Ali Zhang, Jonathan Bramson, Anthony F Rullo.
      A special class of proximity inducing bifunctional molecules/chimeras called molecular glues leverage positive co-operativity to stabilize ternary complex formation and induce a biological response. Despite their utility, molecular glues remain challenging to rationally design. This is particularly true in the context of inducing cell-cell proximity which involve ternary complexes that comprise non- or negatively interacting proteins. In this work, we develop a dual proximity labeling strategy enabling a chimera to covalently crosslink a non-interacting serum antibody to a tumor surface protein, within a ternary complex. The resultant resistance to dissociation, including in the presence of competitor binding ligands, mimics molecular glue stabilization. We demonstrate these covalent glue mimics (CGMs) can induce cell-cell proximity in three distinct model systems of tumor-immune recognition, leading to significant functional enhancements. Collectively, this work underscores the utility of dual proximal covalent labeling as a potential general strategy to stabilize ternary complexes comprising non-interacting proteins and enforce cell-cell interactions.
    DOI:  https://doi.org/10.1038/s41467-025-58083-z
  13. Cell Rep. 2025 Mar 24. pii: S2211-1247(25)00205-0. [Epub ahead of print]44(4): 115434
    SOD2 is a regulator of proteasomal degradation promoting an adaptive cellular starvation response.
    Nurul Khalida Ibrahim, Sabine Schreek, Buesra Cinar, Anna Sophie Stasche, Su Hyun Lee, Andre Zeug, Tim Dolgner, Julia Niessen, Evgeni Ponimaskin, Halyna Shcherbata, Beate Fehlhaber, Jean-Pierre Bourquin, Beat Bornhauser, Martin Stanulla, Andreas Pich, Alejandro Gutierrez, Laura Hinze.
      Adaptation to changes in amino acid availability is crucial for cellular homeostasis, which requires an intricate orchestration of involved pathways. Some cancer cells can maintain cellular fitness upon amino acid shortage, which has a poorly understood mechanistic basis. Leveraging a genome-wide CRISPR-Cas9 screen, we find that superoxide dismutase 2 (SOD2) has a previously unrecognized dismutase-independent function. We demonstrate that SOD2 regulates global proteasomal protein degradation and promotes cell survival under conditions of metabolic stress in malignant cells through the E3 ubiquitin ligases UBR1 and UBR2. Consequently, inhibition of SOD2-mediated protein degradation highly sensitizes different cancer entities, including patient-derived xenografts, to amino acid depletion, highlighting the pathophysiological relevance of our findings. Our study reveals that SOD2 is a regulator of proteasomal protein breakdown upon starvation, which serves as an independent catabolic source of amino acids, a mechanism co-opted by cancer cells to maintain cellular fitness.
    Keywords:  CP: Cancer; CP: Molecular biology; SOD2; UBR1; UBR2; amino acid starvation; cancer; drug resistance; leukemia; protein degradation
    DOI:  https://doi.org/10.1016/j.celrep.2025.115434
  14. Nat Biotechnol. 2025 Mar 26.
    Induced proximity at the cell surface.
    Nicholas A Till, Muthukumar Ramanathan, Carolyn R Bertozzi.
      Molecular proximity is a governing principle of biology that is essential to normal and disease-related biochemical pathways. At the cell surface, protein-protein proximity regulates receptor activation, inhibition and protein recycling and degradation. Induced proximity is a molecular engineering principle in which bifunctional molecules are designed to bring two protein targets into close contact, inducing a desired biological outcome. Researchers use this engineering principle for therapeutic purposes and to interrogate fundamental biological mechanisms. This Review focuses on the use of induced proximity at the cell surface for diverse applications, such as targeted protein degradation, receptor inhibition and activating intracellular signaling cascades. We see a rich future for proximity-based modulation of cell surface protein activity both in basic and translational science.
    DOI:  https://doi.org/10.1038/s41587-025-02592-1
  15. J Cell Biol. 2025 May 05. pii: e202405060. [Epub ahead of print]224(5):
    Ceramide mediates cell-to-cell ER stress transmission by modulating membrane fluidity.
    Yazhen Huo, Xinlu Liu, Chen Lu, Tao Li, Zaili Yang, Fenfen Xu, Si Chen, Kailin Yin, Likun Wang.
      Under endoplasmic reticulum (ER) stress (ERS), cells initiate the unfolded protein response (UPR) to maintain ER homeostasis. Recent studies revealed ERS transmission between cells and tissues, by activating the cell-nonautonomous UPR in cells that do not experience ERS directly. Here, we report that ERS triggers a rapid release of ceramide independent of the UPR, but requiring the acid sphingomyelinase activity. Carried by lipoproteins, ceramide is delivered to receiving cells to induce the UPR and regulate cell functions at multiple aspects, including lipid accumulation, cell death, and cytokine production. Mechanistically, extracellular ceramide stimulates ceramide synthesis at the transcription level in receiving cells, leading to ceramide accumulation in the ER so as to reduce membrane fluidity to disrupt ER calcium homeostasis, thus activating the UPR. Sphingomyelin counterbalanced the effect of ceramide. UPR induction is the frontline response to protect cells from ceramide insult. Our study suggests ceramide-mediated ERS transmission as a universal cell-cell communication model regulating a wide range of physiological events.
    DOI:  https://doi.org/10.1083/jcb.202405060
  16. iScience. 2025 Mar 21. 28(3): 112096
    Ltc1 localization by EMC regulates cell membrane fluidity to facilitate membrane protein biogenesis.
