bims-proteo 2025-07-06 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2025–07–06
forty-one papers selected by
Eric Chevet, INSERM

Mechanism of nascent chain removal by the ribosome-associated quality control complex.
Site-resolved assessment of targeted protein degradation.
SE(3)-equivariant ternary complex prediction towards target protein degradation.
TUG protein acts through a disordered region to organize the early secretory pathway.
Human eIF2A has a minimal role in translation initiation and in uORF-mediated translational control in HeLa cells.
Failed Cellular Surveillance Enables Pathogenic Matrix Deposition in a COL2A1-Related Osteoarthritis.
Mining the CRBN target space redefines rules for molecular glue-induced neosubstrate recognition.
Chemical Proteomics Identifies RBBP7 as a New E3 Ligase Supporting Targeted Protein Degradation.
Decoding polyubiquitin regulation of KV7. 1 (KCNQ1) surface expression with engineered linkage-selective deubiquitinases.
TM9SF3 is a Golgi-resident ATG8-binding protein essential for Golgi-selective autophagy.
Structural insights into the human HRD1 ubiquitin ligase complex.
CALR Type 1-Like Mutations Increase Endoplasmic Reticulum Free Ca2+ and Induce ERK1/2 Activation Independent of Thrombopoietin Receptor Activation.
Signal peptide-independent secretion of keratin-19 by pancreatic cancer cells.
Design and Application of Cereblon-Recruiting Prodegraders.
A privileged ER compartment for posttranslational heteromeric assembly of an ion channel.
ER-related E2-E3 ubiquitin enzyme pair regulates ethylene response by modulating the turnover of ethylene receptors.
Altered Relaxation and Mitochondria-Endoplasmic Reticulum Contacts Precede Major (Mal)Adaptations in Aging Skeletal Muscle and Are Prevented by Exercise.
HIV-1 vif mediates ubiquitination of the proximal protomer in the APOBEC3H dimer to induce degradation.
An adaptive peptide-binding site in ubiquitin receptor hRpn13 revealed by structural studies.
Expanding the reach of molecular glue degraders.
In situ construction of intracellular supramolecular assemblies as an alternative strategy for protein degradation.
Rapid proteasomal degradation of the stress response protein REDD2 is mediated by the E3 ligase HUWE1.
Targeted degradation of α-synuclein by arginine-based PROTACs.
A CRISPR/Cas9 screen reveals proteins at the endosome-Golgi interface that modulate cellular anti-sense oligonucleotide activity.
Cellular and biophysical barriers to lipid nanoparticle mediated delivery of RNA to the cytosol.
Advances in DNA-based proteolysis-targeting chimeras technology: Novel strategies for targeting previously undruggable proteins in cancer therapy.
Sense, plug and seal: Proteins as both rapid responders and constitutive barriers supporting organelle compartmentalization.
Sephin1 reduces TDP-43 cytoplasmic mislocalization and improves motor neuron survival in ALS models.
Immuno-metabolic stress responses control longevity from mitochondrial translation inhibition in C. elegans.
Inferring protein folding mechanisms from natural sequence diversity.
Chaperone Dependency during Primary Protein Biogenesis Does Not Correlate with Chaperone Dependency during in vitro Refolding.
Characterization of E1 enzyme dependencies in mutant-UBA1 human cells reveals UBA6 as a novel therapeutic target in VEXAS syndrome.
Plasma membrane remodeling in GM2 gangliosidoses drives synaptic dysfunction.
DRAM1 promotes the stability of lysosomal VAMP8 to enhance autolysosome formation and facilitates the extravasation.
Trioxane-based MS-cleavable cross-linking mass spectrometry for profiling multimeric interactions of cellular networks.
UFMylation: A supervisor of the HIF1α pathway and a potential therapeutic target for anti-PD-1 combination therapy in hypoxic tumors.
Single-cell signaling network profiling during redox stress reveals dynamic redox regulation in immune cells.
Prosit-XL: enhanced cross-linked peptide identification by fragment intensity prediction to study protein interactions and structures.
Sphingolipid synthesis maintains nuclear membrane integrity and genome stability during cell division.
Live-cell magnetic manipulation of recycling endosomes reveals their direct effect on actin protrusions to promote invasive migration.
STING COPII ER Export Trafficking and Signaling Primed by Phosphorylation Switches.

Nat Commun. 2025 Jul 01. 16(1): 5792

Mechanism of nascent chain removal by the ribosome-associated quality control complex.

Wenyan Li, Talia Scheel, Peter S Shen.

Errors during translation can cause ribosome stalling, leaving incomplete nascent chains attached to large ribosomal subunits. Cells rely on the Ribosome-associated Quality Control (RQC) complex to recognize, process, and remove these aberrant proteins to maintain proteostasis. Despite its importance, the mechanisms by which the RQC orchestrates nascent chain processing and extraction have remained unclear. Here, we present a cryo-EM structure of the RQC complex from budding yeast, revealing how its core components function in nascent chain removal. We show that the Cdc48 ATPase and its Ufd1-Npl4 adaptor are recruited by the Ltn1 E3 ubiquitin ligase to extract ubiquitylated peptides from the 60S ribosome. Additionally, we find that Rqc1 bridges the 60S subunit with ubiquitin and Ltn1, facilitating formation of K48-linked polyubiquitin chains. These findings provide a structural and mechanistic framework for understanding how the RQC complex collaborates to clear stalled translation products, advancing insight into cellular protein quality control.

DOI: https://doi.org/10.1038/s41467-025-61235-w
Cell Chem Biol. 2025 Jul 02. pii: S2451-9456(25)00197-7. [Epub ahead of print]

Site-resolved assessment of targeted protein degradation.

Ricardo Moreno-Ballesteros, Thomas Pembridge, Gaurav Beniwal, Satpal Virdee.

  Induced proximity using small molecules, exemplified by targeted protein degradation (TPD), represents a highly promising therapeutic strategy with significant untapped potential. However, evaluating an induced proximity event that accurately reflects drug binding typically requires the challenging and costly development of specific ligands, which limits the advancement of medicines based on this modality. To overcome this bottleneck, we combine genetic code expansion with ultra-fast bioorthogonal chemistry to sensitize specific protein sites at single-residue resolution to a generic bioorthogonal proximity inducer (BPI) molecule. Mammalian cells expressing sensitized mutants of the ubiquitin E3 ligases VHL and CRBN exhibit neosubstrate degradation in the presence of a BPI equipped with a ligand targeting bromodomain and extraterminal (BET) proteins. Furthermore, we demonstrate E3-independent degradation through recruitment of an upstream E2 conjugating enzyme. We anticipate that this approach will have broad applicability, enabling comprehensive assessment of the scope of induced proximity.

Keywords:  E2 conjugating enzymes; E3 ligases; PROTAC; bioorthogonal chemistry; degrader; genetic code expansion; glues; induced proximity; targeted protein degradation; ubiquitin

DOI:  https://doi.org/10.1016/j.chembiol.2025.06.002
Nat Commun. 2025 Jul 01. 16(1): 5514

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) has rapidly emerged as a powerful modality for drugging previously "undruggable" proteins. TPD employs small molecules like PROTACs and molecular glue degraders (MGD) to induce target protein degradation via the formation of a ternary complex with an E3 ligase. However, the rational design of these degraders is severely hindered by the difficulty of obtaining these ternary structures. Here we introduce DeepTernary, a novel end-to-end deep learning approach using an SE(3)-equivariant encoder and a query-based decoder to accurately and rapidly predict these critical structures. Trained on carefully curated TernaryDB, DeepTernary achieves state-of-the-art performance on PROTAC benchmarks without prior exposure to known PROTACs and shows notable prediction capability on the more challenging MGD benchmark with a blind docking protocol. Remarkably, the buried surface areas calculated from predicted structures correlate with experimental degradation potency metrics. Overall, DeepTernary offers a powerful tool for the development of targeted protein degraders.

DOI: https://doi.org/10.1038/s41467-025-61272-5
Nat Commun. 2025 Jul 01. 16(1): 5518

TUG protein acts through a disordered region to organize the early secretory pathway.

Anup Parchure, Helen Tejada, Zhiqun Xi, Yeongho Kim, Maohan Su, You Yan, Omar Julca-Zevallos, Abel R Alcázar-Román, Marie Villemeur, Xinran Liu, Derek Toomre, Ishier Raote, Jonathan S Bogan.

The Endoplasmic Reticulum (ER)-Golgi Intermediate Compartment (ERGIC) is a network of tubules and vesicles known for producing COPI vesicles and receiving COPII vesicles from the ER. Much about its identity, stability, and regulation remains unknown. Here, we show that TUG (UBXN9, Aspscr1) protein, a central regulator of GLUT4 trafficking, localizes to the ERGIC, and that its deletion enhances anterograde flux of a model soluble cargo protein. TUG deletion redistributes ERGIC markers to the cis-Golgi and alters Golgi morphology. TUG forms biomolecular condensates in vitro and contains a central disordered region that mediates its recruitment to ERGIC membranes. A distinct N-terminal region mediates its oligomerization in cells. TUG deletion disrupts ERGIC-dependent processes, including autophagy and collagen secretion, and alters the targeting of the CFTR chloride channel. We conclude that TUG organizes and stabilizes ERGIC membranes to support their roles in diverse secretory and degradative membrane trafficking pathways.

