bims-proteo Biomed News
on Proteostasis
Issue of 2025–03–16
53 papers selected by
Eric Chevet, INSERM
  1. Structural insights into the coupling between VCP, an essential unfoldase, and a deubiquitinase.
  2. Protein Glycosylation Patterns Shaped By the IRE1-XBP1s Arm of the Unfolded Protein Response.
  3. The E3 ubiquitin ligase RNF126 facilitates quality control of unimported mitochondrial membrane proteins.
  4. SE(3)-Equivariant Ternary Complex Prediction Towards Target Protein Degradation.
  5. Pre-Clinical Model of Dysregulated FicD AMPylation causes diabetes by disrupting pancreatic endocrine homeostasis.
  6. Mechanistic basis for PYROXD1-mediated protection of the human tRNA ligase complex against oxidative inactivation.
  7. An endoplasmic reticulum-associated structure sequesters misassembled FG-Nups to help maintain NPC function.
  8. The deubiquitinase USP45 inhibits autophagy through actin regulation by Coronin 1B.
  9. The TBK1-SCFFBXO3-TMEM192-TAX1BP1 axis: a novel regulatory mechanism for Lysophagy.
  10. Disruption of ER-mitochondria contact sites induces autophagy-dependent loss of P-bodies through the Ca2+-CaMKK2-AMPK pathway.
  11. A mini-hairpin shaped nascent peptide blocks translation termination by a distinct mechanism.
  12. Organizing principles underlying COPII-mediated transport.
  13. Identification of a non-inhibitory aptameric ligand to CRL2ZYG11B E3 ligase for targeted protein degradation.
  14. ATG2A acts as a tether to regulate autophagosome-lysosome fusion in neural cells.
  15. Discovery of RNA-Protein Molecular Clamps Using Proteome-Wide Stability Assays.
  16. Convergent Assembly of Homo- and Heterotypic Ubiquitin Chains from Functionalized, Expressed Monomers via Thiol-Ene Chemistry.
  17. Protein disulfide isomerase is essential for osteoblast differentiation in mice.
  18. Suppression of stress granule formation is a vulnerability imposed by mutant p53.
  19. RNA anchoring of Upf1 facilitates recruitment of Dcp2 in the NMD decapping complex.
  20. Structural basis for anomalous cellular trafficking behavior of glaucoma-associated A427T mutant myocilin.
  21. Yeast poly(A)-binding protein (Pab1) controls translation initiation in vivo primarily by blocking mRNA decapping and decay.
  22. Mechanism and engineering of endoplasmic reticulum-localized membrane protein folding in Saccharomyces cerevisiae.
  23. Structure of human PINK1 at a mitochondrial TOM-VDAC array.
  24. An asymmetric nautilus-like HflK/C assembly controls FtsH proteolysis of membrane proteins.
  25. ATG9A vesicles are a subtype of intracellular nanovesicle.
  26. Molecular grammars of intrinsically disordered regions that span the human proteome.
  27. Unraveling UPR-mediated intercellular crosstalk: Implications for immunotherapy resistance mechanisms.
  28. The translation inhibitors kasugamycin, edeine and GE81112 target distinct steps during 30S initiation complex formation.
  29. Mechanisms and functions of lysosomal lipid homeostasis.
  30. Acetic acid-induced stress granules function as scaffolding complexes for Hog1 activation by Pbs2.
  31. Proteostasis and metabolic dysfunction characterize a subset of storage-induced senescent erythrocytes targeted for post-transfusion clearance.
  32. Integrated multi-omic characterizations of the synapse reveal RNA processing factors and ubiquitin ligases associated with neurodevelopmental disorders.
  33. Selection for somatic escape variants in SERPINA1 in the liver of patients with alpha-1 antitrypsin deficiency.
  34. SUMO4 promotes SUMO deconjugation required for DNA double-strand-break repair.
  35. Neurofilament accumulation disrupts autophagy in giant axonal neuropathy.
  36. Chaperone-mediated autophagy regulates the metastatic state of mesenchymal tumors.
  37. Monoselective Histone Deacetylase 6 PROTAC Degrader Shows In Vivo Tractability.
  38. Cellular homeostatic responses to lysosomal damage.
  39. Microwave-assisted synthesis of pomalidomide building blocks for rapid PROTAC and molecular glue development.
  40. Protein misfolding involving entanglements providesa structural explanation for the origin of stretched-exponential refolding kinetics.
  41. PP2A adapter protein IER5 induces dephosphorylation and degradation of MDM2, thereby stabilizing p53.
  42. Preferential Localization of STIM1 to dendritic subsurface ER structures in Mouse Purkinje Cells.
  43. The DEAD-box helicase eIF4A1/2 acts as RNA chaperone during mitotic exit enabling chromatin decondensation.
  44. Eukaryotic initiation factors eIF4F and eIF4B promote translation termination upon closed-loop formation.
  45. Development of a Calreticulin-targeting Glycan Ligand Based on a Hybrid Binding Concept.
  46. Attenuating ABHD17 isoforms augments the S-acylation and function of NOD2 and a subset of Crohn's disease-associated NOD2 variants.
  47. Sulfinyl Aziridines as Stereoselective Covalent Destabilizing Degraders of the Oncogenic Transcription Factor MYC.
  48. Effective targeting of PDGFRA-altered high-grade glioma with avapritinib.
  49. A versatile toolbox for determining IRES activity in cells and embryonic tissues.
  50. Protein interactions, calcium, phosphorylation, and cholesterol modulate CFTR cluster formation on membranes.
  51. Dual-Action-Only PROTACs.
  52. Targeting both death and paracaspase domains of MALT1 with antisense oligonucleotides overcomes resistance to immune-checkpoint inhibitors.
  53. SH3KBP1 promotes skeletal myofiber formation and functionality through ER/SR architecture integrity.
  1. J Cell Biol. 2025 May 05. pii: e202410148. [Epub ahead of print]224(5):
    Structural insights into the coupling between VCP, an essential unfoldase, and a deubiquitinase.
    Lauren E Vostal, Noa E Dahan, Matthew J Reynolds, Lily I Kronenberg, Tarun M Kapoor.
      Proteostasis involves degradation and recycling of proteins from organelles, membranes, and multiprotein complexes. These processes can depend on protein extraction and unfolding by the essential mechanoenzyme valosin-containing protein (VCP) and on ubiquitin chain remodeling by ubiquitin-specific proteases known as deubiquitinases (DUBs). How the activities of VCP and DUBs are coordinated is poorly understood. Here, we focus on the DUB VCPIP1, a VCP interactor required for post-mitotic Golgi and ER organization. We determine ∼3.3 Å cryogenic electron microscopy structures of VCP-VCPIP1 complexes in the absence of added nucleotide or the presence of an ATP analog. We find that up to 3 VCPIP1 protomers interact with the VCP hexamer to position VCPIP1's catalytic domain at the exit of VCP's central pore, poised to cleave ubiquitin following substrate unfolding. We observe competition between VCPIP1 and other cofactors for VCP binding and show that VCP stimulates VCPIP1's DUB activity. Together, our data suggest how the two enzyme activities can be coordinated to regulate proteostasis.
    DOI:  https://doi.org/10.1083/jcb.202410148
  2. Isr J Chem. 2024 Dec;pii: e202300162. [Epub ahead of print]64(12):
    Protein Glycosylation Patterns Shaped By the IRE1-XBP1s Arm of the Unfolded Protein Response.
    Kenny Chen, Matthew D Shoulders.
      The unfolded protein response (UPR) is a sensing and signaling pathway that surveys the endoplasmic reticulum (ER) for protein folding challenges and responds whenever issues are detected. UPR activation leads to upregulation of secretory pathway chaperones and quality control factors, as well as reduces the nascent protein load on the ER, thereby restoring and maintaining proteostasis. This paradigm-defining view of the role of the UPR is accurate, but it elides additional key functions of the UPR in cell biology. In particular, recent work has revealed that the UPR can shape the structure and function of N- and O-glycans installed on ER client proteins. This crosstalk between the UPR's response to protein misfolding and the regulation of glycosylation remains insufficiently understood. Still, emerging evidence makes it clear that the UPR, and particularly the IRE1-XBP1s arm of the UPR, may be a central regulator of protein glycosylation with important biological consequences. In this review, we discuss the crosstalk between proteostasis, the UPR, and glycosylation, present progress towards understanding biological functions of this crosstalk, and examine potential roles in diseases such as cancer.
    Keywords:  Endoplasmic reticulum stress; N-Glycosylation; O-Glycosylation; Protein folding; Proteostasis
    DOI:  https://doi.org/10.1002/ijch.202300162
  3. J Biol Chem. 2025 Mar 12. pii: S0021-9258(25)00252-2. [Epub ahead of print] 108403
    The E3 ubiquitin ligase RNF126 facilitates quality control of unimported mitochondrial membrane proteins.
    Di Liu, Xin-Yu Huo, Xiaoli Zhang, Zai-Rong Zhang.
      Pathological stress can lead to failure in the translocation of mitochondrial proteins, resulting in accumulation of unimported proteins within the cytosol and upregulation of proteasome for their quality control. Malfunction or delay in protein clearance causes dysregulation of mitochondrial protein homeostasis, cellular toxicity, and diseases. Ubiquilins (UBQLNs) are known to serve as chaperone which associates with unimported mitochondrial membrane protein precursors, and facilitates their proteasomal degradation. However, how UBQLN-engaged proteins are ubiquitinated and efficiently targeted to the proteasome are poorly understood. Here, using mitochondrial membrane protein ATP5G1 as a model substrate, we report that E3 ubiquitin ligase RNF126 interacts with substrate-engaged UBQLN1, thereby promoting ubiquitination and degradation of unimported proteins during mitochondrial stress. We find that UBQLN1's ubiquitin-associated domain (UBA) recruits RNF126 when its middle domain binds to unimported protein substrate. Recombinant RNF126 forms ternary complex with UBQLN1 and pATP5G1 in vitro and catalyzes ubiquitination of UBQLN1-bound ATP5G1. Without RNF126, proteasomal degradation of ATP5G1 was compromised. These results explain how RNF126 and ubiquilins interplay to ensure specific quality control of unimported mitochondrial membrane proteins under pathophysiological conditions.
    Keywords:  ATP synthase F(0) complex subunit C1; RNF126; Ubiquilin; cytosolic quality control; mitochondrial membrane protein degradation
    DOI:  https://doi.org/10.1016/j.jbc.2025.108403
  4. ArXiv. 2025 Feb 26. pii: arXiv:2502.18875v1. [Epub ahead of print]
    SE(3)-Equivariant Ternary Complex Prediction Towards Target Protein Degradation.
    Fanglei Xue, Meihan Zhang, Shuqi Li, Xinyu Gao, James A Wohlschlegel, Wenbing Huang, Yi Yang, Weixian Deng.
      Targeted protein degradation (TPD) induced by small molecules has emerged as a rapidly evolving modality in drug discovery, targeting proteins traditionally considered "undruggable". Proteolysis-targeting chimeras (PROTACs) and molecular glue degraders (MGDs) are the primary small molecules that induce TPD. Both types of molecules form a ternary complex linking an E3 ligase with a target protein, a crucial step for drug discovery. While significant advances have been made in binary structure prediction for proteins and small molecules, ternary structure prediction remains challenging due to obscure interaction mechanisms and insufficient training data. Traditional methods relying on manually assigned rules perform poorly and are computationally demanding due to extensive random sampling. In this work, we introduce DeepTernary, a novel deep learning-based approach that directly predicts ternary structures in an end-to-end manner using an encoder-decoder architecture. DeepTernary leverages an SE(3)-equivariant graph neural network (GNN) with both intra-graph and ternary inter-graph attention mechanisms to capture intricate ternary interactions from our collected high-quality training dataset, TernaryDB. The proposed query-based Pocket Points Decoder extracts the 3D structure of the final binding ternary complex from learned ternary embeddings, demonstrating state-of-the-art accuracy and speed in existing PROTAC benchmarks without prior knowledge from known PROTACs. It also achieves notable accuracy on the more challenging MGD benchmark under the blind docking protocol. Remarkably, our experiments reveal that the buried surface area calculated from predicted structures correlates with experimentally obtained degradation potency-related metrics. Consequently, DeepTernary shows potential in effectively assisting and accelerating the development of TPDs for previously undruggable targets.
