bims-proteo 2025-05-25 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2025–05–25
forty-four papers selected by
Eric Chevet, INSERM

Designing the Proteome with Chemical Tools: Degrons and Beyond.
N-glycan-dependent protein maturation and quality control in the ER.
A Sensitive and Versatile Cell-Based Assay Combines Luminescence and Trapping Approaches to Monitor Unconventional Protein Secretion.
Getting PIKy with the lysosome membrane (again).
Engineering ERα degraders with pleiotropic ubiquitin ligase ligands maximizes therapeutic efficacy by co-opting distinct effector ligases.
Deep learning-guided design of dynamic proteins.
Regulation of the MDM2-p53 Nexus by a Nuclear Phosphoinositide and Small Heat Shock Protein Complex.
Cyclic peptide structure prediction and design using AlphaFold2.
Stepwise ATP translocation into the endoplasmic reticulum by human SLC35B1.
ER tubular body: an ER-derived compartment for redirecting autophagy to secretory functions.
Localizatome: a database for stress-dependent subcellular localization changes in proteins.
Pex30-dependent membrane contact sites maintain ER lipid homeostasis.
Ribosomes modulate transcriptome abundance via generalized frameshift and out-of-frame mRNA decay.
Selective removal of astrocytic PERK protects against glymphatic impairment and decreases toxic aggregation of β-amyloid and tau.
Extracellular Vesicle-Mediated Delivery of 20S Proteasomes Enhances Tau Degradation in Recipient Cells.
S-palmitoylation coordinates the trafficking of ATG9A to mediate autophagy initiation.
An integrated proteomics approach identifies phosphorylation sites on viral and host proteins that regulate West Nile virus infection.
Commissureless acts as a substrate adapter in a conserved Nedd4 E3 ubiquitin ligase pathway to promote axon growth across the midline.
Quality control of ribosome assembly: Kinetic discrimination at work.
Macrophages release neuraminidase and cleaved calreticulin for programmed cell removal.
ATG5 is dispensable for ATG8ylation of cellular proteins.
Trapα deficiency impairs the early events of insulin biosynthesis and glucose homeostasis.
NOD1 deficiency promotes inflammation via autophagic degradation of ASK1.
H/ACA snR30 snoRNP guides independent 18S rRNA subdomain formation.
Linker-free PROTACs efficiently induce the degradation of oncoproteins.
CD83 suppresses endogenous March-I-dependent MHC class II ubiquitination, endocytosis, and degradation.
Single-molecule analysis reveals the mechanism of chromatin ubiquitylation by variant PRC1 complexes.
Phosphatidic acid drives spatiotemporal distribution of Pex30 at ER-LD contact sites.
A new role of RAB21 and VARP in autophagy and autophagic exocytosis of ATP.
Metabolic traits shape responses to LSD1-directed therapy in glioblastoma tumor-initiating cells.
Induction of the ISR by AB5 subtilase cytotoxin drives type-I IFN expression in pDCs via STING activation.
Glutamine catabolism supports amino acid biosynthesis and suppresses the integrated stress response to promote photoreceptor survival.
Protein turnover downstream of the Nipbl/CRL4 axis contributes to abnormal development in zebrafish embryos.
Symmetric stimulation of Hsp90 catalyzed ATP hydrolysis through enhanced active site gate dynamics.
PUS10-induced tRNA fragmentation impacts retrotransposon-driven inflammation.
Disease-associated Kv1.3 variants are energy compromised with impaired nascent chain folding.
mTOR inhibition in Q175 Huntington's disease model mice facilitates neuronal autophagy and mutant huntingtin clearance.
USP37 prevents unscheduled replisome unloading through MCM complex deubiquitination.
Deep-learning-based single-domain and multidomain protein structure prediction with D-I-TASSER.
Dual Ribosome Profiling reveals metabolic limitations of cancer and stromal cells in the tumor microenvironment.
Nuclear ubiquitination permits Hippo-YAP signal for liver development and tumorigenesis.
Goblet cell loss linked to NOD2 and secondary resection in Crohn's disease is induced by dysbiosis and epithelial MyD88.
Type 2 calreticulin mutations activate ATF6 to promote BCL-xL-mediated survival in myeloproliferative neoplasms.
Reengineering of a Proteomimetic Pan-Ras Inhibitor into a Ras Degrader.