DOI: https://doi.org/10.1038/s41467-025-60691-8
Elife. 2025 Jul 02. pii: RP105311. [Epub ahead of print]14

Human eIF2A has a minimal role in translation initiation and in uORF-mediated translational control in HeLa cells.

Mykola Roiuk, Marilena Neff, Aurelio A Teleman.

  Translation initiation in eukaryotes requires a 40 S ribosome loaded with initiator tRNA which scans for an initiation codon. The initiator tRNA is usually recruited to the ribosome as part of a ternary complex composed of initiator tRNA, eIF2, and GTP. Although initiator tRNA recruitment was originally ascribed to another factor, eIF2A, it was later disproven and shown to occur via eIF2. Nonetheless, eIF2A is still considered a translation initiation factor because it binds the ribosome and shows genetic interactions with other initiation factors such as eIF4E. The exact function of eIF2A during translation initiation, however, remains unclear. Here, we use ribosome profiling and luciferase reporter assays to systematically test in HeLa cells the role of eIF2A in translation initiation, including translation of upstream ORFs. Since eIF2A is thought to take over the function of eIF2 when eIF2 is inhibited, we also test conditions where the integrated stress response is activated. In none of our assays, however, could we detect a role of eIF2A in translation initiation. It is possible that eIF2A plays a role in translation regulation in specific conditions that we have not tested here, or that it plays a role in a different aspect of RNA biology.

Keywords:  biochemistry; chemical biology; developmental biology; eIF2; eIF2A; human; translation

DOI:  https://doi.org/10.7554/eLife.105311
J Biol Chem. 2025 Jun 30. pii: S0021-9258(25)02286-0. [Epub ahead of print] 110436

Failed Cellular Surveillance Enables Pathogenic Matrix Deposition in a COL2A1-Related Osteoarthritis.

Kathryn M Yammine, Sophia Mirda Abularach, Michael Xiong, Seo-Yeon Kim, Agata A Bikovtseva, Vincent L Butty, Richard P Schiavoni, John F Bateman, Shireen R Lamandé, Matthew D Shoulders.

Mutations in the COL2A1 gene, encoding procollagen-II, cause various chondrodysplasias, including precocious osteoarthritis with mild spondyloepiphyseal dysplasia engendered by the p.Arg719Cys substitution. The molecular mechanisms underlying these disorders remain incompletely understood, largely owing to the absence of models faithfully recapitulating the human disease. Here, we developed an in vitro human cartilage model using isogenic induced pluripotent stem cell (iPSC) lines carrying either wild-type or Arg719Cys COL2A1. Directed differentiation into chondrocytes yielded cartilage tissues that were analyzed by immunohistochemistry, electron microscopy, SDS-PAGE, and RNA-sequencing. Tissues derived from Arg719Cys heterozygotes displayed a deficient matrix, closely reflecting the human disease phenotype. Arg719Cys procollagen-II was excessively post-translationally modified and partially retained within the endoplasmic reticulum (ER), leading to ER distention. Notably, despite introduction of an aberrant cysteine residue-expected to engage redox-sensitive folding and quality control pathways-Arg719Cys procollagen-II was not detectably recognized by the ER proteostasis network. The resulting inability to mount a quality control response, including activation of the unfolded protein response, indicates a failure in cellular surveillance. As a result, malformed procollagen-II both accumulates intracellularly and is secreted, contributing to the deposition of a structurally compromised extracellular matrix that drives disease pathology. The iPSC-derived cartilage model presented here provides a genetically defined and expandable, human-based system for dissecting the mechanisms of failed proteostasis in collagenopathies. These findings shed light on the types of substitutions in procollagen that cells can or cannot recognize, and underscore the therapeutic potential of targeting cellular surveillance and collagen quality control pathways in COL2A1-related disorders and beyond.

DOI: https://doi.org/10.1016/j.jbc.2025.110436
Science. 2025 Jul 03. 389(6755): eadt6736

Mining the CRBN target space redefines rules for molecular glue-induced neosubstrate recognition.

Georg Petzold, Pablo Gainza, Stefano Annunziato, Ilaria Lamberto, Peter Trenh, Laura A McAllister, Bradley DeMarco, Laura Schwander, Richard D Bunker, Mary Zlotosch, Rohitha SriRamaratnam, Samuel Gilberto, Gerasimos Langousis, Etienne J Donckele, Chao Quan, Vaik Strande, Gian Marco De Donatis, Shanique B Alabi, Jessica Alers, Michelle Matysik, Camille Staehly, Aurélie Dubois, Arnaud Osmont, Mackenzie Garskovas, David Lyon, Lars Wiedmer, Vladimiras Oleinikovas, Raphael Lieberherr, Nooreen T Rubin, Daniel T Lam, Xavier Lucas, Elisa Liardo, Nina Ilic Widlund, Andreas Ritzén, Ramon Miguel Caceres, Dominico Vigil, Jennifer Tsai, Owen Wallace, Marisa Peluso, Amine Sadok, Ralph Tiedt, Alison M Paterson, Vladislav Zarayskiy, Bernhard Fasching, Debora Bonenfant, Markus Warmuth, John C Castle, Sharon A Townson.

The CRL4CRBN E3 ubiquitin ligase is the target of molecular glue degrader compounds that reprogram ligase specificity to induce the degradation of clinically relevant neosubstrate proteins. Known cereblon (CRBN) neosubstrates share a generalizable β-hairpin G-loop recognition motif that allows for the systematic exploration of the CRBN target space. Computational mining approaches using structure- and surface-based matchmaking algorithms predict more than 1600 CRBN-compatible G-loop proteins across the human proteome, including the newly discovered helical G-loop motif, and identify the noncanonical neosubstrate binding mode of VAV1 that engages CRBN through a molecular surface mimicry mechanism. This work broadens the CRBN target space, redefines rules for neosubstrate recognition, and establishes a platform for the elimination of challenging drug targets by repurposing CRL4CRBN through next-generation molecular glue degraders.

DOI: https://doi.org/10.1126/science.adt6736
Angew Chem Int Ed Engl. 2025 Jul 02. e202508538

Chemical Proteomics Identifies RBBP7 as a New E3 Ligase Supporting Targeted Protein Degradation.

Yue Liu, Tao Yang, Lei Huang, Shengrong Li, Tongzheng Liu, Yi Tan, Zhengqiu Li.

  Targeted protein degradation (TPD) has been recognized as a powerful therapeutic strategy for the treatment of a wide range of diseases. However, the application of existing degraders is constrained by their dependence on a limited number of E3 ubiquitin ligases, such as CRBN and VHL. To address this limitation, we developed a suite of novel small-molecule degraders by integrating an ynamide electrophile into protein-targeting ligands. These compounds demonstrated remarkable target degradation capability. Subsequent proteome profiling and functional validation revealed that Cys97 residue of retinoblastoma binding protein 7 (RBBP7) E3 ligase was covalently engaged and responsible for the degradation mechanism. Furthermore, the ynamide motif has proved to be a versatile and transplantable chemical handle, facilitating the development of degraders targeting a wide range of proteins, including CDK4, PDE5, PI3K, AKT, BCR-ABL, BRD4, EGFRL858R, and EGFRL858R/T790M/C797S. Notably, incorporation of ynamide into the "pan-kinase" inhibitor XO44 yielded degraders capable of simultaneously degrading various kinases, such as PI3K, Syk, AKT, and GSK-3β, further highlighting the general feasibility of this approach. Importantly, the ynamide-containing degraders demonstrated significantly enhanced anticancer potency compared to their parent inhibitors.

Keywords:  E3 ubiquitin ligases, covalent probes, chemical proteomics, BTK, anticancer effects

DOI:  https://doi.org/10.1002/anie.202508538
Nat Commun. 2025 Jul 01. 16(1): 5805

Decoding polyubiquitin regulation of KV7. 1 (KCNQ1) surface expression with engineered linkage-selective deubiquitinases.

Sri Karthika Shanmugam, Scott A Kanner, Xinle Zou, Enoch Amarh, Papiya Choudhury, Rajesh Soni, Robert S Kass, Henry M Colecraft.

Polyubiquitin chain diversity generates a 'ubiquitin code' that universally regulates protein abundance, localization, and function. Functions of polyubiquitin diversity are mostly unknown, with lack of progress due to an inability to selectively tune protein polyubiquitin linkages in live cells. We develop linkage-selective engineered deubiquitinases (enDUBs) by fusing linkage-selective DUB catalytic domains to GFP-targeted nanobody and use them to investigate polyubiquitin linkage regulation of an ion channel, YFP-KCNQ1. YFP-KCNQ1 in HEK293 cells has polyubiquitin chains with K48/K63 linkages dominant. EnDUBs yield unique effects on channel surface abundance with a pattern indicating: K11 promotes ER retention/degradation, enhances endocytosis, and reduces recycling; K29/K33 promotes ER retention/degradation; K63 enhances endocytosis and reduces recycling; and K48 is necessary for forward trafficking. EnDUB effects differ in cardiomyocytes and on KCNQ1 disease mutants, emphasizing ubiquitin code mutability. The results reveal distinct polyubiquitin chains control different aspects of KCNQ1 abundance and subcellular localization and introduce linkage-selective enDUBs as potent tools to demystify the polyubiquitin code.