    Modesto Berraquero, Víctor A Tallada, Juan Jimenez.
      The EMC complex, a highly conserved transmembrane chaperone in the endoplasmic reticulum (ER), has been associated in humans with sterol homeostasis and a myriad of different cellular activities, rendering the mechanism of EMC functionality enigmatic. Using fission yeast, we demonstrate that the EMC complex facilitates the biogenesis of the sterol transfer protein Lam6/Ltc1 at ER-plasma membrane and ER-mitochondria contact sites. Cells that lose EMC function sequester unfolded Lam6/Ltc1 and other proteins at the mitochondrial matrix, leading to surplus ergosterol, cold-sensitive growth, and mitochondrial dysfunctions. Remarkably, inhibition of ergosterol biosynthesis, but also fluidization of cell membranes to counteract their rigidizing effects, reduce the ER-unfolded protein response and rescue growth and mitochondrial defects in EMC-deficient cells. These results suggest that EMC-assisted biogenesis of Lam6/Ltc1 may provide, through ergosterol homeostasis, optimal membrane fluidity to facilitate biogenesis of other ER-membrane proteins.
    Keywords:  Biochemistry; Cell biology; Molecular biology
    DOI:  https://doi.org/10.1016/j.isci.2025.112096
  17. Life Sci Alliance. 2025 Jun;pii: e202403114. [Epub ahead of print]8(6):
    Rapid ER remodeling induced by a peptide-lipid complex in dying tumor cells.
    Samudra Sabari, Siddharth Chinchankar, Ines Ambite, Atefeh Nazari, António Pedro Nbm Carneiro, Axel Svenningsson, Catharina Svanborg, Arunima Chaudhuri.
      The membranous ER spans the entire cell, creating a network for the biosynthesis of proteins and lipids, cell-wide communication, and nuclear delivery of molecules, including therapeutic agents. Here, we identify a novel ER response triggered by the tumoricidal complex alpha1-oleate, defined by a loss of peripheral ER structure, extensive ER vesiculation. Alpha1-oleate was present in the ER-derived vesicle membranes, also decorated by ER-resident and ER-interacting proteins, calnexin and ORP3, and in their lumen, also enriched for KDEL, confirming their ER origin distinct from lipid droplets. Rapid nuclear uptake of the complex constituents resulted in diffuse nuclear staining, and the asymmetrical perinuclear enrichment of the collapsing ER with its content of alpha1-oleate created large invaginations lined by the ER, inner nuclear membrane markers, and lamin nucleoskeleton. In parallel, a change in nuclear shape resulted in a volcano-like structure. This newly discovered, potent ER response to alpha1-oleate may have evolved to package ER-associated cellular contents in the nuclei of dying tumor cells, thus sequestering toxic cell debris associated with apoptotic cell death.
    DOI:  https://doi.org/10.26508/lsa.202403114
  18. Nat Commun. 2025 Mar 26. 16(1): 2949
    Evidence supporting a catalytic pentad mechanism for the proteasome and other N-terminal nucleophile enzymes.
    Darlene Fung, Aida Razi, Michael Pandos, Benjamin Velez, Erignacio Fermin Perez, Lea Adams, Shaun Rawson, Richard M Walsh, John Hanna.
      Proteases are defined by their nucleophile but require additional residues to regulate their active sites, most often arranged as catalytic triads that control the generation and resolution of acyl-enzyme intermediates. Threonine N-terminal nucleophiles represent a diverse family of proteases and transferases that possess two active site nucleophiles, the side chain hydroxyl and the free amino-terminus, and require autocatalytic cleavage of their N-terminal propeptides. Here we provide evidence that the proteasome, which mediates intracellular protein degradation and contains three different threonine protease subunits, utilizes a unique catalytic pentad mechanism. In addition to the previously defined lysine/aspartate pair which regulates threonine's side chain, a second serine/aspartate pair appears to regulate threonine's amino-terminus. The pentad is required for substrate proteolysis and assembly-coupled autocatalytic cleavage, the latter triggered by alignment of the full pentad upon fusion of two half-proteasome precursors. A similar pentad mechanism was required by the ornithine acetyltransferase Arg7, suggesting that this may be a general property of threonine N-terminal nucleophiles. Finally, we show that two patient-derived proteasome mutations compromise function of the serine/aspartate unit in yeast, suggesting that defective pentad function may underlie some human proteasomopathies.
    DOI:  https://doi.org/10.1038/s41467-025-58077-x
  19. Bioinform Adv. 2025 ;5(1): vbaf056
    Challenges in predicting PROTAC-mediated protein-protein interfaces with AlphaFold reveal a general limitation on small interfaces.
    Gilberto P Pereira, Corentin Gouzien, Paulo C T Souza, Juliette Martin.
       Motivation: Proteolysis Targeting Chimeras (PROTACs) are heterobifunctional molecules composed by ligands binding to a target protein and a E3-ligase complex, connected by a linker, that induce proximity-based target protein degradation. PROTACs are promising alternatives to conventional drugs against cancer. Predicting PROTAC-mediated complexes is often the first step for in silico PROTAC design pipelines. We previously noted that AlphaFold2 (AF2) fails to predict PROTAC-mediated complexes.
    Results: Here, we investigate the potential causes of this limitation. We consider a set of 326 protein heterodimers orthogonal to the AF2 training set, and evaluate AF2 models focusing on the interface size and presence of interface ligand. Our results show that AF2-multimer predictions are sensitive to the size of the interface to predict even in the absence of ligands, with the majority of models being incorrect for the smallest interfaces. We also benchmark both AF2 and AF3 on a set of 28 PROTAC-mediated dimers and show that AF3 does not significantly improve upon the accuracy of AF2. The low accuracy of AF2 on complexes with small interfaces has strong implications for computational pipelines for PROTAC design, as these stabilize typically small interfaces, and more generally on any prediction task that involves small interfaces.