Chembiochem. 2025 May 21. e202500345

Designing the Proteome with Chemical Tools: Degrons and Beyond.

Laurence John Seabrook, Catherine Ramirez Livelo, Lauren Veronica Albrecht.

  Cell biology relies on precise changes in protein stability, which can be chemically harnessed to transform cell fate. Decades of research have revealed the many intricate systems underlying cellular proteostasis, which can be hijacked by proximity-based degrader compounds. The archetypal degrader, PROteolysis TArgeting Chimera (PROTAC), recruits E3 ligases to protein targets to facilitate their ubiquitination and degradation in the proteasome. Being able to customize the human proteome with chemical tools has great value for fundamental research and for clinical progress through the controlled elimination of disease-causing proteins. Success within the degrader field has reinvigorated interest in mapping the mechanisms underlying native protein degradation, which has platformed new degrader classes capable of advancing the field towards the goal of degrading the entire human proteome. This review discusses ongoing strategies to identify degrons regulating native protein turnover, advances in chemical tools to activate these degrons, and new attempts to streamline degron pathways for simplified targeted protein degradation. The continued discovery and application of degrons has the power to transform human biology and combat disease.

Keywords:  PROTAC; degron; lysosome; proteasome; targeted protein degradation

DOI:  https://doi.org/10.1002/cbic.202500345
Nat Rev Mol Cell Biol. 2025 May 19.

N-glycan-dependent protein maturation and quality control in the ER.

Kevin P Guay, Wen-Chuan Chou, Nathan P Canniff, Kylie B Paul, Daniel N Hebert.

The vast majority of proteins that traverse the mammalian secretory pathway become N-glycosylated in the endoplasmic reticulum (ER). The bulky glycan protein modifications, which are conserved in fungi and humans, act as maturation and quality-control tags. In this Review, we discuss findings published in the past decade that have rapidly expanded our understanding of the transfer and processing of N-glycans, as well as their role in protein maturation, quality control and trafficking in the ER, facilitated by structural insights into the addition of N-glycans by the oligosaccharyltransferases A and B (OST-A and OST-B). These findings suggest that N-glycans serve as reporters of the folding status of secretory proteins as they traverse the ER, enabling the lectin chaperones to guide their maturation. We also explore how the emergence of co-translational glycosylation and the expansion of the glycoproteostasis network in metazoans has expanded the role of N-glycans in early protein-maturation events and quality control.

DOI: https://doi.org/10.1038/s41580-025-00855-y
Traffic. 2025 Apr;26(4-6): e70009

A Sensitive and Versatile Cell-Based Assay Combines Luminescence and Trapping Approaches to Monitor Unconventional Protein Secretion.

Morgane Denus, Aurore Filaquier, William Fargues, Eloïse Néel, Sarah E Stewart, Maëlle Colladant, Thomas Curel, Alexandre Mezghrani, Philippe Marin, Sylvie Claeysen, David C Rubinsztein, Marie-Laure Parmentier, Julien Villeneuve.