DOI: https://doi.org/10.1038/s41467-025-60893-0
Dev Cell. 2025 Jun 27. pii: S1534-5807(25)00372-7. [Epub ahead of print]

TM9SF3 is a Golgi-resident ATG8-binding protein essential for Golgi-selective autophagy.

Jiejie Yang, Yang Dong, Jiaxin Xu, Xuehong Qian, Yang Cai, Yanjun Chen, Pei Zhang, Ding Gao, Zongqiang Cui, Yixian Cui.

  Golgi degradation by selective autophagy (Golgiphagy) requires receptors to direct Golgi fragments into phagophores for sequestration within autophagosomes, followed by lysosomal degradation. Here, we show that the human Golgi transmembrane protein TM9SF3 is a receptor essential for Golgiphagy under nutrient-stress and multiple Golgi-stress conditions. TM9SF3 binds all six mammalian ATG8 proteins through its N-terminal LC3-interacting regions. In U2OS cells, TM9SF3 knockout blocks nutrient-stress-induced Golgi fragmentation and reduces the targeting of Golgi fragments to autophagosomes, resulting in decreased Golgi protein degradation. Beyond nutrient stress, TM9SF3 is required for Golgiphagy induced by monensin, brefeldin A, and disruptions in intra-Golgi protein glycosylation. Knockout of TM9SF3 and mutations in its LC3-interacting regions (LIRs) both compromise protein glycosylation, whereas TM9SF3 overexpression promotes degradation of incompletely glycosylated proteins. Further, we show that TM9SF3 is required for human breast cancer cell proliferation, and high TM9SF3 levels are associated with poor prognosis, implicating its function in breast cancer pathology.

Keywords:  ATG8; Golgi fragmentation; Golgi stress; Golgiphagy; LC3-interacting region; LIR; TM9SF3; breast cancer; lysosomal degradation; protein glycosylation; receptor

DOI:  https://doi.org/10.1016/j.devcel.2025.06.017
Nat Commun. 2025 Jul 01. 16(1): 6007

Structural insights into the human HRD1 ubiquitin ligase complex.

Liling Guo, Guoyun Liu, Jingjing He, Xiaoxiao Jia, Yonglin He, Zhenhua Wang, Hongwu Qian.

In the endoplasmic reticulum (ER), defective proteins are cleaned via the ER-associated protein degradation (ERAD) pathway. The HRD1 ubiquitin ligase complex, with HRD1, SEL1L, XTP3B or OS9 and Derlin family proteins as the core components, plays essential roles in the recognition, retrotranslocation, and ubiquitination of luminal ERAD substrates. However, the molecular basis is unclear. Here, we determine the cryo-EM structure of the human HRD1-SEL1L-XTP3B complex at 3.3 Å resolution. HRD1 is a dimer, but only one protomer carries the SEL1L-XTP3B complex, forming a 2:1:1 complex. Careful inspection of the EM map reveals a trimmed N-glycan sandwiched by XTP3B and SEL1L, and SEL1L may also contribute to the recognition of the trimmed glycan. The complex undergoes dramatic conformational changes when coexpressed with Derlin proteins. The HRD1 dimer is broken, and two HRD1-SEL1L-XTP3B (1:1:1) units are joined together by a four-helix bundle formed by two SEL1L molecules. The four-helix bundle also touches the micelle, resulting in a bent transmembrane region. These findings indicate that Derlins engagement may induce local curvature in the ER membrane. Cell-based functional assays are conducted to verify the structural observations. Our work provides a structural basis for further mechanistic elucidation of mammalian HRD1 complex-mediated ERAD.

DOI: https://doi.org/10.1038/s41467-025-61143-z
Cell Biol Int. 2025 Jun 30.

CALR Type 1-Like Mutations Increase Endoplasmic Reticulum Free Ca2+ and Induce ERK1/2 Activation Independent of Thrombopoietin Receptor Activation.

Mifra Faiz, Caitlin Dunstan-Harrison, Elizabeth C Ledgerwood.

  Calreticulin is a multifunctional protein found in the endoplasmic reticulum lumen that is important for calcium homeostasis and glycoprotein folding. Mutations in exon 9 of the CALR gene are the second most common genetic cause of myeloproliferative neoplasms. CALR-mutated megakaryocyte proliferation in myeloproliferative neoplasms involves cytokine-independent constitutive activation of JAK/STAT signaling caused by binding of mutant calreticulin to the thrombopoietin receptor. However, whether the partial or complete removal of wildtype calreticulin from the endoplasmic reticulum has additional effects on megakaryocyte biology is not clear. To explore the impact of calreticulin mutations independent of thrombopoietin receptor signaling we generated type 1-like CALR mutations in K-562 cells, which do not express the thrombopoietin receptor. We confirmed that the loss of endoplasmic reticulum-retention KDEL motif causes the majority of mutant calreticulin to be secreted from cells. The CALR mutated cells have higher endoplasmic reticulum free Ca2+ but basal cytosolic Ca2+ is unchanged. Cells in which the KDEL endoplasmic reticulum retention motif was lost from all CALR alleles had increased ERp57 expression however the unfolded protein response was not induced. The calreticulin mutated cells also showed elevated basal phosphorylation of ERK1/2. Overall, these results suggest that the phenotype of type 1 CALR mutated myeloproliferative neoplasms is not solely due to cytokine independent activation of the thrombopoietin receptor by the mutant calreticulin, and that increased endoplasmic reticulum Ca2+ and/or basal ERK1/2 activation may contribute to the abnormal megakaryocyte proliferation characteristic of CALR mutant myeloproliferative neoplasms.

Keywords:  ERK1/2; calcium; calreticulin; myeloproliferative neoplasm; unfolded protein response

DOI:  https://doi.org/10.1002/cbin.70053
Proc Natl Acad Sci U S A. 2025 Jul 08. 122(27): e2426218122

Signal peptide-independent secretion of keratin-19 by pancreatic cancer cells.

Philip Moresco, Jonathan P Kastan, Jung-In Yang, Rishvanth Prabakar, Z Larkin Kelley, Francesca Minicozzi, Dexter W Adams, Paolo Cifani, David A Tuveson, Douglas T Fearon.

  The exclusion of T cells causes immune escape of pancreatic ductal adenocarcinoma (PDA). T cell exclusion is mediated by the interaction between CXCR4 on T cells and its ligand, CXCL12, which is complexed to keratin-19 (KRT19) on the surface of PDA cells. KRT19 secretion by PDA cells is essential to this process but is unusual because KRT19 lacks an endoplasmic reticulum (ER)-directing signal peptide (SP). By using biotinylation by an ER-restricted TurboID system and a split-GFP assay in PDA cells, we demonstrate that KRT19 enters the ER via its "head" domain. Additionally, KRT19 is shown to interact with the signal recognition particle and its secretion is sensitive to canonical protein secretion inhibitors. In vivo, mouse tumors formed with ER-TurboID-expressing PDA cells contain biotinylated KRT19. In contrast, keratin-8 (KRT8), which colocalizes with KRT19 on the surface of PDA cells, does not enter the ER. Rather, KRT8 is externalized via secretory autophagy possibly in a complex with KRT19. Thus, despite lacking a classical SP, PDA cells secrete KRT19 to capture CXCL12 and protect against immune attack.

Keywords:  endoplasmic reticulum; pancreatic cancer; tumor immunology; unconventional protein secretion

DOI:  https://doi.org/10.1073/pnas.2426218122
J Am Chem Soc. 2025 Jul 02.

Design and Application of Cereblon-Recruiting Prodegraders.

Davis H Chase, Alicia Stein, Daniel E Grinshpun, Mackenzie W Krone, Craig M Crews.

Cereblon (CRBN) is one of the most widely co-opted E3 ligase substrate receptor proteins in targeted protein degradation (TPD), and several CRBN-recruiting degraders are currently in late-stage preclinical evaluation. While the diversity of CRBN-recruiting moieties has rapidly expanded, the glutarimide ring has remained largely conserved in these ligands. Glutarimides can form during protein cleavage events via the intramolecular cyclization of glutamine within the protein backbone. Inspired by this biological mechanism, we developed CRBN-recruiting prodegraders by replacing the glutarimide in parent degraders with uncyclized glutamine analogs. Prodegraders derived from a potent, cytotoxic GSPT1 degrader exhibited in cellulo cyclization, resulting in GSPT1 degradation. Optimization of the prodegrader scaffold revealed that glutarimide cyclization rates─and consequently, degradation kinetics─were tunable, with the most optimized prodegrader displaying degradation efficacy and cytotoxicity comparable to the parent, glutarimide-containing GSPT1 degrader. Furthermore, this prodegrader strategy can be readily applied to other known CRBN-based molecular glues and PROTACs. In contrast to conventional glutarimide-containing degraders, the amide of the prodegrader scaffold provides an accessible conjugation handle for stimulus-sensitive groups. We show that prodegraders can be conjugated to and released in vitro from a photolabile protecting group and a commonly used cathepsin-cleavable degrader-antibody conjugate (DAC) linker. Therefore, these prodegrader scaffolds introduce a generalizable conjugation strategy for CRBN-recruiting degraders to DAC linkers, eliminating the need for extensive degrader modifications to incorporate a conjugation handle. Overall, these findings establish the feasibility of utilizing glutamine analogs as a CRBN-recruiting prodegrader strategy and highlight their potential application in targeted drug delivery systems.