  5. Mol Metab. 2025 Mar 10. pii: S2212-8778(25)00027-4. [Epub ahead of print] 102120
    Pre-Clinical Model of Dysregulated FicD AMPylation causes diabetes by disrupting pancreatic endocrine homeostasis.
    Amanda K Casey, Nathan M Stewart, Naqi Zaidi, Hillery F Gray, Hazel A Fields, Masahiro Sakurai, Carlos A Pinzon-Arteaga, Bret M Evers, Jun Wu, Kim Orth.
      The bi-functional enzyme FicD catalyzes AMPylation and deAMPylation of the endoplasmic reticulum chaperone BiP to modulate ER homeostasis and the unfolded protein response (UPR). Human hFicD with an arginine-to-serine mutation disrupts FicD deAMPylation activity resulting in severe neonatal diabetes. We generated the mFicDR371S mutation in mice to create a pre-clinical murine model for neonatal diabetes. We observed elevated BiP AMPylation levels across multiple tissues and signature markers for diabetes including glucose intolerance and reduced serum insulin levels. While the pancreas of mFicDR371S mice appeared normal at birth, adult mFicDR371S mice displayed disturbed pancreatic islet organization that progressed with age. mFicDR371S mice provide a preclinical mouse model for the study of UPR associated diabetes and demonstrate the essentiality of FicD for tissue resilience.
    Keywords:  AMPylation; BiP; FicD; Islet biology; Neonatal Diabetes; Unfolded Protein Response; insulin
    DOI:  https://doi.org/10.1016/j.molmet.2025.102120
  6. Nat Struct Mol Biol. 2025 Mar 11.
    Mechanistic basis for PYROXD1-mediated protection of the human tRNA ligase complex against oxidative inactivation.
    Luuk Loeff, Alena Kroupova, Igor Asanović, Franziska M Boneberg, Moritz M Pfleiderer, Luca Riermeier, Alexander Leitner, Andrè Ferdigg, Fabian Ackle, Javier Martinez, Martin Jinek.
      The metazoan tRNA ligase complex (tRNA-LC) has essential roles in tRNA biogenesis and unfolded protein response. Its catalytic subunit RTCB contains a conserved active-site cysteine that is susceptible to metal ion-induced oxidative inactivation. The flavin-containing oxidoreductase PYROXD1 preserves the activity of human tRNA-LC in a NAD(P)H-dependent manner, but its protective mechanism remains elusive. Here, we report a cryogenic electron microscopic structure of the human RTCB-PYROXD1 complex, revealing that PYROXD1 directly interacts with the catalytic center of RTCB through its carboxy-terminal tail. NAD(P)H binding and FAD reduction allosterically control PYROXD1 activity and RTCB recruitment, while reoxidation of PYROXD1 enables timed release of RTCB. PYROXD1 interaction is mutually exclusive with Archease-mediated RTCB guanylylation, and guanylylated RTCB is intrinsically protected from oxidative inactivation. Together, these findings provide a mechanistic framework for the protective function of PYROXD1 that maintains the activity of the tRNA-LC under aerobic conditions.
    DOI:  https://doi.org/10.1038/s41594-025-01516-6
  7. J Cell Sci. 2025 Mar 12. pii: jcs.263659. [Epub ahead of print]
    An endoplasmic reticulum-associated structure sequesters misassembled FG-Nups to help maintain NPC function.
    Madison Pletan, Emily Wang, Luke Gohmann, Billy Tsai.
      Misassembly of nucleoporins (Nups), central components of the nuclear pore complex (NPC), leads to Nup mislocalization outside of the nuclear envelope. Here we elucidate the fate of mislocalized Nups. To impair Nup assembly, we depleted the structural component Nup98 and found that nucleo-cytoplasmic transport by NPC remains largely intact. Under this condition, several phenylalanine-glycine (FG)-rich Nups no longer assemble at the nuclear envelope but instead accumulate at discrete puncta in the endoplasmic reticulum (ER) called foci. Formation of the foci harboring the misassembled FG-Nups requires the ER morphogenic proteins RTN3, ATL3, and LNP. Preventing accumulation of misassembled FG-Nups at the ER-foci impairs NPC nucleo-cytoplasmic transport, likely by allowing the misassembled FG-Nups to reach the nuclear envelope where they disrupt NPC function. Formation of the ER-foci is dependent on the kinesin-1 motor. Our results suggest that the ER can sequester misassembled Nups to help maintain NPC function. Because Nup mislocalization is found in many age-related neurodegenerative diseases, our data should illuminate the molecular basis of these pathologic conditions.
    Keywords:  Endoplasmic reticulum; Nuclear membrane transport; Protein quality control
    DOI:  https://doi.org/10.1242/jcs.263659
  8. J Cell Biol. 2025 May 05. pii: e202407014. [Epub ahead of print]224(5):
    The deubiquitinase USP45 inhibits autophagy through actin regulation by Coronin 1B.
    Yuchieh Jay Lin, Li-Ting Huang, Po-Yuan Ke, Guang-Chao Chen.
      The autophagy-lysosomal system comprises a highly dynamic and interconnected vesicular network that plays a central role in maintaining proteostasis and cellular homeostasis. In this study, we uncovered the deubiquitinating enzyme (DUB), dUsp45/USP45, as a key player in regulating autophagy and lysosomal activity in Drosophila and mammalian cells. Loss of dUsp45/USP45 results in autophagy activation and increased levels of V-ATPase to lysosomes, thus enhancing lysosomal acidification and function. Furthermore, we identified the actin-binding protein Coronin 1B (Coro1B) as a substrate of USP45. USP45 interacts with and deubiquitinates Coro1B, thereby stabilizing Coro1B levels. Notably, the ablation of USP45 or Coro1B promotes the formation of F-actin patches and the translocation of V-ATPase to lysosomes in an N-WASP-dependent manner. Additionally, we observed positive effects of dUsp45 depletion on extending lifespan and ameliorating polyglutamine (polyQ)-induced toxicity in Drosophila. Our findings highlight the important role of dUsp45/USP45 in regulating lysosomal function by modulating actin structures through Coro1B.
    DOI:  https://doi.org/10.1083/jcb.202407014
  9. Autophagy. 2025 Mar 13.
    The TBK1-SCFFBXO3-TMEM192-TAX1BP1 axis: a novel regulatory mechanism for Lysophagy.
    Na Yeon Park, Dong-Hyung Cho.
      Lysophagy, the selective macroautophagic/autophagic clearance of damaged lysosomes, is a critical mechanism for maintaining cellular homeostasis. Our recent study identified a novel regulatory axis involving TBK1, SCFFBXO3, TMEM192, and TAX1BP1 that orchestrates lysophagic flux following lysosomal damage. We demonstrated that TBK1-dependent phosphorylation of FBXO3 facilitates its interaction with TMEM192, promoting its ubiquitination and subsequent recognition by the autophagy receptor TAX1BP1. Perturbing this pathway significantly reduces lysophagic flux and results in accumulation of damaged lysosomes. These findings establish a previously unrecognized mechanistic link between ubiquitination, receptor recruitment, and lysophagic degradation, broadening our understanding of lysosomal quality control.
    Keywords:  FBXO3; TAX1BP1; TBK1; TMEM192; lysophagy; ubiquitination
    DOI:  https://doi.org/10.1080/15548627.2025.2479669
  10. J Cell Sci. 2025 Mar 01. pii: JCS263652. [Epub ahead of print]138(5):
    Disruption of ER-mitochondria contact sites induces autophagy-dependent loss of P-bodies through the Ca2+-CaMKK2-AMPK pathway.
    Nikhil More, Jomon Joseph.
      P-bodies (PBs) and stress granules (SGs) are conserved, non-membranous cytoplasmic condensates of RNA-protein complexes. PBs are implicated in post-transcriptional regulation of gene expression through mRNA decay, translational repression and/or storage. Although much is known about the de novo formation of PBs and SGs involving liquid-liquid phase separation through multiple protein-protein and protein-RNA interactions, their subcellular localization and turnover mechanisms are less understood. Here, we report the presence of a subpopulation of PBs and SGs that are in proximity to ER-mitochondria contact sites (ERMCSs) in mammalian cells. Disruption of ERMCSs, achieved through depletion of ER-mitochondria tethering proteins, leads to the disappearance of PBs but not SGs. This effect can be reversed by inhibiting autophagy through both genetic and pharmacological means. Additionally, we find that the disruption of ERMCSs leads to cytosolic Ca2+-induced activation of CaMKK2 and AMP-activated protein kinase (AMPK), ultimately resulting in an autophagy-dependent decrease in PB abundance. Collectively, our findings unveil a mechanism wherein disturbances in ERMCSs induce autophagy-dependent loss of PBs via activation of the Ca2+-CaMKK2-AMPK pathway, thus potentially linking the dynamics and functions of ERMCS with post-transcriptional gene regulation.
    Keywords:  Autophagy; CaMKK2; ER–mitochondria contact sites; P-bodies; Stress granules
    DOI:  https://doi.org/10.1242/jcs.263652
  11. Nat Commun. 2025 Mar 08. 16(1): 2323
    A mini-hairpin shaped nascent peptide blocks translation termination by a distinct mechanism.
    Yushin Ando, Akinao Kobo, Tatsuya Niwa, Ayako Yamakawa, Suzuna Konoma, Yuki Kobayashi, Osamu Nureki, Hideki Taguchi, Yuzuru Itoh, Yuhei Chadani.
      Protein synthesis by ribosomes produces functional proteins but also serves diverse regulatory functions, which depend on the coding amino acid sequences. Certain nascent peptides interact with the ribosome exit tunnel to arrest translation and modulate themselves or the expression of downstream genes. However, a comprehensive understanding of the mechanisms of such ribosome stalling and its regulation remains elusive. In this study, we systematically screen for unidentified ribosome arrest peptides through phenotypic evaluation, proteomics, and mass spectrometry analyses, leading to the discovery of the arrest peptides PepNL and NanCL in E. coli. Our cryo-EM study on PepNL reveals a distinct arrest mechanism, in which the N-terminus of PepNL folds back towards the tunnel entrance to prevent the catalytic GGQ motif of the release factor from accessing the peptidyl transferase center, causing translation arrest at the UGA stop codon. Furthermore, unlike sensory arrest peptides that require an arrest inducer, PepNL uses tryptophan as an arrest inhibitor, where Trp-tRNATrp reads through the stop codon. Our findings illuminate the mechanism and regulatory framework of nascent peptide-induced translation arrest, paving the way for exploring regulatory nascent peptides.
    DOI:  https://doi.org/10.1038/s41467-025-57659-z
  12. Curr Opin Cell Biol. 2025 Mar 10. pii: S0955-0674(25)00030-4. [Epub ahead of print]94 102492
    Organizing principles underlying COPII-mediated transport.
    Julia R Flood, Caitlin A Mendina, Anjon Audhya.