  In addition to the conventional endoplasmic reticulum (ER)-Golgi secretory pathway, alternative routes are increasingly recognized for their critical roles in exporting a growing number of secreted factors. These alternative processes, collectively referred to as unconventional protein secretion (UcPS), challenge traditional views of protein and membrane trafficking. Unlike the well-characterized molecular machinery of the conventional secretory pathway, the mechanisms underlying UcPS remain poorly understood. Various UcPS pathways may involve direct transport of cytosolic proteins across the plasma membrane or the incorporation of cargo proteins into intracellular compartments redirected for secretion. Identifying the specific chaperones, transporters and fusion machinery involved in UcPS cargo recognition, selection and transport is crucial to decipher how cargo proteins are selectively or synergistically directed through multiple secretory routes. These processes can vary depending on cell type and in response to particular stress conditions or cellular demands, underscoring the need for standardized tools and methods to study UcPS. Here, we combine the sensitivity of split NanoLuc Binary Technology with the versatility of the Retention Using Selective Hooks (RUSH) system to develop a straightforward and reliable cell-based assay for investigating both conventional and unconventional protein secretion. This system allows for the identification of intracellular compartments involved in UcPS cargo trafficking. Additionally, its sensitivity enabled us to demonstrate that disease-associated mutants or variants of Tau and superoxide dismutase-1 (SOD1) show altered secretion via UcPS. Finally, we leveraged this assay to screen for Alzheimer's disease risk factors, revealing a functional link between amyloid-beta production and Tau UcPS. This robust assay provides a powerful tool for increasing our knowledge of protein secretion mechanisms in physiological and pathological contexts.

Keywords:  intercellular communication; intracellular compartments; neurodegenerative diseases; protein trafficking; secretory pathway; split luciferase; tau; unconventional protein secretion

DOI:  https://doi.org/10.1111/tra.70009
Autophagy Rep. 2024 ;3(1): 2412916

Getting PIKy with the lysosome membrane (again).

Alison D Klein, Michael Overholtzer.

  Much is still unknown about microautophagy and its regulators. In our recent paper, one such regulator of microautophagy, the lipid kinase PIKfyve, is described. Previously it was found that treating cells with agents like lysomotropic drugs or proton ionophores, which alter lysosomal osmotic potential and pH, leads to a form of microautophagy that selectively degrades transmembrane proteins. Induction of this type of microautophagy is linked to a lysosomal stress response that involves the targeting of macroautophagy proteins, like ATG8s, to the lysosome membrane, through a mechanism called CASM. We found that CASM-induced microautophagy turns over ATG8s and other lysosomal membrane proteins, and requires PIKfyve activity functioning downstream of ATG8 lipidation. The lysosome biogenesis transcription factor TFEB is induced in parallel to microautophagy, in a CASM-dependent, but PIKfyve-independent manner. These findings demonstrate that stressors that engage CASM cause selective turnover by microautophagy that is coordinated with lysosome biogenesis through a mechanism that is separable through PIKfyve.

Keywords:  ATG8; CASM; LC3; PIKfyve; TFEB; TRPML1; autophagy; lysosome; microautophagy

DOI:  https://doi.org/10.1080/27694127.2024.2412916
Cell Chem Biol. 2025 May 15. pii: S2451-9456(25)00131-X. [Epub ahead of print]32(5): 694-709.e35

Engineering ERα degraders with pleiotropic ubiquitin ligase ligands maximizes therapeutic efficacy by co-opting distinct effector ligases.

Anna Shemorry, Willem den Besten, Melinda M Mulvihill, Curt J Essenburg, Nicole Blaquiere, Tracy Kleinheinz, Elisia Villemure, Frank Peale, Gauri Deshmukh, Danilo Maddalo, Elizabeth Levy, Kebing Yu, Matthew R Steensma, Elizabeth A Tovar, Emily Wolfrum, Karthik Nagapudi, Robert A Blake, William F Forrest, Steven T Staben, Carrie R Graveel, Wayne J Fairbrother, Ingrid E Wertz.