DOI: https://doi.org/10.1021/jacs.5c05036
Proc Natl Acad Sci U S A. 2025 Jul 08. 122(27): e2500218122

A privileged ER compartment for posttranslational heteromeric assembly of an ion channel.

Sudharsan Kannan, William Kasberg, Liliana R Ernandez, Anjon Audhya, Gail A Robertson.

  Mechanisms underlying heterotypic subunit assembly of ion channels and other oligomeric complexes are poorly understood. In the human heart, heteromeric assembly of two isoforms encoded by the human ether-à-go-go related gene (hERG) is essential for the normal function of cardiac IKr in ventricular repolarization, with loss of hERG1b contributing to arrhythmias associated with long QT-syndrome (LQTS). While hERG1a homomers traffic efficiently to the plasma membrane, hERG1b homomers are retained in the endoplasmic reticulum (ER). When expressed together, the two subunits avidly associate during biogenesis. Seeking rules specifying heteromeric association, we characterized the fate of hERG1b proteins using confocal and superresolution imaging in fixed and live HeLa cells. We found hERG1b sequestered in punctate intracellular structures when expressed alone in HeLa cells. These puncta, which depend on the presence of an N-terminal "RXR" ER retention signal, represent a privileged ER subcompartment distinct from that containing ER-retained, type 2 (hERG-based) LQTS mutant proteins, which were rapidly degraded by the proteasome. Introducing hERG1a to cells with preformed hERG1b puncta dissolved these puncta by rescuing extant hERG1b. Rescue occurred by association of fully translated hERG1b with 1a, a surprising finding given previous studies demonstrating cotranslational heteromeric association. We propose that sequestration limits potentially deleterious surface expression of hERG1b homomeric channels while preserving hERG1b for an alternative mode of heteromeric hERG1a/1b channel assembly posttranslationally. These findings reveal a surprising versatility of biosynthetic pathways promoting heteromeric assembly.

Keywords:  arrhythmia; condensate; hERG; long QT syndrome; protein trafficking

DOI:  https://doi.org/10.1073/pnas.2500218122
Nat Commun. 2025 Jul 01. 16(1): 5937

ER-related E2-E3 ubiquitin enzyme pair regulates ethylene response by modulating the turnover of ethylene receptors.

Qingcui Zhao, Xiaofeng Zhou, Qian Chen, Ruoyun Yang, Yonghong Li, Jiaxin Zhao, Xijia Fu, Yan Lin, Yuming Liu, Liangjun Zhao, Cai-Zhong Jiang, Su-Sheng Gan, Qi Xie, Junping Gao, Nan Ma.

Gaseous phytohormone ethylene regulates various aspects of plant development. Ethylene is perceived by ER membrane-localized receptors, which are inactivated upon binding with ethylene molecules, thereby initiating ethylene signal transduction. Here, we report that a novel E3 ligase RING finger for Ethylene receptor Degradation (RED) and its E2 partner UBC32 ubiquitinate ethylene-bound receptors for degradation through an ER associated degradation (ERAD) pathway in both Rosa hybrida and Solanum lycopersicum. The depletion of RED or UBC32 leads to hypersensitivity to ethylene, which is manifested as premature leaf abscission and petal shedding in roses, as well as the dwarf plants and accelerated fruit ripening in tomatoes. Disruption of the conserved ethylene binding site of receptors prevents RED-mediated degradation of the receptors. Our study discovers an ERAD branch that facilitates the ethylene-induced degradation of receptors, and provides insights into how the plant's response to ethylene can be controlled by modulating the turnover of ethylene receptors.

DOI: https://doi.org/10.1038/s41467-025-61066-9
Aging Cell. 2025 Jun 30. e70137

Altered Relaxation and Mitochondria-Endoplasmic Reticulum Contacts Precede Major (Mal)Adaptations in Aging Skeletal Muscle and Are Prevented by Exercise.

Ryan J Allen, Ana Kronemberger, Qian Shi, R Marshall Pope, Elizabeth Cuadra-Muñoz, Wangkuk Son, Long-Sheng Song, Ethan J Anderson, Renata O Pereira, Vitor A Lira.

  Sarcopenia, or age-related muscle dysfunction, contributes to morbidity and mortality. Besides decreases in muscle force, sarcopenia is associated with atrophy and fast-to-slow fiber type switching, which is typically secondary to denervation in humans and rodents. However, very little is known about cellular changes preceding these important (mal)adaptations. To this matter, mitochondria and the sarcoplasmic reticulum are critical for tension generation in myofibers. They physically interact at the boundaries of sarcomeres, forming subcellular hubs called mitochondria-endo/sarcoplasmic reticulum contacts (MERCs). Yet, whether changes at MERCs ultrastructure and proteome occur early in aging is unknown. Here, studying young adult and older mice, we reveal that aging slows muscle relaxation, leading to longer excitation-contraction-relaxation (ECR) cycles before maximal force decreases and fast-to-slow fiber switching takes place. We also demonstrate that muscle MERC ultrastructure and mitochondria-associated ER membrane (MAM) protein composition are affected early in aging and are closely associated with the rate of muscle relaxation. Additionally, we demonstrate that regular exercise preserves muscle relaxation rate and MERC ultrastructure in early aging. Finally, we profile a set of muscle MAM proteins involved in energy metabolism, protein quality control, Ca2+ homeostasis, cytoskeleton integrity, and redox balance that are inversely regulated early in aging and by exercise. These may represent new targets to preserve muscle function in aging individuals.

Keywords:  aging; endoplasmic reticulum; exercise; mitochondria; mitochondrial‐associated ER membranes; sarcopenia; sarcoplasmic reticulum; skeletal muscle

DOI:  https://doi.org/10.1111/acel.70137
Nat Commun. 2025 Jul 01. 16(1): 5879

HIV-1 vif mediates ubiquitination of the proximal protomer in the APOBEC3H dimer to induce degradation.

Katarzyna A Skorupka, Kazuhiro Matsuoka, Bakar Hassan, Rodolfo Ghirlando, Vanivilasini Balachandran, Ting-Hua Chen, Kylie J Walters, Celia A Schiffer, Matthias Wolf, Yasumasa Iwatani, Hiroshi Matsuo.

The APOBEC3 family of cytidine deaminases restricts retroviruses like HIV-1 by mutating viral DNA. HIV-1 evades this restriction by producing Vif, which recruits the Cullin-5 (CUL5) E3 ubiquitin ligase complex to promote APOBEC3 degradation. Here we resolve key aspects of this counter-defense mechanism by determining a 3.6 Å cryo-EM structure of chimpanzee APOBEC3H (cpzA3H) in complex with HIV-1 Vif and three components of the CUL5 E3 ligase-CBFβ, EloB, and EloC (VCBC). The structure captures cpzA3H as an RNA-mediated dimer within the cpzA3H-VCBC complex, allowing us to examine the role of dimerization. We find that ubiquitination occurs specifically at two lysine residues on the Vif-proximal protomer, while the distal protomer remains unmodified. The structural model of the active cpzA3H-Vif-CUL5 E3 ligase holoenzyme reveals spatial preferences for ubiquitin transfer to the targeted lysine residues. These findings enhance our understanding of A3H degradation and suggest new antiviral strategies targeting this host-virus interface.

DOI: https://doi.org/10.1038/s41467-025-60984-y
Nat Commun. 2025 Jul 01. 16(1): 5669

An adaptive peptide-binding site in ubiquitin receptor hRpn13 revealed by structural studies.

Bakar Hassan, Monika Chandravanshi, Martin Y Ng, Hitendra Negi, Brice A P Wilson, Kylie J Walters.

A pleckstrin-like receptor for ubiquitin (Pru) domain in hRpn13 binds ubiquitin and proteasome subunit hRpn2. Here, we report a crystal structure of Pru bound to amino acids at the extreme N-terminus (ENT) of recombinant hRpn13. ENT adopts a U shape with native sequence along one side where M1 is buried in a Pru W108-centered pocket, and non-native sequence along the other with main chain hydrogen bonding to a neighboring Pru of the crystal lattice. These ENT:Pru interactions are stable in molecular dynamics simulations even with inclusion of only one Pru. Our findings suggest that hRpn13 can form bidentate interactions with ubiquitinated substrates by binding to both ubiquitin chains and disordered sequences of substrates. Testing this model by solution nuclear magnetic resonance revealed Pru to bind weakly to various peptides, concurrent binding with ubiquitin, and ENT displacement by hRpn2, the latter required for substrate handoff to the proteasome ATPases.