      The early secretory pathway governs the transport of thousands of secreted and transmembrane proteins and lipids from the endoplasmic reticulum (ER) to juxtaposed ER-Golgi Intermediate Compartments (ERGIC). This process is largely directed by Coat Protein complex II (COPII), which accumulates on distinct, ribosome-free ER subdomains (transitional ER) to generate highly curved transport intermediates of various sizes and shapes. The rate of secretory flux from the ER can vary significantly, depending on cell type, environmental cues, and other factors, but the mechanisms that regulate COPII-mediated trafficking have been slow to emerge. Here, we focus on recent progress that has contributed to our understanding of how the early secretory pathway is structured to facilitate the export of cargoes from the ER into a chasm approximately 300-500-nm in size, prior to fusion with ERGIC membranes without the aid of cytoskeletal elements to guide their journey.
    DOI:  https://doi.org/10.1016/j.ceb.2025.102492
  13. Nat Commun. 2025 Mar 13. 16(1): 2494
    Identification of a non-inhibitory aptameric ligand to CRL2ZYG11B E3 ligase for targeted protein degradation.
    Zhihao Yang, Miao Chen, Ruixin Ge, Ping Zhou, Wei Pan, Jiayi Song, Shuwen Ma, Song Chen, Chenyu Xu, Mengyu Zhou, Wenyi Mi, Hua Ni, He Chen, Xue Yao, Xifeng Dong, Yan Chen, Jun Zhou, Chenghao Xuan, Cheng Dong, Hua Yan, Songbo Xie.
      As a crucial element of proteolysis targeting chimeras (PROTACs), the choice of E3 ubiquitin ligase significantly influences degradation efficacy and selectivity. However, the available arsenal of E3 ligases for PROTAC development remains underexplored, severely limiting the scope of targeted protein degradation. In this study, we identify a non-inhibitory aptamer targeting ZYG11B, a substrate receptor of the Cullin 2-RING ligase complex, as an E3 warhead for targeted protein degradation. This aptamer-based PROTAC platform, termed ZATAC, is facilely produced through bioorthogonal chemistry or self-assembly and shows promise in eliminating several undruggable target proteins, including nucleolin (NCL), SRY-box transcription factor 2 (SOX2), and mutant p53-R175H, underscoring its universality and versatility. To specifically deliver ZATACs into cancer cells, we further develop DNA three-way junction-based ZATACs (3WJ-ZATACs) by integrating an additional aptamer that selectively recognizes the protein overexpressed on the surface of cancer cells. The 3WJ-ZATACs demonstrate in vivo tumor-specific distribution and achieve dual-target degradation, thereby suppressing tumor growth without causing noticeable toxicity. In summary, ZATACs represent a general, modular, and straightforward platform for targeted protein degradation, offering insights into the potential of other untapped E3 ligases.
    DOI:  https://doi.org/10.1038/s41467-025-57823-5
  14. Autophagy. 2025 Mar 13.
    ATG2A acts as a tether to regulate autophagosome-lysosome fusion in neural cells.
    Ze Zheng, Cuicui Ji, Hongyu Zhao, Yan G Zhao.
      The macroautophagy/autophagy proteins ATG2A and ATG2B transfer lipids for phagophore membrane growth. They also form stable complexes with WDR45 and WDR45B. Our previous study demonstrated that WDR45 and WDR45B mediate autophagosome-lysosome fusion in neural cells. Given the defective autophagosome formation in cells lacking both ATG2s, their role in later autophagy stages is hard to explore. Here, we report that in neuroblastoma-derived Neuro-2a (N2a) cells, knocking down (KD) Atg2a, but not Atg2b, results in significant accumulation of SQSTM1/p62 and MAP1LC3/LC3-II, indicating impaired autophagy. Atg2a deficiency does not affect autophagosome formation, but reduces colocalization of autophagosomal LC3 with late endosomal/lysosomal RFP-RAB7, suggesting impaired autophagosome-lysosome fusion. ATG2A interacts with the SNARE proteins STX17, SNAP29, and VAMP8, facilitating their assembly. Overexpression of ATG2A partially rescues the autophagosome-lysosome fusion defects in Wdr45- and Wdr45b-deficient cells. ATG2 and another tether protein, EPG5, function partially redundantly in mediating autophagosome-lysosome fusion. Thus, ATG2A plays a key role in neural autophagy by tethering autophagosomes with lysosomes for fusion.
    Keywords:  ATG2A; WDR45; autophagosome; autophagy; lysosome; tether
    DOI:  https://doi.org/10.1080/15548627.2025.2479427
  15. J Proteome Res. 2025 Mar 12.
    Discovery of RNA-Protein Molecular Clamps Using Proteome-Wide Stability Assays.
    Stanley I Goldstein, Alice C Fan, Zihao Wang, Sai K Naineni, Regina Cencic, Steve B Garcia-Gutierrez, Kesha Patel, Sidong Huang, Lauren E Brown, Andrew Emili, John A Porco.
      Uncompetitive inhibition is an effective strategy for suppressing dysregulated enzymes and their substrates, but discovery of suitable ligands depends on often-unavailable structural knowledge and serendipity. Hence, despite surging interest in mass spectrometry-based target identification, proteomic studies of substrate-dependent target engagement remain sparse. Herein, we describe a strategy for the discovery of substrate-dependent ligand binding. Using proteome integral solubility alteration (PISA) assays, we show that simple biochemical additives can enable detection of RNA-protein-small molecule complexes in native cell lysates. We apply our approach to rocaglates, molecules that specifically clamp RNA to eukaryotic translation initiation factor 4A (eIF4A), DEAD-box helicase 3X (DDX3X), and potentially other members of the DEAD-box (DDX) helicase family. To identify unexpected interactions, we used a target class-specific thermal window and compared ATP analog and RNA base dependencies for key rocaglate-DDX interactions. We report novel DDX targets of high-profile rocaglates-including the clinical candidate Zotatifin-and validate our findings using limited proteolysis-mass spectrometry and fluorescence polarization (FP) experiments. We also provide structural insight into divergent DDX3X affinities between synthetic rocaglates. Taken together, our study provides a model for screening uncompetitive inhibitors using a chemical proteomics approach and uncovers actionable DDX clamping targets, clearing a path toward characterization of novel molecular clamps and associated RNA helicases.
    Keywords:  CETSA; DDX3; DEAD-box helicase; eIF4A; limited proteolysis; proteome integral solubility alteration; rocaglates; target identification; thermal proteome profiling
    DOI:  https://doi.org/10.1021/acs.jproteome.4c01129
  16. Angew Chem Int Ed Engl. 2025 Mar 13. e202502638
    Convergent Assembly of Homo- and Heterotypic Ubiquitin Chains from Functionalized, Expressed Monomers via Thiol-Ene Chemistry.
    Christian F W Becker, Moritz Urschbach, Susanne Huhmann, Dominik Appel, Luca Ferrari, Dominik Vogl, Sascha Martens.
      Nature constructs ubiquitin tags with high spatiotemporal precision to execute defined functions which critically rely on the exact molecular composition of the ubiquitin chain. Deciphering the complex ubiquitin code is of paramount interest in biology and requires flexible access to homogeneous ubiquitin tags. As enzymatic approaches suffer from inherent drawbacks such as hardly controllable chain length or connectivity and substrate-specificity, we apply a combination of expression and chemical tools to assemble ubiquitin chains. Our strategy includes expression of ubiquitin-intein fusion constructs to obtain large quantities of defined ubiquitin monomers with C-terminal modifications such as hydrazides and propargylamides. Linkages between ubiquitins are generated via photoinitiated thiol-ene click (TEC) chemistry resulting in a nearly native isopeptide bond. We demonstrate the generation of homo- and heterotypic ubiquitin oligomers with K27, 29, 48 and 63 linkages up to a K48-linked tetramer. The presented toolbox allows selective installation of ubiquitin on target peptides and proteins with reactive cysteine residues as demonstrated for segments of the microtubule-associated protein tau. Such segments can be implemented into protein semi-syntheses as shown here for ubiquitylated full-length Tau4. The presented work combines minimal synthetic effort with high fidelity linkage chemistry paving the way towards homogeneously ubiquitylated proteins.
    Keywords:  Bioorganic Chemistry; Protein modifications; Protein semi-synthesis; Tau protein; Ubiquitin
    DOI:  https://doi.org/10.1002/anie.202502638
  17. Commun Biol. 2025 Mar 10. 8(1): 402
    Protein disulfide isomerase is essential for osteoblast differentiation in mice.
    Yue Lu, Aizhen Yang, Zhenzhen Zhao, Yue Han, Depei Wu, Yi Wu.
      Protein disulfide isomerase (PDI) is an oxidoreductase responsible for the formation, reduction and isomerization of disulfide bonds of nascent proteins in endoplasmic reticulum (ER). So far, the role of PDI in bone biology has never been characterized using genetically-modified animal models. In this study we generated osteoblast- specific PDI-deficient mice by crossing PDI-floxed (PDIfl/fl) mice with Osx-Cre mice. Compared with their littermate control PDIfl/fl mice, homozygous osteoblast-knockout mice (Osx-Cre/PDIfl/fl) were embryonically lethal, but heterozygous knockout mice (Osx-Cre/PDIfl/wt) displayed significantly pronounced growth retardation and reduced bone length. Besides, the decreases in bone density, osteoblast and osteoclast numbers, collagen fiber content and bone formation rate were observed in Osx-Cre/PDIfl/wt mice. Osteoblast precursors isolated from PDIfl/fl mice were infected with Cre recombinant adenovirus to produce PDI-deficient osteoblasts, followed by induction of differentiation. Osteoblasts deficient of PDI had decreased alkaline phosphatase activity, mineralizing capacity, and differentiation. Quantitative protein mass spectrometry analysis and immunoblotting showed that PDI deficiency markedly decreased the expression of the α-subunits of collagen prolyl 4-hydroxylase (C-P4H), including P4HA1, P4HA2 and P4HA3. These results demonstrate that PDI plays an essential role in osteoblast differentiation and bone formation and is required for the expression of the α-subunit of C-P4H in osteoblasts.
    DOI:  https://doi.org/10.1038/s42003-025-07824-3
  18. Nat Commun. 2025 Mar 10. 16(1): 2365
    Suppression of stress granule formation is a vulnerability imposed by mutant p53.
    Elizabeth Thoenen, Atul Ranjan, Alejandro Parrales, Shigeto Nishikawa, Dan A Dixon, Sugako Oka, Tomoo Iwakuma.
      Missense mutations in the TP53 (p53) gene have been linked to malignant progression. However, our in-silico analyses reveal that hepatocellular carcinoma (HCC) patients with mutant p53 (mutp53) have better overall survival compared to those with p53-null (p53null) HCC, unlike other cancer types. Given the historical use of sorafenib (SOR) monotherapy for advanced HCC, we hypothesize that mutp53 increases sensitivity to SOR, a multikinase inhibitor that induces endoplasmic reticulum (ER) stress. Here we show that mutp53 inhibits stress granule (SG) formation by binding to an ER stress sensor, PKR-like ER kinase (PERK), and a key SG component, GAP SH3 domain-binding protein 1 (G3BP1), contributing to increased sensitivity of SG-competent cells and xenografts to ER stress inducers including SOR. Our study identifies a unique vulnerability imposed by mutp53, suggesting mutp53 as a biomarker for ER stress-inducing agents and highlighting the importance of SG inhibition for cancer treatment.
    DOI:  https://doi.org/10.1038/s41467-025-57539-6
  19. Nucleic Acids Res. 2025 Feb 27. pii: gkaf160. [Epub ahead of print]53(5):
    RNA anchoring of Upf1 facilitates recruitment of Dcp2 in the NMD decapping complex.
    Nadia Ruiz-Gutierrez, Jeanne Dupas, Elvire Auquier, Irène Barbarin-Bocahu, Claudine Gaudon-Plesse, Cosmin Saveanu, Marc Graille, Hervé Le Hir.