    Availability and implementation: All the models analyzed in this article are available in the Zenodo archive https://zenodo.org/records/14810843.
    DOI:  https://doi.org/10.1093/bioadv/vbaf056
  20. Nat Commun. 2025 Mar 21. 16(1): 2814
    ANXA11 biomolecular condensates facilitate protein-lipid phase coupling on lysosomal membranes.
    Jonathon Nixon-Abell, Francesco S Ruggeri, Seema Qamar, Therese W Herling, Magdalena A Czekalska, Yi Shen, Guozhen Wang, Christopher King, Michael S Fernandopulle, Tomas Sneideris, Joseph L Watson, Visakh V S Pillai, William Meadows, James W Henderson, Joseph E Chambers, Jane L Wagstaff, Sioned H Williams, Helena Coyle, Greta Šneiderienė, Yuqian Lu, Shuyuan Zhang, Stefan J Marciniak, Stefan M V Freund, Emmanuel Derivery, Michael E Ward, Michele Vendruscolo, Tuomas P J Knowles, Peter St George-Hyslop.
      Phase transitions of cellular proteins and lipids play a key role in governing the organisation and coordination of intracellular biology. Recent work has raised the intriguing prospect that phase transitions in proteins and lipids can be co-regulated. Here we investigate this possibility in the ribonucleoprotein (RNP) granule-ANXA11-lysosome ensemble, where ANXA11 tethers RNP granules to lysosomal membranes to enable their co-trafficking. We show that changes to the protein phase state within this system, driven by the low complexity ANXA11 N-terminus, induces a coupled phase state change in the lipids of the underlying membrane. We identify the ANXA11 interacting proteins ALG2 and CALC as potent regulators of ANXA11-based phase coupling and demonstrate their influence on the nanomechanical properties of the ANXA11-lysosome ensemble and its capacity to engage RNP granules. The phenomenon of protein-lipid phase coupling we observe within this system serves as a potential regulatory mechanism in RNA trafficking and offers an important template to understand other examples across the cell whereby biomolecular condensates closely juxtapose organellar membranes.
    DOI:  https://doi.org/10.1038/s41467-025-58142-5
  21. Cell Death Differ. 2025 Mar 28.
    The USP43/RNF2 axis negatively regulates antiviral innate immunity by promoting TBK1 ubiquitination and degradation.
    Xibao Zhao, Qianqian Di, Jin Chen, Jing Ling, Jiazheng Quan, Zizhao Zhao, Hongrui Li, Shaoying Chen, Xunwei Li, Xiwei Guo, Han Wu, Yue Xiao, Weilin Chen.
      The E3 ubiquitin ligase usually regulates the substrate proteins ubiquitination and degradation, but the study of itself post-translational modification and stability is still elusive. Here, we reveal that E3 ubiquitin ligase ring finger protein 2 (RNF2) is deubiquitinated and stabilized by ubiquitin specific peptidase 43 (USP43) through interactome and quantitative ubiquitinome mass spectrometry analysis. This study demonstrated that USP43, as a deubiquitinating enzyme, negatively regulates the expression of type I interferon (IFN) and the Usp43 deficient enhances antiviral innate immune response against VSV infection both in vitro and in vivo. Mechanistically, USP43 negatively regulates antiviral immunity by promoting RNF2-mediated TBK1 ubiquitination and degradation. USP43 stabilizes RNF2 by removing K48-linked ubiquitination of RNF2 at Lys239 and Lys249, while RNF2 promotes TBK1 degradation by increasing K48-linked ubiquitination of TBK1 at Lys670. These findings uncover the E3 ubiquitin ligase RNF2 post-translational ubiquitination modification and stability regulation, and reveals a novel mechanism that the USP43/RNF2 axis in regulating antiviral innate immunity.
    DOI:  https://doi.org/10.1038/s41418-025-01491-x
  22. J Pharmacol Exp Ther. 2025 Feb 28. pii: S0022-3565(25)39737-5. [Epub ahead of print]392(4): 103524
    DBeQ derivative targets vacuolar protein sorting 4 functions in cancer cells and suppresses tumor growth in mice.
    Kevin A Fundora, Yan Zhuang, Kouta Hamamoto, Guifang Wang, Longgui Chen, Tatsuya Hattori, Xinwen Liang, Lei Bao, Venugopal Vangala, Fang Tian, Yoshinori Takahashi, Hong-Gang Wang.