DOI: https://doi.org/10.1038/s41467-025-60843-w
Science. 2025 Jul 03. 389(6755): 32-33

Expanding the reach of molecular glue degraders.

Marcus D Hartmann.

An epitope-centric screening approach widens the neosubstrate landscape for targeted protein degradation.

DOI: https://doi.org/10.1126/science.ady4446
Nat Commun. 2025 Jul 01. 16(1): 5974

In situ construction of intracellular supramolecular assemblies as an alternative strategy for protein degradation.

Xiaoqian Hu, Qingxin Yao, Xiaobo Wu, Jiali Chen, Ruijia Zhang, Yan Zhao, Yuan Gao.

Targeted protein degradation has emerged as a promising anticancer strategy. Bringing disease-related proteins into proximity with the degradation system is crucial but often hindered by the availability of suitable ligands for proteins of interest (POIs). In this study, we utilize the interactions between intracellular supramolecular nanofibers and certain guest proteins to establish a ligand-free strategy for protein degradation. As the enterokinase (ENTK)-instructed supramolecular assemblies interact with the histone protein H2B for its translocation, the tetrazine-bearing supramolecular nanofibers conjugate with a cereblon E3 ligase ligand to recruit CRBN and directly degrade wild-type H2B. Using the same bioorthogonal ligation, another reactive oxygen species (ROS)-induced supramolecular assemblies localize to mitochondria and efficiently degrade Cofilin-2. Both in situ formed intracellular supramolecular assemblies are dependent on cancer-related conditions (either overexpressed enzymes or overproduced ROS), owning the merit of cell selectivity. These assemblies synergize with bioorthogonal ligation to exhibit significant biological activities, including chemotherapeutic sensitization and induced apoptosis, thereby inhibiting cancer cell growth.

DOI: https://doi.org/10.1038/s41467-025-61175-5
Biochem Biophys Res Commun. 2025 Jun 28. pii: S0006-291X(25)00985-4. [Epub ahead of print]777 152270

Rapid proteasomal degradation of the stress response protein REDD2 is mediated by the E3 ligase HUWE1.

Ashley M VanCleave, Siddharth Sunilkumar, Christopher M McCurry, Allyson L Toro, Scot R Kimball, Michael D Dennis.

  The stress response proteins regulated in development and DNA damage (REDD)1 and REDD2 act as negative regulators of mechanistic target of rapamycin complex 1 (mTORC1). Prior studies support that REDD1 is rapidly degraded via both chaperone-mediated autophagy (CMA) and the ubiquitin proteasome system (UPS). Compared to REDD1, relatively little is known regarding the regulation of REDD2. The objective here was to investigate the molecular mechanisms that control the cellular abundance of REDD2. Genetic and pharmacologic interventions were used to manipulate protein synthesis and proteolysis. We found that both REDD1 and REDD2 were rapidly degraded with half-lives of <20 min. Interestingly, REDD2 expression reduced the rate of REDD1 degradation, suggesting that the molecular mechanism through which they are degraded overlaps. However, in contrast with REDD1, CMA activation did not promote REDD2 degradation, despite the conservation of a putative KFERQ-like motif sequence in REDD2. Instead, we provide evidence that the rapid degradation of REDD2 was mediated by the UPS, the E3 ligase HUWE1, and K119/K120 of REDD2. The findings support that the cellular abundance of both REDD1 and REDD2 are controlled at the level of protein stability.

Keywords:  DDIT4; DDIT4L; Proteasome; Proteolysis

DOI:  https://doi.org/10.1016/j.bbrc.2025.152270
J Biol Chem. 2025 Jul 02. pii: S0021-9258(25)02299-9. [Epub ahead of print] 110449

Targeted degradation of α-synuclein by arginine-based PROTACs.

Linjing Shen, Jianchao Zhang, Zhaoran Wang, Yaxuan Liu, Shengjin Cui, Hai Rao.

  Parkinson's disease (PD), the second most prevalent neurodegenerative disorder, is associated with α-synuclein (α-syn) overexpression or mutation, leading to harmful aggregates and neuronal apoptosis. Effective drugs that inhibit or reduce α-syn accumulation remain challenging. Targeted protein degradation (TPD) technology offers a novel solution by utilizing the ubiquitin-proteasome pathway to target specific proteins for destruction. Here, we have developed Proteolysis Targeting Chimera (PROTAC) to target α-syn for degradation. Specifically, our PROTACs employ the amino acid arginine (Arg) as the E3 ligase ligand, and a benzothiazole-aniline variant as the warhead for α-syn. The efficacy of these PROTACs in degrading α-syn and its aggregates was tested in mammalian cells and Caenorhabditis elegans (C. elegans) models. Arg-PEG1-Tα-syn shows the highest degradation effect in mammalian cells for both wild-type α-syn and the α-syn (A53T) mutant. UBR1 is the ubiquitin E3 ligase responsible for PROTAC-mediated degradation. Furthermore, Arg-PEG1-Tα-syn significantly reduces α-syn aggregates and associated toxicities in both mammalian cells and C. elegans. These findings highlight the potential of a single amino acid-based PROTAC targeting α-syn for degradation, representing a possible therapeutic approach for PD and other synucleinopathies.

Keywords:  AATac; N-end rule; PROTAC; Parkinson's disease; amino acid; arginine; α-synuclein

DOI:  https://doi.org/10.1016/j.jbc.2025.110449
Nat Commun. 2025 Jun 30. 16(1): 5378

A CRISPR/Cas9 screen reveals proteins at the endosome-Golgi interface that modulate cellular anti-sense oligonucleotide activity.

Liza Malong, Jessica Roskosch, Carolina Hager, Jean-Philippe Fortin, Roland Schmucki, Marinella G Callow, Christian Weile, Valentina Romeo, Christoph Patsch, Scott Martin, Mike Costa, Zora Modrusan, Roberto Villaseñor, Erich Koller, Benjamin Haley, Anne Spang, Filip Roudnicky.

Anti-sense oligonucleotides (ASOs) are modified synthetic single-stranded molecules with enhanced stability, activity, and bioavailability. They associate with RNA through sequence complementarity and can reduce or alter mRNA expression upon binding of splice site positions. To target RNA in the nucleus or cytoplasm, ASOs must cross membranes, a poorly understood process. We performed an unbiased CRISPR/Cas9 knockout screen with a genetic splice reporter to identify genes that can increase or decrease ASO activity, resulting in the most comprehensive catalog of ASO-activity modifier genes. Here we reveal distinct targets, including AP1M1 and TBC1D23, linking ASO activity to transport of cargo between the Golgi and endosomes. AP1M1 absence strongly increases ASO activity by delaying endosome-to-lysosome transport in vitro and in vivo. Prolonged ASO residence time in the endosomal system may increase the likelihood of ASO escape. This insight into AP1M1 role in ASO trafficking suggests a way for enhancing the therapeutic efficacy of ASOs by manipulating the endolysosomal pathways.

DOI: https://doi.org/10.1038/s41467-025-61039-y
Nat Commun. 2025 Jul 01. 16(1): 5354

Cellular and biophysical barriers to lipid nanoparticle mediated delivery of RNA to the cytosol.

Johanna M Johansson, Hampus Du Rietz, Hampus Hedlund, Hanna C Eriksson, Erik Oude Blenke, Aditya Pote, Said Harun, Pontus Nordenfelt, Lennart Lindfors, Anders Wittrup.

Lipid nanoparticles (LNPs) are clinically approved for mRNA-based vaccines and liver-targeted siRNA delivery. However, poor nucleic acid delivery efficiency limits their application in extrahepatic tissues and tumors. Here, using live-cell and super-resolution microscopy, we identify multiple distinct steps of inefficiencies in the cytosolic delivery of both siRNA and mRNA cargoes. Membrane damages marked by galectin recruitment are conducive to cytosolic RNA release, whereas membrane perturbations recruiting the ESCRT machinery do not permit endosomal escape. Notably, only a small fraction of RNA is released from galectin-marked endosomes and, unexpectedly, many damaged endosomes contain no detectable RNA cargo. Using LNPs with both fluorescently labeled ionizable lipid and RNA, we show that these components segregate during endosomal sorting - both within single endosomes and across endosomal compartments. Finally, we visualize localized ionizable lipid enrichment in endosomal membranes and membrane damage in direct proximity to siRNA-LNPs tethered to luminal vesicle membranes. Taken together, our findings reveal multiple mechanistic barriers limiting intracellular RNA delivery by LNPs.

DOI: https://doi.org/10.1038/s41467-025-60959-z
Bioorg Med Chem. 2025 Jun 28. pii: S0968-0896(25)00238-X. [Epub ahead of print]129 118297

Advances in DNA-based proteolysis-targeting chimeras technology: Novel strategies for targeting previously undruggable proteins in cancer therapy.

Min Hou, Mingda Li, Yulin Li, Xiaobo Wu, Die Long, Di Sun, Jincheng Zeng.