  Proximity-inducing compounds that modulate target protein homeostasis represent an emerging therapeutic strategy. While the inherent complexity of these bifunctional compounds presents certain challenges, their unique composition offers opportunities to co-opt specific cellular effectors to enhance therapeutic impact. In this study, we systematically evaluate a series of bifunctional degrader compounds engineered with the estrogen receptor-alpha (ERα) inhibitor endoxifen linked to various bioactive ubiquitin ligase ligands. Notably, ERα degraders containing pan-IAP antagonist ligands significantly reduced the proliferation of ERα-dependent cells compared to clinical-stage ERα degraders. These pan-IAP antagonist-based ERα degraders leverage distinct effector ligases to achieve dual therapeutic effects: They utilize XIAP within tumor cells to promote ERα degradation and activate cIAP1/2 in both tumor and immune cells to induce TNFα, which drives tumor cell death. Our findings illustrate a broader concept that co-opting the discrete functions of selected cellular effectors, while simultaneously modulating therapeutic target protein homeostasis, are dual strategies that can significantly enhance the efficacy of induced proximity therapeutics.

Keywords:  ERα degraders; TNFα; XIAP; cIAP1/2; chemical biology; degrader efficacy; induced proximity; pleiotropic ligands; tumor cell death

DOI:  https://doi.org/10.1016/j.chembiol.2025.04.008
Science. 2025 May 22. 388(6749): eadr7094

Deep learning-guided design of dynamic proteins.

Amy B Guo, Deniz Akpinaroglu, Christina A Stephens, Michael Grabe, Colin A Smith, Mark J S Kelly, Tanja Kortemme.

Deep learning has advanced the design of static protein structures, but the controlled conformational changes that are hallmarks of natural signaling proteins have remained inaccessible to de novo design. Here, we describe a general deep learning-guided approach for de novo design of dynamic changes between intradomain geometries of proteins, similar to switch mechanisms prevalent in nature, with atomic-level precision. We solve four structures that validate the designed conformations, demonstrate modulation of the conformational landscape by orthosteric ligands and allosteric mutations, and show that physics-based simulations are in agreement with deep-learning predictions and experimental data. Our approach demonstrates that new modes of motion can now be realized through de novo design and provides a framework for constructing biology-inspired, tunable, and controllable protein signaling behavior de novo.

DOI: https://doi.org/10.1126/science.adr7094
bioRxiv. 2025 Apr 17. pii: 2025.04.11.648454. [Epub ahead of print]

Regulation of the MDM2-p53 Nexus by a Nuclear Phosphoinositide and Small Heat Shock Protein Complex.

Jeong Hyo Lee, Mo Chen, Tianmu Wen, Richard A Anderson, Vincent L Cryns.

The tumor suppressor p53 maintains genome stability in the setting of cellular stress and is frequently mutated in cancer. The stability of p53 is regulated by its interaction with the oncoprotein MDM2, a ubiquitin E3 ligase. Recently, nuclear phosphoinositides were reported to bind and stabilize p53. Here, we report that genotoxic stress induces the type I phosphatidylinositol phosphate kinase (PIPKIα) and its product phosphatidylinositol 4,5-bisphosphate (PIP 2 ) to bind and regulate the stability and function of MDM2. Following genotoxic stress, nuclear PIPKIα binds to MDM2 to generate a complex of MDM2 and PIP 2 . PIP 2 binding to MDM2 differentially regulates the recruitment of the small heat shock proteins (sHSPs) αB-crystallin (αBC) and HSP27 to the MDM2-PIP 2 complex, acting as an on-off switch that regulates MDM2 stability, downstream targets, ubiquitination activity, and interaction with p53. Our results demonstrate an unexpected role for nuclear phosphoinositides conferring specificity to the MDM2-PIP 2 -sHSPs association. Notably, the differential engagement of αBC and HSP27 reveals that sHSPs are not merely passive chaperones but play active, selective roles in fine-tuning MDM2 function and MDM2-p53 nexus. These findings provide a novel therapeutic strategy for targeting this pathway in cancer.

DOI: https://doi.org/10.1101/2025.04.11.648454
Nat Commun. 2025 May 21. 16(1): 4730

Cyclic peptide structure prediction and design using AlphaFold2.