      Upf1 RNA helicase is a pivotal factor in the conserved nonsense-mediated mRNA decay (NMD) process. Upf1 is responsible for coordinating the recognition of premature termination codons (PTCs) in a translation-dependent manner and subsequently triggering mRNA degradation. Multiple factors assist Upf1 during these two consecutive steps. In Saccharomyces cerevisiae, Upf2 and Upf3 associated with Upf1 (Upf1-2/3) contribute to PTC recognition but are absent from the Upf1-decapping complex that includes Nmd4, Ebs1, Dcp1, and Dcp2. Despite their importance for NMD, the organization and dynamics of these Upf1-containing complexes remain unclear. Using recombinant proteins, here we show how distinct domains of Upf1 make direct contacts with Dcp1/Dcp2, Nmd4, and Ebs1. These proteins also bind to each other, forming an extended network of interactions within the Upf1-decapping complex. Dcp2 and Upf2 compete for the same binding site on the N-terminal CH domain of Upf1, which explains the presence of two mutually exclusive Upf1-containing complexes in cells. Our data demonstrate that Nmd4-assisted recruitment of Upf1 promotes anchoring of the decapping enzyme to NMD targets.
    DOI:  https://doi.org/10.1093/nar/gkaf160
  20. bioRxiv. 2025 Feb 27. pii: 2025.02.26.640437. [Epub ahead of print]
    Structural basis for anomalous cellular trafficking behavior of glaucoma-associated A427T mutant myocilin.
    Kamisha R Hill, Hailee F Scelsi, Hannah A Youngblood, Jennifer A Faralli, Tatsuo Itakura, M Elizabeth Fini, Donna M Peters, Raquel L Lieberman.
      Familial mutations in myocilin cause vision loss in glaucoma due to misfolding and a toxic gain of function in a senescent cell type in the anterior eye. Here we characterize the cellular behavior and structure of the myocilin (myocilin A427T) mutant, of uncertain pathogenicity. Our characterization of A427T demonstrates that even mutations that minimally perturb myocilin structure and stability can present challenges for protein quality control clearance pathways. Namely, when expressed in an inducible immortalized trabecular meshwork cell line, inhibition of the proteasome reroutes wild-type myocilin, but not myocilin A427T, from endoplasmic reticulum associated degradation to lysosomal degradation. Yet, the crystal structure of the A427T myocilin olfactomedin domain shows modest perturbations largely confined to the mutation site. The previously unappreciated range of mutant myocilin behavior correlating with variable stability and structure provides a rationale for why it is challenging to predict causal pathogenicity of a given myocilin mutation, even in the presence of clinical data for members of an affected family. Comprehending the continuum of mutant myocilin behavior in the laboratory supports emerging efforts to use genetics to assess glaucoma risk in the clinic. In addition, the study supports a therapeutic strategy aimed at enhancing autophagic clearance of mutant myocilin.
    DOI:  https://doi.org/10.1101/2025.02.26.640437
  21. Nucleic Acids Res. 2025 Feb 27. pii: gkaf143. [Epub ahead of print]53(5):
    Yeast poly(A)-binding protein (Pab1) controls translation initiation in vivo primarily by blocking mRNA decapping and decay.
    Poonam Poonia, Vishalini Valabhoju, Tianwei Li, James Iben, Xiao Niu, Zhenguo Lin, Alan G Hinnebusch.
      Poly(A)-binding protein (Pab1 in yeast) is involved in mRNA decay and translation initiation, but its molecular functions are incompletely understood. We found that auxin-induced degradation of Pab1 reduced bulk mRNA and polysome abundance in WT but not in a mutant lacking the catalytic subunit of decapping enzyme (Dcp2), suggesting that enhanced decapping/degradation is a major driver of reduced translation at limiting Pab1. An increased median poly(A) tail length conferred by Pab1 depletion was likewise not observed in the dcp2Δ mutant, suggesting that mRNA isoforms with shorter tails are preferentially decapped/degraded at limiting Pab1. In contrast to findings on mammalian cells, the translational efficiencies (TEs) of many mRNAs were altered by Pab1 depletion; however, these changes were diminished in dcp2Δ cells, suggesting that reduced mRNA abundance is also a major driver of translational reprogramming at limiting Pab1. Thus, assembly of the closed-loop mRNP via PABP-eIF4G interaction appears to be dispensable for wild-type translation of most transcripts at normal mRNA levels. Interestingly, histone mRNAs and proteins were preferentially diminished on Pab1 depletion in DCP2 but not dcp2Δ cells, accompanied by activation of internal cryptic promoters in the manner expected for reduced nucleosome occupancies, implicating Pab1 in post-transcriptional control of histone gene expression.
    DOI:  https://doi.org/10.1093/nar/gkaf143
  22. Metab Eng. 2025 Mar 08. pii: S1096-7176(25)00042-4. [Epub ahead of print]90 43-56
    Mechanism and engineering of endoplasmic reticulum-localized membrane protein folding in Saccharomyces cerevisiae.
    Yuhuan Luo, Jian-Jiang Zhong, Han Xiao.
      Correct folding of endoplasmic reticulum (ER)-localized membrane proteins, such as cytochrome P450, endows a synthetic biology host with crucial catalytic functions, which is of vital importance in the field of metabolic engineering and synthetic biology. However, due to complexed interaction with cellular membrane environment and other proteins (e.g., molecular chaperone) regulation, a substantial proportion of heterologous membrane proteins cannot be properly folded in the ER of Saccharomyces cerevisiae, a widely used synthetic biology host. In this review, we first introduce the four steps in membrane protein folding process and the affecting factors including the amino acid sequence of membrane protein, the folding process, molecular chaperones, quality control mechanism, and lipid environment in S. cerevisiae. Then, we summarize the metabolic engineering strategies to enhance the correct folding of ER-localized membrane proteins, such as by engineering and de novel design of membrane protein, regulation of the co-translational folding process, co-expression of molecular chaperones, modulation of ER quality, and lipids engineering. Finally, we discuss the limitations of current strategies and propose future research directions to address the key issues.
    Keywords:  Chaperones; Cytochrome P450; Endoplasmic reticulum; Lipid microenvironment engineering; Membrane protein folding; Protein design
    DOI:  https://doi.org/10.1016/j.ymben.2025.03.006
  23. Science. 2025 Mar 13. eadu6445
    Structure of human PINK1 at a mitochondrial TOM-VDAC array.
    Sylvie Callegari, Nicholas S Kirk, Zhong Yan Gan, Toby Dite, Simon A Cobbold, Andrew Leis, Laura F Dagley, Alisa Glukhova, David Komander.
      Mutations in the ubiquitin kinase PINK1 cause early onset Parkinson's Disease, but how PINK1 is stabilized at depolarized mitochondrial translocase complexes has remained poorly understood. We determined a 3.1-Å resolution cryo-electron microscopy structure of dimeric human PINK1 stabilized at an endogenous array of mitochondrial TOM and VDAC complexes. Symmetric arrangement of two TOM core complexes around a central VDAC2 dimer is facilitated by TOM5 and TOM20, both of which also bind PINK1 kinase C-lobes. PINK1 enters mitochondria through the proximal TOM40 barrel of the TOM core complex, guided by TOM7 and TOM22. Our structure explains how human PINK1 is stabilized at the TOM complex and regulated by oxidation, uncovers a previously unknown TOM-VDAC assembly, and reveals how a physiological substrate traverses TOM40 during translocation.
    DOI:  https://doi.org/10.1126/science.adu6445
  24. EMBO J. 2025 Mar 13.
    An asymmetric nautilus-like HflK/C assembly controls FtsH proteolysis of membrane proteins.
    Alireza Ghanbarpour, Bertina Telusma, Barrett M Powell, Jia Jia Zhang, Isabella Bolstad, Carolyn Vargas, Sandro Keller, Tania A Baker, Robert T Sauer, Joseph H Davis.
      The AAA protease FtsH associates with HflK/C subunits to form a megadalton-size complex that spans the inner membrane and extends into the periplasm of E. coli. How this bacterial complex and homologous assemblies in eukaryotic organelles recruit, extract, and degrade membrane-embedded substrates is unclear. Following the overproduction of protein components, recent cryo-EM structures showed symmetric HflK/C cages surrounding FtsH in a manner proposed to inhibit the degradation of membrane-embedded substrates. Here, we present structures of native protein complexes, in which HflK/C instead forms an asymmetric nautilus-shaped assembly with an entryway for membrane-embedded substrates to reach and be engaged by FtsH. Consistent with this nautilus-like structure, proteomic assays suggest that HflK/C enhances FtsH degradation of certain membrane-embedded substrates. Membrane curvature in our FtsH•HflK/C complexes is opposite that of surrounding membrane regions, a property that correlates with lipid scramblase activity and possibly with FtsH's function in the degradation of membrane-embedded proteins.
    Keywords:  AAA Protease; Cryo-EM; Macromolecular Complexes; Proteostasis
    DOI:  https://doi.org/10.1038/s44318-025-00408-1
  25. J Cell Sci. 2025 Mar 07. pii: jcs.263852. [Epub ahead of print]
    ATG9A vesicles are a subtype of intracellular nanovesicle.
    Mary Fesenko, Daniel J Moore, Peyton Ewbank, Elizabeth Courthold, Stephen J Royle.
      Cells are filled with thousands of vesicles, which mediate protein transport and ensure homeostasis of the endomembrane system. Distinguishing these vesicles functionally and molecularly represents a major challenge. Intracellular nanovesicles (INVs) are a large class of transport vesicles that likely comprises of multiple subtypes. Here, we define the INV proteome and find that it is molecularly heterogeneous, and enriched for transmembrane cargo molecules including integrins, transporters, and ATG9A, a lipid scramblase associated with autophagy. ATG9A is known to reside in 'ATG9A vesicles': small vesicles that contribute to autophagosome formation. Using in-cell vesicle capture assays we found that ATG9A, as well as other ATG9A vesicle cargos, were in INVs. Quantitative analysis showed that virtually all ATG9A vesicles are INVs, but that only ∼20% of INVs are ATG9A vesicles, suggesting that ATG9A vesicles are in fact a subtype of INV, which we term ATG9A-flavor INVs. Finally, we show that perturbing ATG9A-flavor INVs impaired the autophagy response induced by starvation.
    Keywords:  Autophagy; Cargo; Membrane traffic; Proteomics; Transport vesicle
    DOI:  https://doi.org/10.1242/jcs.263852
  26. bioRxiv. 2025 Feb 28. pii: 2025.02.27.640591. [Epub ahead of print]
    Molecular grammars of intrinsically disordered regions that span the human proteome.
    Kiersten M Ruff, Matthew R King, Alexander W Ying, Vicky Liu, Avnika Pant, Whitney E Lieberman, Min Kyung Shinn, Xiaolei Su, Cigall Kadoch, Rohit V Pappu.
      Intrinsically disordered regions (IDRs) of proteins are defined by functionally relevant molecular grammars. This refers to IDR-specific non-random amino acid compositions and non-random patterning of distinct pairs of amino acid types. Here, we introduce GIN (Grammars Inferred using NARDINI+) as a resource, which we have used to extract the molecular grammars of all human IDRs and classified them into thirty distinct clusters. Unbiased analyses of IDRome-spanning grammars reveals that specialized IDR grammar features direct biological processes, cellular localization preferences, and molecular functions. IDRs with exceptional grammars, defined as sequences with high-scoring non-random features, are harbored in proteins and complexes that enable spatial and temporal sorting of biochemical activities. Protein complexes within the nucleus recruit specific factors through top-scoring IDRs. These IDRs are frequently disrupted via cancer-associated mutations and fusion oncoproteins. Overall, GIN enables the decoding of sequence-function relationships of IDRs and can be deployed in IDR-specific and IDRome-wide analyses.
    DOI:  https://doi.org/10.1101/2025.02.27.640591
  27. Cancer Lett. 2025 Mar 05. pii: S0304-3835(25)00177-6. [Epub ahead of print]617 217613
    Unraveling UPR-mediated intercellular crosstalk: Implications for immunotherapy resistance mechanisms.