  Targeted protein degradation (TPD) technology has emerged as a transformative therapeutic strategy for selectively eliminating aberrant proteins across diverse pathological conditions. This comprehensive review systematically examines recent advances in DNA-based proteolysis-targeting chimeras (DNA-PROTACs), which harness the exceptional specificity and binding affinity of DNA to substantially expand the targetable protein repertoire beyond conventional small molecule PROTACs. Through extensive literature analysis encompassing mechanistic studies, preclinical evaluations, and clinical investigations, we demonstrate that DNA-PROTACs effectively target previously undruggable proteins, including transcription factors, cell membrane proteins, and DNA damage response mediators. These innovative chimeric constructs exhibit superior catalytic efficiency through E3 ubiquitin ligase recruitment via the proteasomal degradation pathway, with unique advantages in linker optimization enabled by precise nucleotide-level control during DNA synthesis. Cell-based assays consistently reveal enhanced selectivity profiles and expanded therapeutic windows compared to traditional PROTAC modalities and alternative RNA-based approaches. Despite promising preclinical outcomes and advancing clinical development timelines, challenges in delivery optimization, molecular stability enhancement, and clinical translation persist. The integration of artificial intelligence-assisted drug design platforms and in vivo aptamer evolution technologies presents unprecedented opportunities for accelerating DNA-PROTACs development toward sub-nanomolar potency targets, positioning this technology as a paradigm-shifting approach in precision medicine across oncology, immunotherapy, and neurodegeneration therapeutics.

Keywords:  Aptamer-based therapeutics; DNA-PROTAC; Targeted protein degradation; Undruggable proteins; cancer therapy

DOI:  https://doi.org/10.1016/j.bmc.2025.118297
Mol Biol Cell. 2025 Jul 02. mbcE23080307

Sense, plug and seal: Proteins as both rapid responders and constitutive barriers supporting organelle compartmentalization.

Megan C King, C Patrick Lusk, Nicholas R Ader.

While organellar compartmentalization is primarily established by the delimiting phospholipid bilayer membranes, the contribution of proteins has been less appreciated. Recently, studies across many realms of cell biology have put new focus on the role of proteins in acting as diffusion barriers in contexts where there are constitutive, regulated, or pathological discontinuities in membranes. Here, we synthesize longstanding observations of proteins acting as both barriers to lateral diffusion on membranes and diffusion in three-dimensional space. In particular, we focus on an emerging, conserved two-step paradigm of protein diffusion barriers that rapidly assemble in response to membranous organelle damage: a first phase of coincident sensing and stopgap "plugging" by responding repair proteins followed by a second phase of membrane sealing. We highlight recent work exemplifying this sense, plug, and seal paradigm at the post-mitotic nuclear envelope and at ruptures of the interphase nuclear envelope, lysosomes, and the plasma membrane. Taken together, we highlight how cells employ a variety of constitutive and induced proteinaceous barriers that support the role of biological membranes in defining organelle compartmentalization. Determining the biophysical nature of these barriers, and their means of "sensing" membrane rupture, will be an exciting avenue of future investigations.

DOI: https://doi.org/10.1091/mbc.E23-08-0307
Life Sci Alliance. 2025 Sep;pii: e202403195. [Epub ahead of print]8(9):

Sephin1 reduces TDP-43 cytoplasmic mislocalization and improves motor neuron survival in ALS models.

Emmanuelle Abgueguen, Massimo Tortarolo, Laura Rouviere, Stefania Marcuzzo, Laura Camporeale, Alexandre Henriques, Laura Pasetto, Georgia R Culley, Valentina Bonetto, Anca Marian, Beatrice L Lejeune, Anne Visbecq, Giuseppe Lauria, Edor Kabashi, Noëlle Callizot, Caterina Bendotti, Pierre Y Miniou.

A pathological hallmark of ALS is the abnormal accumulation of misfolded proteins (e.g., TDP-43) and enlarged endoplasmic reticulum (ER), indicating ER stress. To resolve this stress, cells initiate the Unfolded Protein Response (UPR). However, unresolved stress leads to apoptosis. In ALS, UPR activation fails to resolve proteostasis impairment. UPR activation modulators, among them Sephin1, reduce protein aggregates and improve motor neuron survival in ALS models. We demonstrate that following glutamate intoxication, Sephin1 increases motor neuron survival by reducing mitochondria ROS production and extranuclear TDP-43. Sephin1 reduces abnormal splicing because of TDP-43 nuclear loss of function following oxidative stress. In SOD1G93A mice, Sephin1 treatment decreases TDP-43 in triton-insoluble fraction, improving motor neuron survival in spinal cord. Sephin1 improves motor neurons survival, motor function and survival of mutated TDP-43 transgenic zebrafish. Sephin1 improves motor neuron survival in ALS models by reducing TDP-43 cytoplasmic mislocalization and its toxicity. These findings open new therapeutic opportunities for Sephin1 in neurodegenerative pathologies with TDP-43 proteinopathy, including ALS.

DOI: https://doi.org/10.26508/lsa.202403195
Nat Commun. 2025 Jul 02. 16(1): 6083

Immuno-metabolic stress responses control longevity from mitochondrial translation inhibition in C. elegans.

Iman Man Hu, Marte Molenaars, Yorrick R J Jaspers, Bauke V Schomakers, Michel van Weeghel, Amber Bakker, Melanie Modder, Joseph P Dewulf, Guido T Bommer, Arwen W Gao, Georges E Janssens, Riekelt H Houtkooper.

Perturbing mitochondrial translation represents a conserved longevity intervention, with proteostasis processes proposed to mediate the resulting lifespan extension. Here, we explore whether other mechanisms may contribute to lifespan extension upon mitochondrial translation inhibition. Using multi-omics and functional in vivo screening, we identify the ethylmalonyl-CoA decarboxylase orthologue C32E8.9 in C. elegans as an essential factor for longevity induced by mitochondrial translation inhibition. Reducing C32E8.9 completely abolishes lifespan extension from mitochondrial translation inhibition, while mitochondrial unfolded protein response activation remains unaffected. We show that C32E8.9 mediates immune responses and lipid remodeling, which play crucial roles in the observed lifespan extension. Mechanistically, sma-4 (a TGF-β co-transcription factor) serves as an effector of C32E8.9, responsible for the immune response triggered by mitochondrial translation inhibition. Collectively, these findings underline the importance of the "immuno-metabolic stress responses" in longevity upon mitochondrial translation inhibition and identify C32E8.9 as a central factor orchestrating these responses.

DOI: https://doi.org/10.1038/s41467-025-61433-6
Biophys J. 2025 Jun 26. pii: S0006-3495(25)00411-4. [Epub ahead of print]

Inferring protein folding mechanisms from natural sequence diversity.

Ezequiel A Galpern, Ernesto A Roman, Diego U Ferreiro.

  Protein sequences serve as a natural record of the evolutionary constraints that shape their functional structures. We show that it is possible to use only sequence information to go beyond predicting native structures and global stability to infer the folding mechanisms of globular proteins. The one- and two-body evolutionary energy fields at the amino-acid level are mapped to a coarse-grained description of folding, where proteins are divided into contiguous folding elements, commonly referred to as foldons. For 15 diverse protein families, we calculated the folding mechanisms of hundreds of proteins by simulating an Ising chain of foldons, with their energetics determined by the amino acid sequences. We show that protein topology imposes limits on the variability of folding cooperativity within a family. While most beta and alpha/beta structures exhibit only a few possible mechanisms despite high sequence diversity, alpha topologies allow for diverse folding scenarios among family members. We show that both the stability and cooperativity changes induced by mutations can be computed directly using sequence-based evolutionary models.

Keywords:  Evolution; Foldon; Ising; Potts

DOI:  https://doi.org/10.1016/j.bpj.2025.06.034
bioRxiv. 2025 Jun 19. pii: 2025.06.16.659923. [Epub ahead of print]

Chaperone Dependency during Primary Protein Biogenesis Does Not Correlate with Chaperone Dependency during in vitro Refolding.

Divya Yadav, İdil I Demiralp, Mark Fakler, Stephen D Fried.

Many proteins require molecular chaperones to fold into their functional native forms. Previously we used limited proteolysis mass-spectrometry (LiP-MS) to find that ca. 40% of the E. coli proteome do not efficiently refold spontaneously following dilution from denaturation, a frequency that drops to ca. 15% once molecular chaperones like DnaK or GroEL are provided. However, the roles of chaperones during primary biogenesis in vivo can differ from the functions they play during in vitro refolding experiments. Here, we used LiP-MS to probe structural changes incurred by the E. coli proteome when two key chaperones, trigger factor and DnaKJ, are deleted. While knocking out DnaKJ induces pervasive structural perturbations across the soluble E. coli proteome, trigger factor deletion only impacts a small number of proteins' structures. Overall, proteins which cannot spontaneously refold (or require chaperones to refold in vitro ) are not more likely to be dependent on chaperones to fold in vivo . For instance, the glycolytic enzyme, phosphoglycerate kinase (PGK), cannot refold to its native form in vitro following denaturation (even with chaperones), but by LiP-MS we find that its structure is unperturbed upon DnaKJ or Tig deletion, which is further supported with biochemical and biophysical assays. Thus, PGK folds to its native structure most efficiently during co-translational folding and does so without chaperone assistance. This behaviour is generally found among chaperone-nonrefolders (proteins that cannot refold even with chaperone assistance), strengthening the view that chaperone-nonrefolders are obligate co-translational folders. Hence, for some E. coli proteins, the vectorial nature of co-translational folding is the most important "chaperone."
Highlights: LiP-MS is used to identify which proteins in E. coli are structurally perturbed when DnaKJ or trigger factor is deleted Very few proteins require trigger factor to assume their native structures The proteome's dependence on DnaKJ is increased at lower growth temperature The enzyme PGK does not need chaperones to fold, but it cannot refold from denaturant, even with chaperone assistance For some E. coli proteins (such as PGK) co-translational folding during primary biogenesis is the most important "chaperone".