      Vacuolar protein sorting 4 (VPS4) is an AAA-ATPase that catalyzes the endosomal sorting complex required for transport-III disassembly, mediating various cellular membrane-remodeling processes including endolysosomal membrane repair and autophagosome closure. Humans have 2 VPS4 paralogs, VPS4A and VPS4B, and the loss of either paralog has been identified in a significant proportion of cancers, rendering them dependent on the remaining paralog for survival. In this study, we explored VPS4 inhibition as an anticancer strategy by investigating the mechanisms of VPS4 inhibition-induced cell death and developing small-molecule compounds that target VPS4 functions. We found that genetic inhibition of VPS4 triggered both caspase-8 (CASP8)-dependent apoptosis and caspase-independent cell death in osteosarcoma cells. We synthesized approximately 100 derivatives of the VPS4 and related AAA-ATPase valosin-containing protein inhibitor DBeQ and screened for their inhibitory effects on VPS4 ATPase activity using the EnzChek phosphate assay and a high-content assay monitoring GFP-CHMP4B puncta formation. In cells, the lead compound 4-107 caused endolysosomal damage, disrupted subsequent membrane repair, inhibited autophagy, and led to the accumulation of the endosomal sorting complex required for transport on membranes. These effects were accompanied by the stabilization of CASP8 on autophagosomal membranes, leading to the induction of CASP8-mediated apoptosis. Notably, the CASP8-mediated cell death induced by 4-107 was further enhanced by the loss of either VPS4 paralog. Moreover, 4-107 exhibited antitumor activity in a syngeneic mouse model of neuroblastoma. Our findings provide an important step for targeting VPS4 in cancer and developing VPS4 inhibitors as a cancer treatment strategy. SIGNIFICANCE STATEMENT: VPS4A and VPS4B, paralogs of the AAA-ATPase VPS4, are critical for cancer cell survival. This study reports that 4-107, a DBeQ derivative, inhibits VPS4 ATPase activity, induces CASP8-mediated apoptosis, and suppresses tumor growth in mice. This study supports the further development of VPS4A/B inhibitors as a promising anticancer treatment strategy.
    Keywords:  Apoptosis; Autophagy; ESCRT; Lysosomal membrane integrity; VPS4
    DOI:  https://doi.org/10.1016/j.jpet.2025.103524
  23. Cell Rep. 2025 Mar 27. pii: S2211-1247(25)00259-1. [Epub ahead of print]44(4): 115488
    The impact of Coronavirus Nsp1 on host mRNA degradation is independent of its role in translation inhibition.
    Emilie Bäumlin, Dominic Andenmatten, Jonas Luginbühl, Aurélien Lalou, Nino Schwaller, Evangelos D Karousis.
      When host cells are infected with coronaviruses, the first viral protein produced is non-structural protein 1 (Nsp1). This protein inhibits host protein synthesis and induces host mRNA degradation to enhance viral proliferation. Despite its critical role, the mechanism by which Nsp1 mediates cellular mRNA degradation remains unclear. In this study, we use cell-free translation to address how host mRNA stability is regulated by Nsp1. We reveal that SARS-CoV-2 Nsp1 binding to the ribosome is enough to trigger mRNA degradation independently of ribosome collisions or active translation. MERS-CoV Nsp1 inhibits translation without triggering degradation, highlighting mechanistic differences between the two Nsp1 counterparts. Nsp1 and viral mRNAs appear to co-evolve, rendering viral mRNAs immune to Nsp1-mediated degradation in SARS-CoV-2, MERS-CoV, and Bat-Hp viruses. By providing insights into the mode of action of Nsp1, our study helps to understand the biology of Nsp1 better and find strategies for therapeutic targeting against coronaviral infections.
    Keywords:  COVID-19; CP: Microbiology; CP: Molecular biology; MERS-CoV; Nsp1; SARS-CoV-2; cell-free translation; coronaviruses; host-viral interactions; mRNA decay; mRNA degradation; mRNA translation
    DOI:  https://doi.org/10.1016/j.celrep.2025.115488
  24. Sci Adv. 2025 Mar 28. 11(13): eads2086
    Drosophila and human Headcase define a new family of ribonucleotide granule proteins required for stress response.
    Delia Ricolo, Jordi Casanova, Panagiotis Giannios.
      Cells have means to adapt to environmental stresses such as temperature fluctuations, toxins, or nutrient availability. Stress responses, being dynamic, extend beyond transcriptional control and encompass post-transcriptional mechanisms allowing for rapid changes in protein synthesis. Previous research has established headcase as a fundamental gene for stress responses and survival of the Drosophila adult progenitor cells (APCs). However, the molecular role of Headcase has remained elusive. Here, we identify Headcase as a component of ribonucleoprotein (RNP) granules. We also show that, Headcase is required for proper RNP granule formation and remodeling upon stress and is crucial for translation control. Likewise, the human Headcase homolog (HECA) is identified as a component of RNP granules and has similar roles in translational regulation and stress protection. Thus, Headcase proteins define a new family contributing to specific roles among the RNP heterogeneous network.
    DOI:  https://doi.org/10.1126/sciadv.ads2086
  25. EMBO J. 2025 Mar 24.
    S-palmitoylation modulates ATG2-dependent non-vesicular lipid transport during starvation-induced autophagy.
    Wenhui Zheng, Maomao Pu, Sai Zeng, Hongtao Zhang, Qian Wang, Tao Chen, Tianhua Zhou, Chunmei Chang, Dante Neculai, Wei Liu.
      Lipid transfer proteins mediate the non-vesicular transport of lipids at membrane contact sites to regulate the lipid composition of organelle membranes. Despite significant recent advances in our understanding of the structural basis for lipid transfer, its functional regulation remains unclear. In this study, we report that S-palmitoylation modulates the cellular function of ATG2, a rod-like lipid transfer protein responsible for transporting phospholipids from the endoplasmic reticulum (ER) to phagophores during autophagosome formation. During starvation-induced autophagy, ATG2A undergoes depalmitoylation as the balance between ZDHHC11-mediated palmitoylation and APT1-mediated depalmitoylation. Inhibition of ATG2A depalmitoylation leads to impaired autophagosome formation and disrupted autophagic flux. Further, in cell and in vitro analyses demonstrate that S-palmitoylation at the C-terminus of ATG2A anchors the C-terminus to the ER. Depalmitoylation detaches the C-terminus from the ER membrane, enabling it to interact with phagophores and promoting their growth. These findings elucidate a S-palmitoylation-dependent regulatory mechanism of cellular ATG2, which may represent a broad regulatory strategy for lipid transport mediated by bridge-like transporters within cells.