Stephen A Rettie, Katelyn V Campbell, Asim K Bera, Alex Kang, Simon Kozlov, Yensi Flores Bueso, Joshmyn De La Cruz, Maggie Ahlrichs, Suna Cheng, Stacey R Gerben, Mila Lamb, Analisa Murray, Victor Adebomi, Guangfeng Zhou, Frank DiMaio, Sergey Ovchinnikov, Gaurav Bhardwaj.

Small cyclic peptides have gained significant traction as a therapeutic modality; however, the development of deep learning methods for accurately designing such peptides has been slow, mostly due to the lack of sufficiently large training sets. Here, we introduce AfCycDesign, a deep learning approach for accurate structure prediction, sequence redesign, and de novo hallucination of cyclic peptides. Using AfCycDesign, we identified over 10,000 structurally-diverse designs predicted to fold into the designed structures with high confidence. X-ray crystal structures for eight tested de novo designed sequences match very closely with the design models (RMSD < 1.0 Å), highlighting the atomic level accuracy in our approach. Further, we used the set of hallucinated peptides as starting scaffolds to design binders with nanomolar IC50 against MDM2 and Keap1. The computational methods and scaffolds developed here provide the basis for the custom design of peptides for diverse protein targets and therapeutic applications.

DOI: https://doi.org/10.1038/s41467-025-59940-7
Nature. 2025 May 21.

Stepwise ATP translocation into the endoplasmic reticulum by human SLC35B1.

Ashutosh Gulati, Do-Hwan Ahn, Albert Suades, Yurie Hult, Gernot Wolf, So Iwata, Giulio Superti-Furga, Norimichi Nomura, David Drew.

ATP generated in the mitochondria is exported by an ADP/ATP carrier of the SLC25 family1. The endoplasmic reticulum (ER) cannot synthesize ATP but must import cytoplasmic ATP to energize protein folding, quality control and trafficking2,3. It was recently proposed that a member of the nucleotide sugar transporter family, termed SLC35B1 (also known as AXER), is not a nucleotide sugar transporter but a long-sought-after ER importer of ATP4. Here we report that human SLC35B1 does not bind nucleotide sugars but indeed executes strict ATP/ADP exchange with uptake kinetics consistent with the import of ATP into crude ER microsomes. A CRISPR-Cas9 cell-line knockout demonstrated that SLC35B1 clusters with the most essential SLC transporters for cell growth, consistent with its proposed physiological function. We have further determined seven cryogenic electron microscopy structures of human SLC35B1 in complex with an Fv fragment and either bound to an ATP analogue or ADP in all major conformations of the transport cycle. We observed that nucleotides were vertically repositioned up to approximately 6.5 Å during translocation while retaining key interactions with a flexible substrate-binding site. We conclude that SLC35B1 operates by a stepwise ATP translocation mechanism, which is a previously undescribed model for substrate translocation by an SLC transporter.

DOI: https://doi.org/10.1038/s41586-025-09069-w
Autophagy. 2025 May 19.

ER tubular body: an ER-derived compartment for redirecting autophagy to secretory functions.

Min Seok Song, Hun Ju Sim, Shin Hye Noh, Vivek Malhotra, Min Goo Lee.

  The secretion of proteins that do not follow the well-characterized endoplasmic reticulum (ER)-Golgi apparatus pathway, known as unconventional protein secretion (UCPS), is gradually revealing its complexities. Our study has identified an ER-based tubulovesicular network, termed ER tubular body (ER-TB), as a central compartment in this process. We demonstrate that ER-TBs are formed by two reticulophagy receptors, ATL3 and RTN3L, under conditions of cellular stress. In addition to their role in stress-induced secretion, the activation of UCPS via ER-TBs facilitates cell surface trafficking of trafficking-deficient transmembrane proteins such as ΔF508-CFTR. Furthermore, their involvement in ER remodeling and vesicle trafficking suggests a potential role in viral replication, particularly in the formation of membrane compartments utilized by positive-strand RNA viruses. By uncovering ER-TBs as key cellular structures in stress-induced UCPS and demonstrating their regulation by autophagy-related factors, our findings offer valuable insights into protein homeostasis, viral pathogenesis, and potential therapeutic strategies for diseases linked to trafficking defects.