    Si Lu, Qimin Zhou, Rongjie Zhao, Lei Xie, Wen-Ming Cao, Yu-Xiong Feng.
      Endoplasmic reticulum (ER) is the critical organelle that regulates essential cellular processes, including protein synthesis, folding, and post-translational modification, as well as lipid metabolism and calcium homeostasis. Disruption in ER homeostasis leads to a condition known as ER stress, characterized by the accumulation of misfolded or unfolded proteins. This triggers the unfolded protein response (UPR), an adaptive pathway mediated by three ER-resident sensors: inositol-requiring enzyme 1α (IRE1α), protein kinase R-like ER kinase (PERK), and activating transcription factor 6 (ATF6). Increasing evidence highlights sustained UPR activation in malignant and immune cells within the tumor microenvironment (TME), which promotes tumor progression and metastasis while simultaneously impairing antitumor immunity. This review explores how UPR-driven intercellular signaling influences immunotherapy resistance, focusing on the alterations occurring in tumor cells as well as in the surrounding immune environment. By providing insights into these mechanisms, we aim to highlight the therapeutic potential of targeting the UPR pathways in modulating cancer immunity.
    Keywords:  Immunotherapy resistance; Tumor microenvironment; UPR
    DOI:  https://doi.org/10.1016/j.canlet.2025.217613
  28. Nat Commun. 2025 Mar 12. 16(1): 2470
    The translation inhibitors kasugamycin, edeine and GE81112 target distinct steps during 30S initiation complex formation.
    Haaris A Safdari, Martino Morici, Ana Sanchez-Castro, Andrea Dallapè, Helge Paternoga, Anna Maria Giuliodori, Attilio Fabbretti, Pohl Milón, Daniel N Wilson.
      During bacterial translation initiation, the 30S ribosomal subunit, initiation factors, and initiator tRNA define the reading frame of the mRNA. This process is inhibited by kasugamycin, edeine and GE81112, however, their mechanisms of action have not been fully elucidated. Here we present cryo-electron microscopy structures of 30S initiation intermediate complexes formed in the presence of kasugamycin, edeine and GE81112 at resolutions of 2.0-2.9 Å. The structures reveal that all three antibiotics bind within the E-site of the 30S and preclude 30S initiation complex formation. While kasugamycin and edeine affect early steps of 30S pre-initiation complex formation, GE81112 stalls pre-initiation complex formation at a further step by allowing start codon recognition, but impeding IF3 departure. Collectively, our work highlights how chemically distinct compounds binding at a conserved site on the 30S can interfere with translation initiation in a unique manner.
    DOI:  https://doi.org/10.1038/s41467-025-57731-8
  29. Cell Chem Biol. 2025 Feb 28. pii: S2451-9456(25)00035-2. [Epub ahead of print]
    Mechanisms and functions of lysosomal lipid homeostasis.
    Michael Ebner, Florian Fröhlich, Volker Haucke.
      Lysosomes are the central degradative organelle of mammalian cells and have emerged as major intersections of cellular metabolite flux. Macromolecules derived from dietary and intracellular sources are delivered to the acidic lysosomal lumen where they are subjected to degradation by acid hydrolases. Lipids derived from lipoproteins, autophagy cargo, or autophagosomal membranes themselves constitute major lysosomal substrates. Dysregulation of lysosomal lipid processing, defective export of lipid catabolites, and lysosomal membrane permeabilization underly diseases ranging from neurodegeneration to metabolic syndromes and lysosomal storage disorders. Mammalian cells are equipped with sophisticated homeostatic control mechanisms that protect the lysosomal limiting membrane from excessive damage, prevent the spillage of luminal hydrolases into the cytoplasm, and preserve the lysosomal membrane composition in the face of constant fusion with heterotypic organelles such as endosomes and autophagosomes. In this review we discuss the molecular mechanisms that govern lysosomal lipid homeostasis and, thereby, lysosome function in health and disease.
    Keywords:  contact sites; lipids; lysosomes; membrane homeostasis; phosphoinositides; signalling
    DOI:  https://doi.org/10.1016/j.chembiol.2025.02.003
  30. J Cell Biol. 2025 May 05. pii: e202409072. [Epub ahead of print]224(5):
    Acetic acid-induced stress granules function as scaffolding complexes for Hog1 activation by Pbs2.
    Jongmin Lee, Kazuo Tatebayashi, David E Levin.
      Stress-activated protein kinases (SAPKs) respond to a wide variety of stressors. In most cases, the pathways through which specific stress signals are transmitted to the SAPK are not known. We show that the yeast SAPK Hog1 is activated by acetic acid through an intracellular mechanism that does not involve stimulation of the high osmolarity glycerol (HOG) signaling pathway beyond its basal level. Rather, acetic acid treatment drives the formation of stress granules, which function as a scaffold to bring Hog1 together with Pbs2, its immediately upstream activating kinase, in a stable assembly that leverages the basal activity of Pbs2 to phosphorylate Hog1. Deletion analysis of stress granule components revealed that the assembly is critical for both the acetic acid-induced activation of Hog1 and its association with Pbs2. Activated Hog1 remains associated with stress granules, which may have implications for its targeting.
    DOI:  https://doi.org/10.1083/jcb.202409072
  31. J Clin Invest. 2025 Mar 11. pii: e183099. [Epub ahead of print]
    Proteostasis and metabolic dysfunction characterize a subset of storage-induced senescent erythrocytes targeted for post-transfusion clearance.
    Sandy Peltier, Mickaël Marin, Monika Dzieciatkowska, Michaël Dussiot, Micaela Kalani Roy, Johanna Bruce, Louise Leblanc, Youcef Hadjou, Sonia Georgeault, Aurélie Fricot, Camille Roussel, Daniel Stephenson, Madeleine Casimir, Abdoulaye Sissoko, François Paye, Safi Dokmak, Papa Alioune Ndour, Philippe Roingeard, Emilie-Fleur Gautier, Steven L Spitalnik, Olivier Hermine, Pierre A Buffet, Angelo D'Alessandro, Pascal Amireault.
      Although refrigerated storage slows the metabolism of volunteer donor RBCs, which is essential in transfusion medicine, cellular aging still occurs throughout this in vitro process. Storage-induced microerythrocytes (SMEs) are morphologically-altered senescent RBCs that accumulate during storage and are cleared from circulation following transfusion. However, the molecular and cellular alterations that trigger clearance of this RBC subset remain to be identified. Using a staining protocol that sorts long-stored SMEs (i.e., CFSEhigh) and morphologically-normal RBCs (CFSElow), these in vitro aged cells were characterized. Metabolomics analysis identified depletion of energy, lipid-repair, and antioxidant metabolites in CFSEhigh RBCs. By redox proteomics, irreversible protein oxidation primarily affected CFSEhigh RBCs. By proteomics, 96 proteins, mostly in the proteostasis family, had relocated to CFSEhigh RBC membranes. CFSEhigh RBCs exhibited decreased proteasome activity and deformability; increased phosphatidylserine exposure, osmotic fragility, and endothelial cell adherence; and were cleared from the circulation during human spleen perfusion ex vivo. Conversely, molecular, cellular, and circulatory properties of long-stored CFSElow RBCs resembled those of short-stored RBCs. CFSEhigh RBCs are morphologically and metabolically altered, have irreversibly oxidized and membrane-relocated proteins, and exhibit decreased proteasome activity. In vitro aging during storage selectively alters metabolism and proteostasis in these storage-induced senescent RBCs targeted for clearance.
    Keywords:  Cell biology; Cellular senescence; Hematology; Proteomics; Ubiquitin-proteosome system
    DOI:  https://doi.org/10.1172/JCI183099
  32. Cell Syst. 2025 Mar 03. pii: S2405-4712(25)00037-7. [Epub ahead of print] 101204
    Integrated multi-omic characterizations of the synapse reveal RNA processing factors and ubiquitin ligases associated with neurodevelopmental disorders.
    Yuan Mei, Maya L Gosztyla, Xinzhu Tan, Lara E Dozier, Brent Wilkinson, Justin McKetney, John Lee, Michael Chen, Dorothy Tsai, Hema Kopalle, Marina A Gritsenko, Nicolas Hartel, Nicholas A Graham, Ilse Flores, Stephen K Gilmore-Hall, Shuhao Xu, Charlotte A Marquez, Sophie N Liu, Dylan Fong, Jing Chen, Kate Licon, Derek Hong, Sarah N Wright, Jason F Kreisberg, Alexi Nott, Richard D Smith, Wei-Jun Qian, Danielle L Swaney, Lilia M Iakoucheva, Nevan J Krogan, Gentry N Patrick, Yang Zhou, Guoping Feng, Marcelo P Coba, Gene W Yeo, Trey Ideker.
      The molecular composition of the excitatory synapse is incompletely defined due to its dynamic nature across developmental stages and neuronal populations. To address this gap, we apply proteomic mass spectrometry to characterize the synapse in multiple biological models, including the fetal human brain and human induced pluripotent stem cell (hiPSC)-derived neurons. To prioritize the identified proteins, we develop an orthogonal multi-omic screen of genomic, transcriptomic, interactomic, and structural data. This data-driven framework identifies proteins with key molecular features intrinsic to the synapse, including characteristic patterns of biophysical interactions and cross-tissue expression. The multi-omic analysis captures synaptic proteins across developmental stages and experimental systems, including 493 synaptic candidates supported by proteomics. We further investigate three such proteins that are associated with neurodevelopmental disorders-Cullin 3 (CUL3), DEAD-box helicase 3 X-linked (DDX3X), and Y-box binding protein-1 (YBX1)-by mapping their networks of physically interacting synapse proteins or transcripts. Our study demonstrates the potential of an integrated multi-omic approach to more comprehensively resolve the synaptic architecture.
    Keywords:  CUL3; DDX3X; YBX1; machine learning; multi-omics; proteomics; synapse
    DOI:  https://doi.org/10.1016/j.cels.2025.101204
  33. Nat Genet. 2025 Mar 10.
    Selection for somatic escape variants in SERPINA1 in the liver of patients with alpha-1 antitrypsin deficiency.
    Natalia Brzozowska, Lily Y D Wu, Vera Khodzhaeva, William J Griffiths, Adam Duckworth, Hyunchul Jung, Tim H H Coorens, Yvette Hooks, Joseph E Chambers, Peter J Campbell, Stefan J Marciniak, Matthew Hoare.
      Somatic variants accumulate in non-malignant tissues with age. Functional variants, leading to clonal advantage of hepatocytes, accumulate in the liver of patients with acquired chronic liver disease (CLD). Whether somatic variants are common to CLD from differing etiologies is unknown. We analyzed liver somatic variants in patients with genetic CLD from alpha-1 antitrypsin (A1AT) deficiency or hemochromatosis. We show that somatic variants in SERPINA1, the gene encoding A1AT, are strongly selected for in A1AT deficiency, with evidence of convergent evolution. Acquired SERPINA1 variants are clustered at the carboxyl terminus of A1AT, leading to truncation. In vitro and in vivo, C-terminal truncation variants reduce disease-associated Z-A1AT polymer accumulation and disruption of the endoplasmic reticulum, supporting the C-terminal domain swap mechanism. Therefore, somatic escape variants from a deleterious germline variant are selected for in A1AT deficiency, suggesting that functional somatic variants are disease-specific in CLD and point to disease-associated mechanisms.
    DOI:  https://doi.org/10.1038/s41588-025-02125-1
  34. Mol Cell. 2025 Mar 06. pii: S1097-2765(25)00109-1. [Epub ahead of print]85(5): 877-893.e9
    SUMO4 promotes SUMO deconjugation required for DNA double-strand-break repair.