    Keywords:  ATG2; Autophagy; Lipid Transfer Protein; S-palmitoylation
    DOI:  https://doi.org/10.1038/s44318-025-00410-7
  26. EMBO Rep. 2025 Mar 27.
    Praja2 controls P-body assembly and translation in glioblastoma by non-proteolytic ubiquitylation of DDX6.
    Emanuela Senatore, Rosario Avolio, Laura Rinaldi, Francesco Chiuso, Maria A Oliva, Chiara D'Ambrosio, Antonio Giuseppe Bianco, Emiliano Dalla, Stefano Maria Pagnotta, Raffaella Flammia, Concetta Ambrosino, Domenico Memoli, Gabriele Turacchio, Sonia Ines Mimoune, Yves Toiron, Stephane Audebert, Luc Camoin, Luca Lignitto, Andrea Scaloni, Antonietta Arcella, Antonio Feliciello.
      Glioblastoma multiforme (GBM) is the most lethal form of malignant brain tumor in adults. Dysregulation of protein synthesis contributes to cancer cell plasticity, driving GBM cell heterogeneity, metastatic behavior, and drug resistance. Understanding the complex network and signaling pathways governing protein translation, is therefore an important goal for GBM treatment. Here we identify a novel signaling network centered on the E3 ubiquitin ligase praja2 that controls protein translation in GBM. Praja2 forms a multimeric complex with the RNA helicase DDX6, which inhibits translation of target RNAs within processing bodies (P-bodies). Stimulation of cAMP signaling through activation of G-protein-coupled receptors induces P-body assembly through praja2-mediated non-proteolytic polyubiquitylation of DDX6. Genetic inactivation of praja2 reshapes DDX6/mRNA complexes and translating polysomes and promotes cellular senescence and GBM growth arrest. Expression of an ubiquitylation-defective DDX6 mutant suppresses the assembly of P-bodies and sustains GBM growth. Taken together, our findings identify a cAMP-driven network that controls translation in P-bodies and GBM growth.
    Keywords:  Glioblastoma; P-body; PKA; Praja2; cAMP
    DOI:  https://doi.org/10.1038/s44319-025-00425-5
  27. J Biol Chem. 2025 Mar 22. pii: S0021-9258(25)00287-X. [Epub ahead of print] 108438
    Melatonin-mediated cGAS-STING signal in senescent macrophages promote TNBC chemotherapy resistance and drive the SASP.
    Xiaoqiang Zhang, Minyu Zhuang, Hongfei Zhang, Yanhui Zhu, Junzhe Yang, Xian Wu, Xiafei Yu, Jing Tao, Xiaoan Liu.
      The build-up of senescent cells in tissues is a key indicator of aging, associated with negative prognosis and therapy resistance. Despite immune dysfunction related to aging, also known as immunosenescence, is recognized as a factor in this process, the exact mechanisms are still unclear. In this study, we reported that melatonin deficiency accelerated macrophage senescence in triple-negative breast cancer (TNBC), whereas, melatonin could defend macrophages against senescence through the Nfatc1-Trim26-cgas-Sting pathway. Mechanistically, melatonin enhanced the nuclear translocation of Nfatc1 and elevated Trim26 transcription levels. Trim26, functioning as an E3 ligase, ubiquitinates cgas, thereby inhibiting the activation of the cgas-Sing pathway and consequently preventing cell senescence. Conversely, melatonin deficiency induced cgas-Sting pathway activation to promote macrophage aging. Our results show that melatonin inhibited macrophage senescence and improved chemotherapy responsiveness, with further enhancement when combined with the cgas inhibitor (G150). Overall, our findings indicated that melatonin protects macrophages from immunosenescence, suggesting its therapeutic potential for enhancing chemotherapy response.
    Keywords:  TNBC; chemotherapy; macrophage; melatonin; senescence
    DOI:  https://doi.org/10.1016/j.jbc.2025.108438
  28. JCI Insight. 2025 Mar 25. pii: e173484. [Epub ahead of print]
    SEC24C deficiency causes trafficking and glycosylation abnormalities in an epileptic encephalopathy with cataracts and dyserythropoeisis.
    Nina Bögershausen, Büsranur Cavdarli, Taylor Nagai, Miroslav P Milev, Alexander Wolff, Mahsa Mehranfar, Julia Schmidt, Dharmendra Choudhary, Óscar Gutiérrez-Gutiérrez, Lukas Cyganek, Djenann Saint-Dic, Arne Zibat, Karl Köhrer, Tassilo E Wollenweber, Dagmar Wieczorek, Janine Altmüller, Tatiana Borodina, Dilek Kaçar, Göknur Haliloğlu, Yun Li, Christian Thiel, Michael Sacher, Ela W Knapik, Gökhan Yigit, Bernd Wollnik.