DOI: https://doi.org/10.1101/2025.06.16.659923
Leukemia. 2025 Jun 30.

Characterization of E1 enzyme dependencies in mutant-UBA1 human cells reveals UBA6 as a novel therapeutic target in VEXAS syndrome.

Courtnee A Clough, Claire Cunningham, Sophia Y Philbrook, Kathleen M Hueneman, Avery M Sampson, Kwangmin Choi, Kenneth D Greis, Daniel Starczynowski.

VEXAS syndrome is a clonal hematopoietic disorder characterized by hyperinflammation, bone marrow failure, and high mortality. The molecular hallmark of VEXAS is somatic mutations at methionine 41 (M41) in the E1 ubiquitin enzyme, UBA1. These mutations induce a protein isoform switch, but the mechanisms underlying disease pathogenesis remain unclear. Here, we developed a human cell model of VEXAS syndrome by engineering the male monocytic THP1 cell line to express the common UBA1M41V mutation. We found that mutant UBA1M41V cells exhibit aberrant UBA1 isoform expression, increased vacuolization, and upregulation of the unfolded protein response, recapitulating key features of VEXAS. Moreover, proteomic analyses revealed dysregulated ubiquitination and proteotoxic stress in UBA1M41V cells, with alterations in inflammatory and stress-response pathways. Functional studies demonstrated that UBA1M41V cells were highly sensitive to genetic or pharmacological inhibition of E1 ubiquitin enzymes. Treatment with the E1 enzyme inhibitor TAK-243 preferentially suppressed colony formation of UBA1M41V cells as compared to WT cells. Moreover, UBA1M41V cells exhibited greater sensitivity to TAK-243 in competition assays and showed increased apoptosis. Interestingly, TAK-243 preferentially inhibited UBA6 activity over UBA1, suggesting that UBA6 may compensate for UBA1 dysfunction in UBA1M41V cells. Targeting UBA6 using shRNA or the UBA6-specific inhibitor phytic acid further revealed an acquired dependency on UBA6 in UBA1M41V cells. Phytic acid selectively impaired growth and colony formation in UBA1M41V cells while sparing WT cells, highlighting a potential therapeutic vulnerability. Together, these findings establish a novel human model of VEXAS syndrome, identify key roles for UBA1 and UBA6 in disease pathogenesis, and demonstrate that UBA6 inhibition represents a promising therapeutic strategy for selectively targeting UBA1 mutant clones.

DOI: https://doi.org/10.1038/s41375-025-02671-x
PLoS Biol. 2025 Jul 03. 23(7): e3003265

Plasma membrane remodeling in GM2 gangliosidoses drives synaptic dysfunction.

Alex S Nicholson, David A Priestman, Robin Antrobus, James C Williamson, Reuben Bush, Shannon J McKie, Henry G Barrow, Emily Smith, Kostantin Dobrenis, Nicholas A Bright, Frances M Platt, Janet E Deane.

Glycosphingolipids (GSL) are important bioactive membrane components. GSLs containing sialic acids, known as gangliosides, are highly abundant in the brain and diseases of ganglioside metabolism cause severe early-onset neurodegeneration. The ganglioside GM2 is processed by β-hexosaminidase A and when non-functional GM2 accumulates causing Tay-Sachs and Sandhoff diseases. We have developed i3Neuron-based disease models demonstrating storage of GM2 and severe endolysosomal dysfunction. Additionally, the plasma membrane (PM) is significantly altered in its lipid and protein composition. These changes are driven in part by lysosomal exocytosis causing inappropriate accumulation of lysosomal proteins on the cell surface. There are also significant changes in synaptic protein abundances with direct functional impact on neuronal activity. Lysosomal proteins are also enriched at the PM in GM1 gangliosidosis supporting that lysosomal exocytosis is a conserved mechanism of PM proteome change in these diseases. This work provides mechanistic insights into neuronal dysfunction in gangliosidoses highlighting that these are severe PM disorders with implications for other lysosomal and neurodegenerative diseases.

DOI: https://doi.org/10.1371/journal.pbio.3003265
Nat Commun. 2025 Jul 01. 16(1): 5826

DRAM1 promotes the stability of lysosomal VAMP8 to enhance autolysosome formation and facilitates the extravasation.

Rui Zhang, Xin Zhang, Hua Bai, Qiuyu Cheng, Xia Yao, Shi Li, Vincenzo Torraca, Chaojun Yan, Xueying Dong, Siyi Miao, Xueyuan Hu, Yeping Yu, Yueyan Wu, Hongfei Tan, Xin Chen, Shicheng Liu, Hao Lyu, Shuai Xiao, Dong Guo, Qi Zhang, Xing-Zhen Chen, Zhiyin Song, Cefan Zhou, Jingfeng Tang.

Autophagy classically functions to protect cells and organisms during stressful conditions by catabolizing intracellular components to maintain energy homeostasis. Lysosome-autophagosome fusion is a critical step in emptying degraded unwanted contents. However, the mechanism of autophagosome fusion with lysosomes is still not fully understood. Here, we report that DNA Damage-Regulated Autophagy Modulator 1 (DRAM1) interacts with Vesicle Associated Membrane Protein 8 (VAMP8) to mediate the fusion of autophagosomes with lysosomes. This DRAM1-VAMP8 interaction is enhanced upon stimulation of autophagy. However, DRAM1 preferentially mediates the fusion between autophagosomes and lysosomes by enhancing the assembly of the STX17-SNAP29-VAMP8 complex. Moreover, we reveal that DRAM1 specifically promotes the stability of lysosomal VAMP8 via inhibiting VAMP8 degradation by CHIP mediating ubiquitination. We also identify that DRAM1 inhibits the ubiquitination of VAMP8 at Lys 68,72, and 75 via competitively binding with CHIP. Furthermore, we demonstrate that DRAM1 promotes the extravasation of Hepatocellular Carcinoma (HCC) cells, and this process relies on enhanced autophagosome degradation. Our study reveals a mechanism for regulating autolysosome formation by DRAM1-VAMP8 association and suggests a potential strategy to inhibit the extravasation of HCC.

DOI: https://doi.org/10.1038/s41467-025-60887-y
Nat Commun. 2025 Jul 01. 16(1): 5585

Trioxane-based MS-cleavable cross-linking mass spectrometry for profiling multimeric interactions of cellular networks.

Clinton Yu, Eric Novitsky, Sree Ganesh Balasubramani, Xiaorong Wang, Xiyu Shen, Qin Yang, Scott Rychnovsky, Ignacia Echeverria, Lan Huang.

Cross-linking mass spectrometry (XL-MS) is a powerful technology for mapping protein-protein interactions (PPIs) at the systems level. While bivalent cross-links are effective for defining protein interactions and structures, multivalent cross-links offer enhanced spatial resolution to facilitate characterization of heterogeneous protein complexes. However, their identification remains challenging due to fragmentation complexity and the vast expansion of database search space. Here, we present tris-succinimidyl trioxane (TSTO), a novel trioxane-based, MS-cleavable homotrifunctional cross-linker capable of targeting three proximal lysines simultaneously. TSTO's unique MS-cleavability enables concurrent release of cross-linked peptide constituents during collision-induced dissociation, permitting their unambiguous identification. The TSTO-based XL-MS platform is effective for mapping cellular networks from intact cells and tissues, illustrating its versatility for complex biological systems. Trimeric interactions captured by TSTO reveal structural details inaccessible to bifunctional reagents, enhancing modeling accuracy and precision. Furthermore, this development opens a new avenue for designing multifunctional MS-cleavable cross-linkers to further advance structural systems biology.

DOI: https://doi.org/10.1038/s41467-025-60642-3
Proc Natl Acad Sci U S A. 2025 Jul 08. 122(27): e2500562122

UFMylation: A supervisor of the HIF1α pathway and a potential therapeutic target for anti-PD-1 combination therapy in hypoxic tumors.

Yongkang Zou, Zhaoxiang Wang, Qiang Jiang, Xia Kong, Xiaohe Ma, Zhengyan Liang, Zhiguo Wang, Beiying Chen, Jiao Yuan, Jiayue Wen, Sheng Ye, Yubin Yan, Binbin Li, Xing-Dong Xiong, Xin-Guang Liu, Zhiwei He, Yafei Cai, Junzhi Zhou.