    Alexander J Garvin, Alexander J Lanz, George E Ronson, Matthew J W Mackintosh, Katarzyna Starowicz, Alexandra K Walker, Yara Aghabi, Hannah MacKay, Ruth M Densham, Jai S Bhachoo, Aneika C Leney, Joanna R Morris.
      The amplitudes of small-modifier protein signaling through ubiquitin and the small ubiquitin-like modifiers, SUMO1-3, are critical to the correct phasing of DNA repair protein accumulation, activity, and clearance and for the completion of mammalian DNA double-strand-break (DSB) repair. However, how SUMO-conjugate signaling in the response is delineated is poorly understood. At the same time, the role of the non-conjugated SUMO protein, SUMO4, has remained enigmatic. Here, we reveal that human SUMO4 is required to prevent excessive DNA-damage-induced SUMOylation and deleterious over-accumulation of RAP80. Mechanistically we show that SUMO4 acts independently of its conjugation and potentiates SENP1 catalytic activity. These data identify SUMO4 as a SUMO deconjugation component and show that SUMO4:SENP1 are critical regulators of DNA-damage-induced SUMO signaling.
    Keywords:  DNA repair; RAP80; SENP1; SUMO; SUMO4; homologous recombination
    DOI:  https://doi.org/10.1016/j.molcel.2025.02.004
  35. JCI Insight. 2025 Mar 10. pii: e177999. [Epub ahead of print]10(5):
    Neurofilament accumulation disrupts autophagy in giant axonal neuropathy.
    Jean-Michel Paumier, James Zewe, Chiranjit Panja, Melissa R Pergande, Meghana Venkatesan, Eitan Israeli, Shikha Prasad, Natasha Snider, Jeffrey N Savas, Puneet Opal.
      Neurofilament accumulation is associated with many neurodegenerative diseases, but it is the primary pathology in giant axonal neuropathy (GAN). This childhood-onset autosomal recessive disease is caused by loss-of-function mutations in gigaxonin, the E3 adaptor protein that enables neurofilament degradation. Using a combination of genetic and RNA interference approaches, we found that dorsal root ganglia from mice lacking gigaxonin have impaired autophagy and lysosomal degradation through 2 mechanisms. First, neurofilament accumulations interfere with the distribution of autophagic organelles, impairing their maturation and fusion with lysosomes. Second, the accumulations attract the chaperone 14-3-3, which is responsible for the proper localization of the key autophagy regulator transcription factor EB (TFEB). We propose that this dual disruption of autophagy contributes to the pathogenesis of other neurodegenerative diseases involving neurofilament accumulations.
    Keywords:  Autophagy; Cell biology; Neurological disorders; Neuroscience; Ubiquitin-proteosome system
    DOI:  https://doi.org/10.1172/jci.insight.177999
  36. EMBO Mol Med. 2025 Mar 07.
    Chaperone-mediated autophagy regulates the metastatic state of mesenchymal tumors.
    Xun Zhou, Eva Berenger, Yong Shi, Vera Shirokova, Elena Kochetkova, Tina Becirovic, Boxi Zhang, Vitaliy O Kaminskyy, Yashar Esmaeilian, Kayoko Hosaka, Cecilia Lindskog, Per Hydbring, Simon Ekman, Yihai Cao, Maria Genander, Marcin Iwanicki, Erik Norberg, Helin Vakifahmetoglu-Norberg.
      Tumors often recapitulate programs to acquire invasive and dissemination abilities, during which pro-metastatic proteins are distinctively stabilized in cancer cells to drive further progression. Whether failed protein degradation affects the metastatic programs of cancer remains unknown. Here, we show that the human cancer cell-specific knockout (KO) of LAMP-2A, a limiting protein for chaperone-mediated autophagy (CMA), promotes the aggressiveness of mesenchymal tumors. Deficient CMA resulted in widespread tumor cell dissemination, invasion into the vasculature and cancer metastasis. In clinical samples, metastatic lesions showed suppressed LAMP-2A expression compared to primary tumors from the same cancer patients. Mechanistically, while stimulating TGFβ signaling dampens LAMP-2A levels, genetic suppression of CMA aggravated TGFβ signaling in cancer cells and tumors. Conversely, pharmacological inhibition of TGFβ signaling repressed the growth of LAMP-2A KO-driven tumors. Furthermore, we found that multiple EMT-driving proteins, such as TGFβR2, are degraded by CMA. Our study demonstrates that the tumor suppressive function of CMA involves negative regulation of TGFβ-driven EMT and uncovers a mechanistic link between CMA and a major feature of metastatic invasiveness.
    Keywords:  Cancer; Chaperone-mediated Autophagy; EMT; Metastasis; TGFβ
    DOI:  https://doi.org/10.1038/s44321-025-00210-w
  37. J Med Chem. 2025 Mar 10.
    Monoselective Histone Deacetylase 6 PROTAC Degrader Shows In Vivo Tractability.
    Harsimran K Garcha, Olasunkanmi O Olaoye, Abootaleb Sedighi, Daniel Pölöske, Pearla Hariri, Wenlong Yu, Diaaeldin I Abdallah, Richard Moriggl, Elvin D de Araujo, Patrick T Gunning.
      Herein, we report a potent HDAC6 PROTAC, TO-1187, which selectively degrades HDAC6 in cellulo and demonstrates in vivo efficacy. The design of TO-1187 was achieved by linking our previously reported HDAC6 inhibitor, TO-317, to the cereblon (CRBN) E3 ligase ligand, pomalidomide. TO-1187 achieved monoselective HDAC6 degradation in human multiple myeloma cells, MM.1S, with a Dmax of 94% and a DC50 of 5.81 nM after 6 h. Importantly, at concentrations up to 25 μM, TO-1187 exhibited no cellular degradation of other HDACs. Proteomic evaluation confirmed a highly selective proteome-wide degradation profile, with HDAC6 the only protein observed to be depleted. Notably, TO-1187 did not impact the abundance of well-known CRBN neosubstrates, like IKZF1, IKZF3, CK1α, SALL4, and GSPT1. In vivo evaluation confirmed that TO-1187 efficiently degraded HDAC6 in mouse tissues, measured 6 h after intravenous injection. In summary, TO-1187 represents a viable candidate for advanced preclinical evaluation of HDAC6 biology.
    DOI:  https://doi.org/10.1021/acs.jmedchem.4c02021
  38. Trends Cell Biol. 2025 Mar 10. pii: S0962-8924(25)00041-8. [Epub ahead of print]
    Cellular homeostatic responses to lysosomal damage.
    Jingyue Jia, Suttinee Poolsup, Jay E Salinas.
      Lysosomes are essential membrane-bound organelles that control cellular homeostasis by integrating intracellular functions with external signals. Their critical roles make lysosomal membranes vulnerable to rupture under various stressors, leading to cellular dysfunction. However, the mechanisms by which cells respond to lysosomal damage have only recently begun to be explored. In this review, we summarize the cellular mechanisms activated by lysosomal damage, emphasizing those that restore lysosomal integrity and sustain homeostasis, including recognition, repair, removal, replacement, and remodeling. Drawing on our expertise, we provide an in-depth focus on the remodeling process involved in these responses, including metabolic signaling and stress granule formation. Finally, we discuss the implications of lysosomal damage in human diseases, underscoring potential therapeutic strategies to preserve lysosomal function and alleviate related disorders.
    Keywords:  damaged lysosomes; recognition; remodeling; removal; repair; replacement
    DOI:  https://doi.org/10.1016/j.tcb.2025.02.007
  39. Chem Commun (Camb). 2025 Mar 10.
    Microwave-assisted synthesis of pomalidomide building blocks for rapid PROTAC and molecular glue development.
    Diaaeldin I Abdallah, Johan Israelian, Lina S Hasan, Naman H Patel, Jeffrey W Keillor, Timothy B Wright, Bilal Saqib, Elvin D de Araujo, Patrick T Gunning.
      Targeted protein degradation (TPD) is revolutionizing drug discovery, but PROTAC synthesis remains challenging due to multi-step synthesis and slow linker installation via SNAr on 4-fluorothalidomide. Here, we optimize a microwave-assisted synthesis (MAS) of pomalidomide building blocks, achieving high yields within 15 min - boosting yield by at least 14% at gram scale without the need for purification. Unlike conventional oil bath heating and overnight reactions, MAS streamlines degrader development. The method's utility was demonstrated by synthesizing ARV-110, highlighting MAS as a powerful tool for accelerating pomalidomide PROTAC and molecular glue discovery programs.
    DOI:  https://doi.org/10.1039/d4cc03435j
  40. Sci Adv. 2025 Mar 14. 11(11): eads7379
    Protein misfolding involving entanglements providesa structural explanation for the origin of stretched-exponential refolding kinetics.
    Yang Jiang, Yingzi Xia, Ian Sitarik, Piyoosh Sharma, Hyebin Song, Stephen D Fried, Edward P O'Brien.
      Stretched-exponential protein refolding kinetics, first observed decades ago, were attributed to a nonnative ensemble of structures with parallel, non-interconverting folding pathways. However, the structural origin of the large energy barriers preventing interconversion between these folding pathways is unknown. Here, we combine simulations with limited proteolysis (LiP) and cross-linking (XL) mass spectrometry (MS) to study the protein phosphoglycerate kinase (PGK). Simulations recapitulate its stretched-exponential folding kinetics and reveal that misfolded states involving changes of entanglement underlie this behavior: either formation of a nonnative, noncovalent lasso entanglement or failure to form a native entanglement. These misfolded states act as kinetic traps, requiring extensive unfolding to escape, which results in a distribution of free energy barriers and pathway partitioning. Using LiP-MS and XL-MS, we propose heterogeneous structural ensembles consistent with these data that represent the potential long-lived misfolded states PGK populates. This structural and energetic heterogeneity creates a hierarchy of refolding timescales, explaining stretched-exponential kinetics.
    DOI:  https://doi.org/10.1126/sciadv.ads7379
  41. Cell Signal. 2025 Mar 11. pii: S0898-6568(25)00152-4. [Epub ahead of print]131 111739
    PP2A adapter protein IER5 induces dephosphorylation and degradation of MDM2, thereby stabilizing p53.
    Taisei Nakada, Mayuko Koga, Hiroto Takeuchi, Kuriko Doi, Haruka Sugiyama, Hiroshi Sakurai.
      The tumor suppressor p53 activates transcription of the IER5 gene, which encodes an adapter protein of protein phosphatase PP2A. IER5 binds to both the B55 regulatory subunit of PP2A and PP2A's target proteins, facilitating PP2A/B55-catalyzed dephosphorylation of these proteins. Here, we show that IER5 functions as a positive regulator of p53 by inhibiting its ubiquitination, thereby increasing cellular p53 levels. Mechanistically, this effect of IER5 requires its nuclear localization and binding to both PP2A/B55 and the p53 ubiquitin E3 ligase MDM2. Importantly, IER5 fails to inhibit p53 ubiquitination in cells treated with the MDM2 inhibitor Nutlin-3. The IER5-PP2A/B55 complex dephosphorylates MDM2 at Ser166, leading to MDM2 ubiquitination and a reduction in nuclear MDM2. Altogether, our data provide evidence that IER5-PP2A/B55 regulates the nuclear balance between MDM2 and p53 via MDM2 dephosphorylation.
    Keywords:  IER5; MDM2; PP2A; Phosphorylation; Ubiquitination; p53
    DOI:  https://doi.org/10.1016/j.cellsig.2025.111739
  42. J Neurosci. 2025 Mar 14. pii: e1829242025. [Epub ahead of print]
    Preferential Localization of STIM1 to dendritic subsurface ER structures in Mouse Purkinje Cells.
    Sakyo Nomura, Miwako Yamasaki, Taisuke Miyazaki, Kohtarou Konno, Masahiko Watanabe.