      As a major component of intracellular trafficking, the coat protein complex II (COPII) is indispensable for cellular function during embryonic development and throughout life. The four SEC24 proteins (A-D) are essential COPII components involved in cargo selection and packaging. A human disorder corresponding to alterations of SEC24 function is currently only known for SEC24D. Here, we report that biallelic loss of SEC24C leads to a syndrome characterized by primary microcephaly, brain anomalies, epilepsy, hearing loss, liver dysfunction, anemia, and cataracts in an extended consanguineous family with four affected individuals. We show that knockout of sec24C in zebrafish recapitulates important aspects of the human phenotype. SEC24C-deficient fibroblasts display alterations in the expression of several COPII components as well as impaired anterograde trafficking to the Golgi, indicating a severe impact on COPII function. Transcriptome analysis revealed that SEC24C deficiency also impacts the proteasome and autophagy pathways. Moreover, a shift in the N-glycosylation pattern and deregulation of the N-glycosylation pathway suggest a possible secondary alteration of protein glycosylation, linking the described disorder with the congenital disorders of glycosylation.
    Keywords:  Epilepsy; Genetics; Glycobiology; Neuroscience; Protein traffic
    DOI:  https://doi.org/10.1172/jci.insight.173484
  29. Nature. 2025 Mar 26.
    Plasticity of the mammalian integrated stress response.
    Chien-Wen Chen, David Papadopoli, Krzysztof J Szkop, Bo-Jhih Guan, Mohammed Alzahrani, Jing Wu, Raul Jobava, Mais M Asraf, Dawid Krokowski, Anastasios Vourekas, William C Merrick, Anton A Komar, Antonis E Koromilas, Myriam Gorospe, Matthew J Payea, Fangfang Wang, Benjamin L L Clayton, Paul J Tesar, Ashleigh Schaffer, Alexander Miron, Ilya Bederman, Eckhard Jankowsky, Christine Vogel, Leoš Shivaya Valášek, Jonathan D Dinman, Youwei Zhang, Boaz Tirosh, Ola Larsson, Ivan Topisirovic, Maria Hatzoglou.
      An increased level of phosphorylation of eukaryotic translation initiation factor 2 subunit-α (eIF2α, encoded by EIF2S1; eIF2α-p) coupled with decreased guanine nucleotide exchange activity of eIF2B is a hallmark of the 'canonical' integrated stress response (c-ISR)1. It is unclear whether impaired eIF2B activity in human diseases including leukodystrophies2, which occurs in the absence of eIF2α-p induction, is synonymous with the c-ISR. Here we describe a mechanism triggered by decreased eIF2B activity, distinct from the c-ISR, which we term the split ISR (s-ISR). The s-ISR is characterized by translational and transcriptional programs that are different from those observed in the c-ISR. Opposite to the c-ISR, the s-ISR requires eIF4E-dependent translation of the upstream open reading frame 1 and subsequent stabilization of ATF4 mRNA. This is followed by altered expression of a subset of metabolic genes (for example, PCK2), resulting in metabolic rewiring required to maintain cellular bioenergetics when eIF2B activity is attenuated. Overall, these data demonstrate a plasticity of the mammalian ISR, whereby the loss of eIF2B activity in the absence of eIF2α-p induction activates the eIF4E-ATF4-PCK2 axis to maintain energy homeostasis.
    DOI:  https://doi.org/10.1038/s41586-025-08794-6
  30. J Am Chem Soc. 2025 Mar 27.
    Capturing the Conformational Heterogeneity of HSPB1 Chaperone Oligomers at Atomic Resolution.
    Raymond F Berkeley, Alexander P Plonski, Tien M Phan, Kristof Grohe, Lukas Becker, Sebastian Wegner, Mark A Herzik, Jeetain Mittal, Galia T Debelouchina.
      Small heat shock proteins (sHSPs), including HSPB1, are essential regulators of cellular proteostasis that interact with unfolded and partially folded proteins to prevent aberrant misfolding and aggregation. These proteins fulfill a similar role in biological condensates, where they interact with intrinsically disordered proteins to modulate their liquid-liquid and liquid-to-solid phase transitions. Characterizing the sHSP structure, dynamics, and client interactions is challenging due to their partially disordered nature, their tendency to form polydisperse oligomers, and their diverse range of clients. In this work, we leverage various biophysical methods, including fast 1H-based magic angle spinning (MAS) NMR spectroscopy, molecular dynamics (MD) simulations, and modeling, to shed new light on the structure and dynamics of HSPB1 oligomers. Using split-intein-mediated segmental labeling, we provide unambiguous evidence that in the oligomer context, the N-terminal domain (NTD) of HSPB1 is rigid and adopts an ensemble of heterogeneous conformations, the α-Crystallin domain (ACD) forms dimers and experiences multiple distinct local environments, while the C-terminal domain (CTD) remains highly dynamic. Our computational models suggest that the NTDs participate in extensive NTD-NTD and NTD-ACD interactions and are sequestered within the oligomer interior. We further demonstrate that HSPB1 higher order oligomers disassemble into smaller oligomeric species in the presence of a client protein and that an accessible NTD is essential for HSPB1 partitioning into condensates and interactions with client proteins. Our integrated approach provides a high-resolution view of the complex oligomeric landscape of HSPB1 and sheds light on the elusive network of interactions that underlies the function of HSPB1 in biological condensates.
    DOI:  https://doi.org/10.1021/jacs.4c18668
  31. Nature. 2025 Mar 26.
    irCLIP-RNP and Re-CLIP reveal patterns of dynamic protein assemblies on RNA.
    Luca Ducoli, Brian J Zarnegar, Douglas F Porter, Robin M Meyers, Weili Miao, Nicholas M Riley, Suhas Srinivasan, Leandra V Jackrazi, Yen-Yu Yang, Zhouxian Li, Yinsheng Wang, Carolyn R Bertozzi, Ryan A Flynn, Paul A Khavari.