Keywords:  ATL3; ER stress; ER tubular body; RTN3L; compartment for unconventional protein secretion; unconventional protein secretion

DOI:  https://doi.org/10.1080/15548627.2025.2508935
Database (Oxford). 2025 Apr 21. pii: baaf028. [Epub ahead of print]2025

Localizatome: a database for stress-dependent subcellular localization changes in proteins.

Takahide Matsushima, Yuki Naito, Tomoki Chiba, Ryota Kurimoto, Keiko Itano, Koji Ochiai, Koichi Takahashi, Naoki Goshima, Hiroshi Asahara.

Understanding protein subcellular localization and its dynamic changes is crucial for elucidating cellular function and disease mechanisms, particularly under stress conditions, where protein localization changes can modulate cellular responses. Currently available databases provide insights into protein localization under steady-state conditions; however, stress-related dynamic localization changes remain poorly understood. Here, we present the Localizatome, a comprehensive database that captures stress-induced protein localization dynamics in living cells. Using an original high-throughput microscopy system and machine learning algorithms, we analysed the localization patterns of 10 287 fluorescent protein-fused human proteins in HeLa cells before and after exposure to oxidative stress. Our analysis revealed that 1910 proteins exhibited oxidative stress-dependent localization changes, particularly forming distinct foci. Among them, there were stress granule assembly factors and autophagy-related proteins, as well as components of various signalling pathways. Subsequent characterization identified some specific amino acid motifs and intrinsically disordered regions associated with stress-induced protein redistribution. The Localizatome provides open access to these data through a web-based interface, supporting a wide range of studies on cellular stress response and disease mechanisms. Database URL https://localizatome.embrys.jp/.

DOI: https://doi.org/10.1093/database/baaf028
J Cell Biol. 2025 Jul 07. pii: e202409039. [Epub ahead of print]224(7):

Pex30-dependent membrane contact sites maintain ER lipid homeostasis.

Joana Veríssimo Ferreira, Yara Ahmed, Tiaan Heunis, Aamna Jain, Errin Johnson, Markus Räschle, Robert Ernst, Stefano Vanni, Pedro Carvalho.

In eukaryotic cells, communication between organelles and the coordination of their activities depend on membrane contact sites (MCS). How MCS are regulated under the dynamic cellular environment remains poorly understood. Here, we investigate how Pex30, a membrane protein localized to the endoplasmic reticulum (ER), regulates multiple MCS in budding yeast. We show that Pex30 is critical for the integrity of ER MCS with peroxisomes and vacuoles. This requires the dysferlin (DysF) domain on the Pex30 cytosolic tail. This domain binds to phosphatidic acid (PA) both in vitro and in silico, and it is important for normal PA metabolism in vivo. The DysF domain is evolutionarily conserved and may play a general role in PA homeostasis across eukaryotes. We further show that the ER-vacuole MCS requires a Pex30 C-terminal domain of unknown function and that its activity is controlled by phosphorylation in response to metabolic cues. These findings provide new insights into the dynamic nature of MCS and their coordination with cellular metabolism.

DOI: https://doi.org/10.1083/jcb.202409039
Mol Cell. 2025 May 15. pii: S1097-2765(25)00364-8. [Epub ahead of print]85(10): 2017-2031.e7

Ribosomes modulate transcriptome abundance via generalized frameshift and out-of-frame mRNA decay.

Yujie Zhang, Lilit Nersisyan, Eliska Fürst, Ioannis Alexopoulos, Carlos Santolaria, Susanne Huch, Claudio Bassot, Elena Garre, Per Sunnerhagen, Ilaria Piazza, Vicent Pelechano.