      The endoplasmic reticulum (ER) is the largest intracellular Ca2+ store, serving as the source and sink of intracellular Ca2+ The ER Ca2+ store is continuous yet organized into distinct subcompartments with spatial and functional heterogeneity. In cerebellar Purkinje cells (PCs), glutamatergic inputs trigger Ca2+ release from specific ER domains via inositol 1,4,5-trisphosphate receptors (IP3Rs) or ryanodine receptors (RyRs). Upon ER store depletion, refilling occurs through store-operated Ca2+ entry mediated by stromal interaction molecule-1 (STIM1). Although the significance of STIM1-mediated Ca2+ regulation within PCs is established, STIM1 localization in ER subcompartments in PCs for Ca2+ release and refilling remains elusive. Using validated antibodies, we demonstrated that STIM1 was predominantly localized as intense puncta along dendritic shafts in male and female mice, colocalizing with IP3R1 but not with RyR1. Immunoelectron microscopy revealed that STIM1 was accumulated in the subsurface ER in the dendritic shaft but excluded from those in the dendritic spine, the primary site of metabotropic glutamate receptor 1 (mGluR1)-IP3R-mediated Ca²⁺ signaling. Ca²⁺ imaging from control and STIM1-knockdown (STIM1-KD) PCs demonstrated that mGluR1-mediated Ca²⁺ release is more critically dependent on STIM1 than RyR-mediated Ca²⁺ release. These findings reveal a spatially organized ER network in PCs, where specialized ER subcompartments differentially regulate Ca²⁺ release and refilling. These findings suggest that STIM1 preferentially regulates Ca²⁺ dynamics associated with mGluR1-IP3R signaling, supporting specialized ER subcompartments for Ca²⁺ release and refilling. These findings highlight the intricate molecular-anatomical organization of dendritic ER Ca2+ signaling in PCs, which is crucial for synaptic plasticity and motor learning.Significance statement Intracellular calcium (Ca²⁺) signaling is essential for neuronal function, yet the organization of endoplasmic reticulum (ER) subcompartments that coordinate Ca²⁺ release and refilling remains unclear. This study demonstrates that stromal interaction molecule-1 (STIM1), a key regulator of store-operated Ca²⁺ entry, is predominantly localized to the subsurface ER in Purkinje cell dendrites, which had not been previously identified. STIM1 colocalizes with inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) and sarco/endoplasmic reticulum Ca²⁺-ATPase 2 (SERCA2) but is segregated from ryanodine receptor 1 (RyR1), highlighting specialized ER subdomains for Ca²⁺ release and refilling. These findings provide new insights into the molecular-anatomical organization of Ca²⁺ signaling in Purkinje cells, which plays key roles in synaptic plasticity, motor learning, and the pathophysiology of neurodegenerative diseases.
    DOI:  https://doi.org/10.1523/JNEUROSCI.1829-24.2025
  43. Nat Commun. 2025 Mar 11. 16(1): 2434
    The DEAD-box helicase eIF4A1/2 acts as RNA chaperone during mitotic exit enabling chromatin decondensation.
    Ramona Jühlen, Sabine C Wiesmann, Anja Scheufen, Thilo Stausberg, Isabel Braun, Chantal Strobel, Carmen Llera-Brandt, Sabrina Rappold, Rabia Suluyayla, Marianna Tatarek-Nossol, Birgitt Lennartz, Hongqi Lue, Maximilian W G Schneider, Juan-Felipe Perez-Correa, Daniel Moreno-Andrés, Wolfram Antonin.
      During mitosis, chromosomes condense and decondense to segregate faithfully and undamaged. The exact molecular mechanisms are not well understood. We identify the DEAD-box helicase eIF4A1/2 as a critical factor in this process. In a cell-free condensation assay eIF4A1/2 is crucial for this process, relying on its RNA-binding ability but not its ATPase activity. Reducing eIF4A1/2 levels in cells consistently slows down chromatin decondensation during nuclear reformation. Conversely, increasing eIF4A1/2 concentration on mitotic chromosomes accelerates their decondensation. The absence of eIF4A1/2 affects the perichromatin layer, which surrounds the chromosomes during mitosis and consists of RNA and mainly nucleolar proteins. In vitro, eIF4A1/2 acts as an RNA chaperone, dissociating biomolecular condensates of RNA and perichromatin proteins. During mitosis, the chaperone activity of eIF4A1/2 is required to regulate the composition and fluidity of the perichromatin layer, which is crucial for the dynamic reorganization of chromatin as cells exit mitosis.
    DOI:  https://doi.org/10.1038/s41467-025-57592-1
  44. Nucleic Acids Res. 2025 Feb 27. pii: gkaf161. [Epub ahead of print]53(5):
    Eukaryotic initiation factors eIF4F and eIF4B promote translation termination upon closed-loop formation.
    Ekaterina Shuvalova, Alexey Shuvalov, Walaa Al Sheikh, Alexander V Ivanov, Nikita Biziaev, Tatiana V Egorova, Sergey E Dmitriev, Ilya M Terenin, Elena Alkalaeva.
      Eukaryotic translation initiation factor 4F (eIF4F), comprising subunits eIF4G, eIF4E, and eIF4A, plays a pivotal role in the 48S preinitiation complex assembly and ribosomal scanning. Additionally, eIF4B enhances the helicase activity of eIF4A. eIF4F also interacts with poly (A)-binding protein (PABP) bound to the poly (A) tail of messenger RNA (mRNA), thereby forming a closed-loop structure. PABP, in turn, interacts with eukaryotic release factor 3 (eRF3), stimulating translation termination. Here, we employed a reconstituted mammalian system to directly demonstrate that eIF4F potently enhances translation termination. Specifically, eIF4A and eIF4B promote the loading of eRF1 into the A site of the ribosome, while eIF4G1 stimulates the GTPase activity of eRF3 and facilitates the dissociation of release factors following peptide release. We also identified MIF4G as the minimal domain required for this activity and showed that eIF4G2/DAP5 can also promote termination. Our findings provide compelling evidence that the closed-loop mRNA structure facilitates translation termination, with PABP and eIF4F directly involved in this process.
    DOI:  https://doi.org/10.1093/nar/gkaf161
  45. Glycobiology. 2025 Mar 13. pii: cwaf015. [Epub ahead of print]
    Development of a Calreticulin-targeting Glycan Ligand Based on a Hybrid Binding Concept.
    Taiki Kuribara, Taiga Kojima, Yuka Kobayashi, Mitsuaki Hirose, Keita Shibayama, Yoichi Takeda, Kiichiro Totani.
      Calreticulin (CRT), a chaperone that possesses both lectin and chaperone domains, is localized in the endoplasmic reticulum (ER). CRT has diverse functions and localizations; thus, CRT is a multifunctional protein. Particularly in the ER, CRT mainly aids in the proper folding of nascent glycoproteins as lectin chaperones. Approximately one-third of cellular proteins, including disease-related proteins, are synthesized in the ER. The lectin chaperones CRT and calnexin facilitate the correct folding of these glycoproteins; hence, these chaperones are essential for cells. Various CRT ligands have been reported, mainly composed of Glc1Man9GlcNAc2-type glycan. However, it remains problematic for the complicated synthesis and preparation, and it interacts with glycoprotein folding-related proteins in the ER other than CRT. This suggests that the development of CRT ligands still can be improved. In this study, we developed a hybrid binding concept, which encompasses concurrent binding of ligands to CRT lectin and chaperone domains. We synthesized a CRT-targeting glycan ligand with a glycan and hydrophobic aglycone for CRT lectin and chaperone domain binding, respectively. The thermal shift assay with the CRT-targeting glycan demonstrated that binding was enhanced by simultaneous glycan and hydrophobic aglycone binding. The affinity of the CRT-targeting ligand showed isothermal titration calorimetry approximately 50-fold stronger than that of the glycan alone, thereby supporting the hybrid binding concept. In addition, the CRT-targeting ligand inhibited chaperone function. Overall, these results indicate that the hybrid binding concept may be useful as a novel strategy for the development of CRT ligands and inhibitors.
    Keywords:  Calreticulin; chaperone inhibitor; glycan ligand; hybrid binding
    DOI:  https://doi.org/10.1093/glycob/cwaf015
  46. Cell Mol Gastroenterol Hepatol. 2025 Mar 05. pii: S2352-345X(25)00032-3. [Epub ahead of print] 101491
    Attenuating ABHD17 isoforms augments the S-acylation and function of NOD2 and a subset of Crohn's disease-associated NOD2 variants.
    Charneal L Dixon, Noah R Martin, Micah J Niphakis, Benjamin F Cravatt, Gregory D Fairn.
       BACKGROUND AND AIMS: NOD2 is an intracellular innate immune receptor that detects bacterial peptidoglycan fragments. Although nominally soluble, some NOD2 is associated with the plasma membrane and endosomal compartments for microbial surveillance. This membrane targeting is achieved through post-translational S-acylation of NOD2 by the protein acyltransferase ZDHHC5. Membrane attachment is necessary to initiate a signaling cascade in response to cytosolic peptidoglycan fragments. Ultimately, this signaling results in the production of antimicrobial peptides and proinflammatory cytokines. In most cases, S-acylation is a reversible post-translational modification with removal of the fatty acyl chain catalyzed by one of several acyl protein thioesterases. Deacylation of NOD2 by such an enzyme will displace it from the plasma membrane and endosomes, thus preventing signaling.
    METHODS: To identify the enzymes responsible for NOD2 deacylation, we used engineered cell lines with RNA interference and small-molecule inhibitors. These approaches were combined with confocal microscopy, acyl-resin-assisted capture, immunoblotting, and cytokine multiplex assays.
    RESULTS: We identified α/β-hydrolase domain-containing protein 17 isoforms (ABHD17A, ABHD17B, and ABHD17C) as the acyl protein thioesterases responsible for NOD2 deacylation. Inhibiting ABHD17 increased the plasma membrane localization of wild-type NOD2 and a subset of poorly acylated Crohn's disease-associated variants. This enhanced NOD2 activity, increasing NF-κB activation and pro-inflammatory cytokine production in epithelial cells.
    CONCLUSIONS: These findings demonstrate that ABHD17 isoforms are negative regulators of NOD2. The results also suggest that targeting ABHD17 isoforms could restore functionality to specific Crohn's disease-associated NOD2 variants, offering a potential therapeutic strategy.
    Keywords:  ABHD17; Acyl Protein Thioesterase; Crohn’s disease; IL-8; Inflammation; NOD2; S-acylation
    DOI:  https://doi.org/10.1016/j.jcmgh.2025.101491
  47. bioRxiv. 2025 Feb 27. pii: 2025.02.24.639755. [Epub ahead of print]
    Sulfinyl Aziridines as Stereoselective Covalent Destabilizing Degraders of the Oncogenic Transcription Factor MYC.
    Hannah T Rosen, Kelvin Li, Erin L Li, Brynne Currier, Scott M Brittain, Francisco J Garcia, Diana C Beard, Sandra Haenni-Holzinger, Dustin Dovala, Jeffrey M McKenna, Markus Schirle, Thomas J Maimone, Daniel K Nomura.
      While MYC is a significant oncogenic transcription factor driver of cancer, directly targeting MYC has remained challenging due to its intrinsic disorder and poorly defined structure, deeming it "undruggable." Whether transient pockets formed within intrinsically disordered and unstructured regions of proteins can be selectively targeted with small molecules remains an outstanding challenge. Here, we developed a bespoke stereochemically-paired spirocyclic oxindole aziridine covalent library and screened this library for degradation of MYC. Through this screen, we identified a hit covalent ligand KL2-236, bearing a unique sulfinyl aziridine warhead, that engaged MYC in vitro as pure MYC/MAX protein complex and in situ in cancer cells to destabilize MYC, inhibit MYC transcriptional activity and degrade MYC in a proteasome-dependent manner through targeting intrinsically disordered C203 and D205 residues. Notably, this reactivity was most pronounced for specific stereoisomers of KL2-236 with a diastereomer KL4-019 that was largely inactive. Mutagenesis of both C203 and D205 completely attenuated KL2-236-mediated MYC degradation. We have also optimized our initial KL2-236 hit compound to generate a more durable MYC degrader KL4-219A in cancer cells. Our results reveal a novel ligandable site within MYC and indicate that certain intrinsically disordered regions within high-value protein targets, such as MYC, can be interrogated by isomerically unique chiral small molecules, leading to destabilization and degradation.