      RNA-binding proteins (RBPs) control varied processes, including RNA splicing, stability, transport and translation1-3. Dysfunctional RNA-RBP interactions contribute to the pathogenesis of human disease1,4,5; however, characterizing the nature and dynamics of multiprotein assemblies on RNA has been challenging. Here, to address this, non-isotopic ligation-based ultraviolet-light-induced cross-linking and immunoprecipitation6 was combined with mass spectrometry (irCLIP-RNP) to identify RNA-dependent associated proteins (RDAPs) co-bound to RNA with any RBP of interest. irCLIP-RNP defined landscapes of multimeric protein assemblies on RNA, revealing patterns of RBP-RNA associations, including cell-type-selective combinatorial relationships between RDAPs and primary RBPs. irCLIP-RNP also defined dynamic RDAP remodelling in response to epidermal growth factor (EGF), revealing that EGF-induced recruitment of UPF1 adjacent to HNRNPC promotes splicing surveillance of cell proliferation mRNAs. To identify the RNAs simultaneously co-bound by multiple studied RBPs, a sequential immunoprecipitation irCLIP (Re-CLIP) method was also developed. Re-CLIP confirmed binding relationships observed in irCLIP-RNP and identified HNRNPC and UPF1 RBP co-binding on RND3 and DDX3X mRNAs. irCLIP-RNP and Re-CLIP provide a framework to identify and characterize dynamic RNA-protein assemblies in living cells.
    DOI:  https://doi.org/10.1038/s41586-025-08787-5
  32. EMBO Rep. 2025 Mar 24.
    Induction of a transcriptional adaptation response by RNA destabilization events.
    Lihan Xie, Gabrielius Jakutis, Christopher M Dooley, Stefan Guenther, Zacharias Kontarakis, Sarah P Howard, Thomas Juan, Didier Y R Stainier.
      Transcriptional adaptation (TA) is a cellular process whereby mRNA-destabilizing mutations are associated with the transcriptional upregulation of so-called adapting genes. The nature of the TA-triggering factor(s) remains unclear, namely whether an mRNA-borne premature termination codon or the subsequent mRNA decay process, and/or its products, elicits TA. Here, working with mouse Actg1, we first establish two types of perturbations that lead to mRNA destabilization: Cas9-induced mutations predicted to lead to mutant mRNA decay, and Cas13d-mediated mRNA cleavage. We find that both types of perturbations are effective in degrading Actg1 mRNA, and that they both upregulate Actg2. Notably, increased chromatin accessibility at the Actg2 locus was observed only in the Cas9-induced mutant cells but not in the Cas13d-targeted cells, suggesting that chromatin remodeling is not required for Actg2 upregulation. We further show that ribozyme-mediated Actg1 pre-mRNA cleavage also leads to a robust upregulation of Actg2, and that this upregulation is again independent of chromatin remodeling. Together, these data highlight the critical role of RNA destabilization events as a trigger for TA, or at least a TA-like response.
    Keywords:  CRISPR/Cas13d; CRISPR/Cas9; Self-cleaving Ribozyme; Transcriptional Adaptation; mRNA Decay
    DOI:  https://doi.org/10.1038/s44319-025-00427-3
  33. J Med Chem. 2025 Mar 26.
    Deubiquitinase-Targeting Chimeras (DUBTACs) as a Potential Paradigm-Shifting Drug Discovery Approach.
    Zonghui Ma, Mingxiang Zhou, Haiying Chen, Qiang Shen, Jia Zhou.
      Developing proteolysis-targeting chimeras (PROTACs) is well recognized through target protein degradation (TPD) toward promising therapeutics. While a variety of diseases are driven by aberrant ubiquitination and degradation of critical proteins with protective functions, target protein stabilization (TPS) rather than TPD is emerging as a unique therapeutic modality. Deubiquitinase-targeting chimeras (DUBTACs), a class of heterobifunctional protein stabilizers consisting of deubiquitinase (DUB) and protein-of-interest (POI) targeting ligands conjugated with a linker, can rescue such proteins from aberrant elimination. DUBTACs stabilize the levels of POIs in a DUB-dependent manner, removing ubiquitin from polyubiquitylated and degraded proteins. DUBTACs can induce a new interaction between POI and DUB by forming a POI-DUBTAC-DUB ternary complex. Herein, therapeutic benefits of TPS approaches for human diseases are introduced, and recent advances in developing DUBTACs are summarized. Relevant challenges, opportunities, and future perspectives are also discussed.
    DOI:  https://doi.org/10.1021/acs.jmedchem.4c02975
  34. Autophagy. 2025 Mar 26.
    RHOD mediates ATG9A trafficking to promote autophagosome formation during autophagy in cancer.
    Sijia Wang, Jing Ren, Jinghan Chi, Yifei Guan, Ran Zheng, Juan Wang, Xinhui Liu, Hua Huang.
      ATG9A is a transmembrane protein essential for macroautophagy/autophagy that drives autophagosome formation by delivering essential proteins and lipids to the phagophore through vesicle trafficking. Here, we demonstrate that the atypical Rho GTPase RHOD is required for ATG9A trafficking and stimulates autophagosome formation. Upon starvation, RHOD interacted with ATG9A and accompanied ATG9A trafficking from the Golgi toward phagophores. In addition, starvation-induced high levels of RHOD resulted in Golgi fragmentation to further promote ATG9A vesicle export from the trans-Golgi network to the peripheral region. Loss of RHOD suppressed ATG9A trafficking and reduced the distribution of ATG9A on the phagophore. Moreover, WHAMM (WASP homolog associated with actin, golgi membranes and microtubules) forms a complex with RHOD and participates in this process in a RHOD-dependent manner. Importantly, RHOD mutants, which lack the exon II-containing effector region motif that is required for ATG9A binding or lack the CAAX box that is responsible for membrane targeting, fail to stimulate ATG9A trafficking and autophagosome formation. Furthermore, RHOD plays a distinct suppressor role in tumor development, partly associated with its regulatory effect on autophagy. These findings reveal the important roles of RHOD in autophagy and tumor development.