  Activation of hypoxia signaling has been identified as an innate resistance signature against anti-PD-1 therapy, suggesting its potential as a target for combination treatments. Here, we demonstrate that UFMylation modification of HIF1α stabilizes the protein by antagonizing its ubiquitination and proteasomal degradation under hypoxic conditions. Mechanistically, depletion of UFL1 or defective UFMylation increases HIF1α binding to p53, promoting its degradation. Depletion of UFL1 or UBA5, or defective UFMylation of HIF1α, destabilizes HIF1α, significantly inhibiting tumor growth and development in vitro and in xenograft mouse models. Defective UFMylation of HIF1α enhances the response to anti-PD-1 therapy in xenograft models. Clinically, UBA5 expression is upregulated in breast cancer tissues, and a selective UBA5 inhibitor reduces UFMylation activity and HIF1α protein levels, thereby enhancing anti-PD-1 combination therapy in mouse tumor models. Our findings highlight UFMylation as a critical posttranslational modification for the HIF1α pathway and a promising therapeutic target in hypoxic tumors.

Keywords:  UFMylation; hypoxia; posttranslational modification; tumor microenvironment

DOI:  https://doi.org/10.1073/pnas.2500562122
Nat Commun. 2025 Jul 01. 16(1): 5600

Single-cell signaling network profiling during redox stress reveals dynamic redox regulation in immune cells.

Yi-Chuan Wang, Ping-Hsun Wu, Wen-Chieh Ting, Yi-Fu Wang, Ming-Han Yang, Tung-Hung Su, Jia-Ying Su, Hsun-I Sun, Wei-Min Huang, Pei-Ling Tsai, Gerlinde Wernig, Ping-Chih Ho, Limei Wang, Chen-Tu Wu, Yih-Leong Chang, Tseng-Cheng Chen, Tzu-Ching Meng, Yao-Ming Chang, Shih-Lei Lai, Chia-Wei Li, Tai-Ming Ko, Kai-Chien Yang, Ya-Jen Chang, Yijuang Chern, Mei-Chuan Kuo, Yen-Tsung Huang, Yi-Shiuan Tzeng, Jih-Luh Tang, Shih-Yu Chen.

In eukaryotic cells, reactive oxygen species (ROS) serve as crucial signaling components. ROS are potentially toxic, so constant adjustments are needed to maintain cellular health. Here we describe a single-cell, mass cytometry-based method that we call signaling network under redox stress profiling (SN-ROP) to monitor dynamic changes in redox-related pathways during redox stress. SN-ROP quantifies ROS transporters, enzymes, oxidative stress products and associated signaling pathways to provide information on cellular redox regulation. Applied to diverse cell types and conditions, SN-ROP reveals unique redox patterns and dynamics including coordinated shifts in CD8+ T cells upon antigen stimulation as well as variations in CAR-T cell persistence. Furthermore, SN-ROP analysis uncovers environmental factors such as hypoxia and T cell exhaustion for influencing redox balance, and also reveals distinct features in patients on hemodialysis. Our findings thus support the use of SN-ROP to elucidate intricate redox networks and their implications in immune cell function and disease.

DOI: https://doi.org/10.1038/s41467-025-60727-z
Nat Commun. 2025 Jul 01. 16(1): 5429

Prosit-XL: enhanced cross-linked peptide identification by fragment intensity prediction to study protein interactions and structures.

Mostafa Kalhor, Cemil Can Saylan, Mario Picciani, Lutz Fischer, Falk Boudewijn Schimweg, Joel Lapin, Juri Rappsilber, Mathias Wilhelm.

It has been shown that integrating peptide property predictions such as fragment intensity into the scoring process of peptide spectrum match can greatly increase the number of confidently identified peptides compared to using traditional scoring methods. Here, we introduce Prosit-XL, a robust and accurate fragment intensity predictor covering the cleavable (DSSO/DSBU) and non-cleavable cross-linkers (DSS/BS3), achieving high accuracy on various holdout sets with consistent performance on external datasets without fine-tuning. Due to the complex nature of false positives in XL-MS, an approach to data-driven rescoring was developed that benefits from Prosit-XL's predictions while limiting the overestimation of the false discovery rate (FDR). After validating this approach using two ground truth datasets consisting of synthetic peptides and proteins, we applied Prosit-XL on a proteome-scale dataset, demonstrating an up to ~3.4-fold improvement in PPI discovery compared to classic approaches. Finally, Prosit-XL was used to increase the coverage and depth of a spatially resolved interactome map of intact human cytomegalovirus virions, leading to the discovery of previously unobserved interactions between human and cytomegalovirus proteins.

DOI: https://doi.org/10.1038/s41467-025-61203-4
J Cell Biol. 2025 Aug 04. pii: e202407209. [Epub ahead of print]224(8):

Sphingolipid synthesis maintains nuclear membrane integrity and genome stability during cell division.

Sunyoung Hwang, William Russo, Jaylah Cormier, Jillian Johnson, Sara Martin, Marica Rosaria Ippolito, Sara Cordone, Rui Li, Lihua Julie Zhu, Stefano Santaguida, Eduardo M Torres.

Lipid synthesis must be precisely regulated to support membrane growth and organelle biogenesis during cell division, yet little is known about how this process is coordinated with other cell cycle events. Here, we show that de novo synthesis of sphingolipids during the S and G2 phases of the cell cycle is essential to increasing nuclear membranes. Indeed, the products of serine palmitoyltransferase (SPT), long-chain bases, localize to the nucleus and are integral components of nuclear membranes in yeast and human cells. Importantly, inhibition of SPT fails to induce cell cycle arrest, causing nuclear membrane collapse and loss of viability in yeast cells. In human cells, this causes abnormal nuclear morphology and genomic instability, evidenced by the increased incidence of nuclear blebs, micronuclei, anaphase bridges, and multipolar mitosis. These results indicate that dysregulated cell division under low sphingolipid availability can drive several disease-associated phenotypes, including aberrant nuclear morphologies and genomic instability.

DOI: https://doi.org/10.1083/jcb.202407209
Sci Adv. 2025 Jul 04. 11(27): eadu6361

Live-cell magnetic manipulation of recycling endosomes reveals their direct effect on actin protrusions to promote invasive migration.

Jakub Gemperle, Domenik Liße, Marie Kappen, Emilie Secret, Mathieu Coppey, Martin Gregor, Christine Menager, Jacob Piehler, Patrick Caswell.

Endocytic recycling pathways play key roles in a broad range of cellular processes, and many vesicle trafficking regulators are implicated in progression of disease such as cancer. The Rab11 family (Rab11a, Rab11b, and Rab25) controls the return of internalized cargos to the plasma membrane, and Rab25 has been implicated in the aggressiveness of cancer by promoting invasive migration. However, while Rab25 vesicles distribute to the leading edge of moving cells, how directly they contribute to cell protrusion is not clear. Here, we adopt a magnetogenetic approach that allows direct manipulation of Rab25 positioning to show that localization to the cell periphery drives the formation of F-actin protrusions. We demonstrate that endogenous Rab25 vesicles coordinate the positioning of key cargos, including the actin regulator FMNL1 and integrin β1, with the activation of Rho guanosine triphosphatases at the plasma membrane to generate and maintain F-actin-rich filopodium-like protrusions and promote cancer cell invasive migration in the three-dimensional matrix.

DOI: https://doi.org/10.1126/sciadv.adu6361
Adv Sci (Weinh). 2025 Jul 01. e03660

STING COPII ER Export Trafficking and Signaling Primed by Phosphorylation Switches.

Yanan Nan, Dongxiao Cui, Jiajian Guo, Xiaojing Ma, Jiaming Wang, Linyue Guo, Tianyu Li, Mingrui Yang, Guangrui Huang, Anlong Xu, Wenfu Ma.

  Despite advances in understanding the STING signaling pathway, mechanisms governing cyclic GMP-AMP (cGAMP)-induced STING trafficking out of the endoplasmic reticulum (ER) remain unclear. This study reveals that STING localization is regulated by the balance between coat protein II (COPII)- and coat protein I (COPI)-mediated trafficking, maintaining ER residency in the inactive state or promoting transport to the cis-Golgi via enhanced COPII-mediated export upon activation. Two novel TANK-binding kinase 1 (TBK1)-regulated phosphorylated COPII sorting signals on STING-a conserved pSGME motif and a primate-specific pFS motif-are biochemically and structurally identified. These cGAMP-induced signals drive activated STING toward the ER-Golgi intermediate compartment (ERGIC) and the cis-Golgi complex. Using a cell-free COPII vesicle reconstitution system, TBK1 activation is shown to occur on COPII vesicles, while IRF3 phosphorylation is confined to the ERGIC or the cis-Golgi complex post-uncoating, due to the competitive binding of COPII Sec24 and IRF3 to phosphorylated STING. A class of compounds is also identified that attenuates IRF3 phosphorylation by inhibiting phosphorylated STING packaging into COPII vesicles. These findings elucidate STING trafficking mechanisms and offer therapeutic potential for diseases linked to dysregulated STING activation.

Keywords:  COPII vesicle trafficking; ER export; IRF3; STING; TBK1; interferon; phosphorylated motifs

DOI:  https://doi.org/10.1002/advs.202503660