    DOI:  https://doi.org/10.1101/2025.02.24.639755
  48. Cancer Cell. 2025 Mar 04. pii: S1535-6108(25)00070-4. [Epub ahead of print]
    Effective targeting of PDGFRA-altered high-grade glioma with avapritinib.
    Lisa Mayr, Sina Neyazi, Kallen Schwark, Maria Trissal, Alexander Beck, Jenna Labelle, Sebastian K Eder, Liesa Weiler-Wichtl, Joana G Marques, Carlos A O de Biagi-Junior, Costanza Lo Cascio, Owen Chapman, Sunita Sridhar, Rishaan Kenkre, Aditi Dutta, Shanqing Wang, Jessica Wang, Olivia Hack, Andrezza Nascimento, Cuong M Nguyen, Sophia Castellani, Jacob S Rozowsky, Andrew Groves, Eshini Panditharatna, Gustavo Alencastro Veiga Cruzeiro, Rebecca D Haase, Kuscha Tabatabai, Sibylle Madlener, Jack Wadden, Tiffany Adam, Seongbae Kong, Madeline Miclea, Tirth Patel, Katharina Bruckner, Daniel Senfter, Anna Lämmerer, Jeffrey Supko, Armin S Guntner, Hana Palova, Jakub Neradil, Natalia Stepien, Daniela Lötsch-Gojo, Walter Berger, Ulrike Leiss, Verena Rosenmayr, Christian Dorfer, Karin Dieckmann, Andreas Peyrl, Amedeo A Azizi, Alicia Baumgartner, Ondrej Slaby, Petra Pokorna, Louise M Clark, Amy Cameron, Quang-De Nguyen, Hiroaki Wakimoto, Frank Dubois, Noah F Greenwald, Pratiti Bandopadhayay, Rameen Beroukhim, Keith Ligon, Christof Kramm, Annika Bronsema, Simon Bailey, Ana Guerreiro Stucklin, Sabine Mueller, Mary Skrypek, Nina Martinez, Daniel C Bowers, David T W Jones, Chris Jones, Natalie Jäger, Jaroslav Sterba, Leonhard Müllauer, Christine Haberler, Chandan Kumar-Sinha, Arul Chinnaiyan, Rajen Mody, Lukas Chavez, Julia Furtner, Carl Koschmann, Johannes Gojo, Mariella G Filbin.
      PDGFRA is crucial to tumorigenesis and frequently genomically altered in high-grade glioma (HGG). In a comprehensive dataset of pediatric HGG (n = 261), we detect PDGFRA mutations and/or amplifications in 15% of cases, suggesting PDGFRA as a therapeutic target. We reveal that the PDGFRA/KIT inhibitor avapritinib shows (1) selectivity for PDGFRA inhibition, (2) distinct patterns of subcellular effects, (3) in vitro and in vivo activity in patient-derived HGG models, and (4) effective blood-brain barrier penetration in mice and humans. Furthermore, we report preliminary clinical real-world experience using avapritinib in pediatric and young adult patients with predominantly recurrent/refractory PDGFRA-altered HGG (n = 8). Our early data demonstrate that avapritinib is well tolerated and results in radiographic response in 3/7 cases, suggesting a potential role for avapritinib in the treatment of HGG with specific PDGFRA alterations. Overall, these translational results underscore the therapeutic potential of PDGFRA inhibition with avapritinib in HGG.
    Keywords:  PDGFRA alteration; PDGFRA amplification; PDGFRA inhibitor; PDGFRA mutation; avapritinib; brain penetrance; diffuse midline glioma; glioblastoma; high-grade glioma; tyrosine kinase inhibitor
    DOI:  https://doi.org/10.1016/j.ccell.2025.02.018
  49. EMBO J. 2025 Mar 13.
    A versatile toolbox for determining IRES activity in cells and embryonic tissues.
    Philipp Koch, Zijian Zhang, Naomi R Genuth, Teodorus Theo Susanto, Martin Haimann, Alena Khmelinskaia, Gun Woo Byeon, Saurabh Dey, Maria Barna, Kathrin Leppek.
      Widespread control of gene expression through translation has emerged as a key level of spatiotemporal regulation of protein expression. A prominent mechanism by which ribosomes can confer gene regulation is via internal ribosomal entry sites (IRESes), whose functions have however, remained difficult to rigorously characterize. Here we present a set of technologies in embryos and cells, including IRES-mediated translation of circular RNA (circRNA) reporters, single-molecule messenger (m)RNA isoform imaging, PacBio long-read sequencing, and isoform-sensitive mRNA quantification along polysome profiles as a new toolbox for understanding IRES regulation. Using these techniques, we investigate a broad range of cellular IRES RNA elements including Hox IRESes. We show IRES-dependent translation in circRNAs, as well as the relative expression, localization, and translation of an IRES-containing mRNA isoform in specific embryonic tissues. We thereby provide a new resource of technologies to elucidate the roles of versatile IRES elements in gene regulation and embryonic development.
    Keywords:  Embryo Development; Internal Ribosome Entry Site; cellular IRES; mRNA Isoforms; mRNA Translation Regulation
    DOI:  https://doi.org/10.1038/s44318-025-00404-5
  50. Proc Natl Acad Sci U S A. 2025 Mar 18. 122(11): e2424470122
    Protein interactions, calcium, phosphorylation, and cholesterol modulate CFTR cluster formation on membranes.
    Yimei Wan, Rhea Hudson, Jordyn Smith, Julie D Forman-Kay, Jonathon A Ditlev.
      The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel whose dysfunction leads to intracellular accumulation of chloride ions, dehydration of cell surfaces, and subsequent damage to airway and ductal organs. Beyond its function as a chloride channel, interactions between CFTR, epithelium sodium channel, and solute carrier (SLC) transporter family membrane proteins and cytoplasmic proteins, including calmodulin and Na+/H+ exchanger regulatory factor-1 (NHERF-1), coregulate ion homeostasis. CFTR has also been observed to form mesoscale membrane clusters. However, the contributions of multivalent protein and lipid interactions to cluster formation are not well understood. Using a combination of computational modeling and biochemical reconstitution assays, we demonstrate that multivalent interactions with CFTR protein binding partners, calcium, and membrane cholesterol can induce mesoscale CFTR cluster formation on model membranes. Phosphorylation of the intracellular domains of CFTR also promotes mesoscale cluster formation in the absence of calcium, indicating that multiple mechanisms can contribute to CFTR cluster formation. Our findings reveal that coupling of multivalent protein and lipid interactions promotes CFTR cluster formation consistent with membrane-associated biological phase separation.
    Keywords:  biomolecular condensates; cystic fibrosis; cystic fibrosis transmembrane conductance regulator; phase separation
    DOI:  https://doi.org/10.1073/pnas.2424470122
  51. J Am Chem Soc. 2025 Mar 10.
    Dual-Action-Only PROTACs.
    Ranit Dutta, Anirudh Devarajan, Amelia Talluri, Ritam Das, S Thayumanavan.
      Proteolysis targeting chimera (PROTAC)-based degraders are highly potent pseudocatalytic drugs, but on-target off-site homing could yield undesirable consequences. We report here a generalizable AND-logic gated PROTAC, where the concurrent presence of two different disease-relevant endogenous stimuli liberates an active protein degrader. We design Dual-Action-Only PROTAC (DAO-PROTAC) molecules that are dormant and can only be activated in the presence of both hypoxia and cathepsin-L to degrade the protein of interest (POI). We also show that the dormancy of DAO-PROTACs translates to considerable mitigation of cytotoxicity, demonstrating the potential advantages over the corresponding free PROTAC and single-stimulus triggerable pro-PROTACs.
    DOI:  https://doi.org/10.1021/jacs.5c00131
  52. Nat Cancer. 2025 Mar 12.
    Targeting both death and paracaspase domains of MALT1 with antisense oligonucleotides overcomes resistance to immune-checkpoint inhibitors.
    Yuwei Tao, Chen Tian, Shaolong Qi, Ziqi Jia, Zheng Xu, Jingjing Meng, Guoyuan Xu, Haitian Hu, Xuxiang Wang, Tengjiang Zhang, Huiwen You, Xun Lan, Xin Lin, Guocan Yu, Haitao Zhou, Jiaqi Liu, Hanqiu Zheng.
      Targeting MALT1's paracaspase activity has been explored for B cell lymphoma and solid tumors. While the role of MALT1 in promoting cancer cell proliferation has been investigated, its involvement in immune evasion is unclear. Here we report that MALT1 promotes immune evasion through its paracaspase and death domain. In a paracaspase-dependent manner, MALT1 protects CD274 mRNA from degradation by its cleavage of ROQUIN1 and ROQUIN2. In a death-domain-dependent manner, MALT1 promotes the proliferation and polarization of tumor-associated macrophages to generate an immunosuppressive tumor microenvironment. Targeting MALT1 with antisense oligonucleotides inhibits PD-L1 expression in patient-derived tumor cells and suppresses the proliferation and M2-like polarization of tumor-associated macrophages isolated from patients with cancer. In preclinical models of solid tumors in female mice, treatment with MALT1 antisense oligonucleotides overcomes resistance to immune-checkpoint inhibitors. Together, our study demonstrates that targeting MALT1 is a potential strategy to overcome immune-checkpoint inhibitor resistance.
    DOI:  https://doi.org/10.1038/s43018-025-00930-5
  53. EMBO Rep. 2025 Mar 10.
    SH3KBP1 promotes skeletal myofiber formation and functionality through ER/SR architecture integrity.
    Alexandre Guiraud, Nathalie Couturier, Emilie Christin, Léa Castellano, Marine Daura, Carole Kretz-Remy, Alexandre Janin, Alireza Ghasemizadeh, Peggy Del Carmine, Laloe Monteiro, Ludivine Rotard, Colline Sanchez, Vincent Jacquemond, Claire Burny, Stéphane Janczarski, Anne-Cécile Durieux, David Arnould, Norma Beatriz Romero, Mai Thao Bui, Vladimir L Buchman, Laura Julien, Marc Bitoun, Vincent Gache.
      Dynamic changes in the arrangement of myonuclei and the organization of the sarcoplasmic reticulum are important determinants of myofiber formation and muscle function. To find factors associated with muscle integrity, we perform an siRNA screen and identify SH3KBP1 as a new factor controlling myoblast fusion, myonuclear positioning, and myotube elongation. We find that the N-terminus of SH3KBP1 binds to dynamin-2 while the C-terminus associates with the endoplasmic reticulum through calnexin, which in turn control myonuclei dynamics and ER integrity, respectively. Additionally, in mature muscle fibers, SH3KBP1 contributes to the formation of triads and modulates the Excitation-Contraction Coupling process efficiency. In Dnm2R465W/+ mice, a model for centronuclear myopathy (CNM), depletion of Sh3kbp1 expression aggravates CNM-related atrophic phenotypes and impaired autophagic flux in mutant skeletal muscle fiber. Altogether, our results identify SH3KBP1 as a new regulator of myofiber integrity and function.
    Keywords:  Centronuclear Myopathies; Endoplasmic Reticulum; Myonuclear Positioning; Triads
    DOI:  https://doi.org/10.1038/s44319-025-00413-9
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