    Keywords:  ATG9A; RHOD; WHAMM; autophagy; lung cancer; vesicle trafficking
    DOI:  https://doi.org/10.1080/15548627.2025.2484604
  35. iScience. 2025 Mar 21. 28(3): 111884
    A large-scale sORF screen identifies putative microproteins involved in cancer cell fitness.
    Dörte Schlesinger, Christopher Dirks, Carmen Navarro, Lorenzo Lafranchi, Anna Spinner, Glancis Luzeena Raja, Gregory Mun-Sum Tong, Jürgen Eirich, Thomas Farid Martinez, Simon Johannes Elsässer.
      The human genome contains thousands of potentially coding short open reading frames (sORFs). While a growing set of microproteins translated from these sORFs have been demonstrated to mediate important cellular functions, the majority remains uncharacterized. In our study, we performed a high-throughput CRISPR-Cas9 knock-out screen targeting 11,776 sORFs to identify microproteins essential for cancer cell line growth. We show that the CENPBD2P gene encodes a translated sORF and promotes cell fitness. We selected five additional candidate sORFs encoding microproteins between 11 and 63 amino acids in length for further functional assessment. Green fluorescent protein fusion constructs of these microproteins localized to distinct subcellular compartments, and the majority showed reproducible biochemical interaction partners. Studying the fitness and transcriptome of sORF knock-outs and complementation with the corresponding microprotein, we identify rescuable phenotypes while also illustrating the limitations and caveats of our pipeline for sORF functional screening and characterization.
    Keywords:  cancer; cell biology; classification of proteins; methodology in biological sciences; molecular genetics
    DOI:  https://doi.org/10.1016/j.isci.2025.111884
  36. Nature. 2025 Mar 26.
    STING agonist-based ER-targeting molecules boost antigen cross-presentation.
    Xiafeng Wang, Zhangping Huang, Lixiao Xing, Liru Shang, Juan Jiang, Caiguanxi Deng, Wei Yu, Lin Peng, Hao Yang, Xiaohong Zheng, Xinmin Liu, Haolan Yang, Yixin Chen, Yongyong Li, Jing Liu, Xi Xie, Wei Xu, Xiaojun Xia, Zezhong Liu, Wanli Liu, Shibo Jiang, Yingyue Zeng, Lu Lu, Ji Wang.
      CD8+ T cell immune responses are critical for combating infectious diseases and tumours1-3. Antigen cross-presentation, primarily occurring at the endoplasmic reticulum (ER) of dendritic cells, is essential for protein-based vaccines to induce CD8+ T cell responses4. Current efforts have focused on antigen delivery at the tissue and cellular levels, whereas subcellular delivery has been limited to facilitating antigen escape from lysosomes into the cytosol. In the absence of a small-sized high-affinity ER-targeting molecule, the importance of the 'last mile' from the cytosol to the ER remains elusive. Here we developed stimulator of interferon genes (STING) agonist-based ER-targeting molecules (SABER), which effectively deliver antigens to the ER and cluster key machinery in cross-presentation to form microreactors by folding the ER membrane. Conjugation of SABER to various antigens substantially enhances the induction of CD8+ T cell immune responses to tumour neoantigens and conserved viral epitopes, far exceeding that achieved by mixtures of antigens with STING agonists or conventional adjuvants. SABER also retains a potent adjuvant effect, effectively enhancing the ability of a SARS-CoV-2 subunit vaccine to induce broadly neutralizing antibodies. This study provides a high-affinity ER-targeting delivery system and vaccine adjuvant, demonstrating that precise subcellular delivery targeting the last mile of cross-presentation can lead to a qualitative leap.
    DOI:  https://doi.org/10.1038/s41586-025-08758-w
  37. Oncogene. 2025 Mar 27.
    SUMOylation facilitates the stability of BCR-ABL to promote chronic myeloid leukemia progression.
    Lu Zhang, Xuefeng Wang, Dongmei Hu, Shijie Li, Mingshan Sun, Qian Liu, Huimin Feng, Minran Zhou, Chunyan Chen, Huan Zhou, Sai Ma.
      Tyrosine kinase inhibitors (TKIs) targeting the oncoprotein BCR-ABL have improved the prognosis for patients with chronic myeloid leukemia (CML). However, TKI resistance and persistent expression of BCR-ABL are responsible for the relapse and progression of CML. Here, we describe a novel approach to induce BCR-ABL protein degradation by small ubiquitin-like modifier (SUMO) modification. The E3 SUMO ligase TRIM28, upregulated during the progression of CML, promoted SUMOylation of BCR-ABL, thereby inhibiting its binding to the autophagy receptor P62 and repressing its autophagic degradation. Accordingly, genetic and pharmacological inhibition of TRIM28 or SUMOylation suppressed progression in both the CML mouse model and patient-derived xenograft model. Furthermore, targeting SUMOylation of BCR-ABL restrained the proliferation of TKI-resistant CML cells. These results identify the mechanism by which TRIM28 maintains BCR-ABL stability to promote CML progression and suggest SUMOylation as a target for CML treatment.
    DOI:  https://doi.org/10.1038/s41388-025-03350-y
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