  Cells need to adapt their transcriptome to quickly match cellular needs in changing environments. mRNA abundance can be controlled by altering both its synthesis and decay. Here, we show how, in response to poor nutritional conditions, the bulk of the S. cerevisiae transcriptome undergoes -1 ribosome frameshifts and experiences an accelerated out-of-frame co-translational mRNA decay. Using RNA metabolic labeling, we demonstrate that in poor nutritional conditions, nonsense-mediated mRNA decay (NMD)-dependent degradation represents at least one-third of the total mRNA decay. We further characterize this mechanism and identify low codon optimality as a key factor for ribosomes to induce out-of-frame mRNA decay. Finally, we show that this phenomenon is conserved from bacteria to humans. Our work provides evidence for a direct regulatory feedback mechanism coupling protein demand with the control of mRNA abundance to limit cellular growth and broadens the functional landscape of mRNA quality control.

Keywords:  NMD; codon optimality; frameshift; mRNA decay; out-of-frame

DOI:  https://doi.org/10.1016/j.molcel.2025.04.022
Neuron. 2025 May 19. pii: S0896-6273(25)00310-1. [Epub ahead of print]

Selective removal of astrocytic PERK protects against glymphatic impairment and decreases toxic aggregation of β-amyloid and tau.

Kai Chen, Yosuke M Morizawa, Tal Nuriel, Osama Al-Dalahmah, Zhongcong Xie, Guang Yang.

  Dysfunction of the glymphatic system, a brain-wide waste clearance network, is strongly linked to Alzheimer's disease (AD) and the accumulation of β-amyloid (Aβ) and tau proteins. Here, we identify an astrocytic signaling pathway that can be targeted to preserve glymphatic function and mitigate neurotoxic protein buildup. Analysis of astrocytes from both human AD brains and two transgenic mouse models (5XFAD and PS19) reveals robust activation of the protein kinase RNA-like endoplasmic reticulum (ER) kinase (PERK)-α subunit of eukaryotic initiation factor 2 (eIF2α) branch of the unfolded protein response. Chronic PERK activation suppresses astrocytic protein synthesis and, through casein kinase 2 (CK2)-dependent mechanisms, disrupts the perivascular localization of aquaporin-4 (AQP4), a water channel essential for glymphatic flow. Importantly, astrocyte-specific PERK deletion or pharmacological inhibition restores AQP4 localization, enhances glymphatic clearance, reduces Aβ and tau pathology, and improves cognitive performance in mice. These findings highlight the critical role of the astrocytic PERK-CK2-AQP4 axis in glymphatic dysfunction and AD pathogenesis, positioning this pathway as a promising therapeutic target.

Keywords:  AD; AQP4; CK2; PERK; astrocyte; glymphatic function; protein synthesis; unfolded protein response

DOI:  https://doi.org/10.1016/j.neuron.2025.04.027
J Extracell Vesicles. 2025 May;14(5): e70086

Extracellular Vesicle-Mediated Delivery of 20S Proteasomes Enhances Tau Degradation in Recipient Cells.

Jiseong Kim, Yuping Zhao, Hyun Young Kim, Sumin Kim, Yanxialei Jiang, Min Jae Lee.

  The 26S proteasome holoenzyme comprises 20S catalytic and 19S regulatory complexes. Accumulating evidence suggests that the majority of proteasomes in the extracellular space exist as free 20S proteasomes; however, their origin and pathophysiological function remain to be determined. Here, we report that cellular proteasomes are effectively packaged into the lumen of extracellular vesicles (EVs) and secreted in a structurally intact and enzymatically active 20S form. We further demonstrate that EV-encapsulated 20S proteasomes are delivered to recipient cells and facilitate the degradation of overexpressed tau proteins without disrupting global proteolytic pathways. These findings highlight a novel cell-to-cell communication system that transports the proteasomes to target cells for the clearance of proteotoxic substrates. Further characterisation of this homeostatic mechanism will improve our understanding of organismal stress response mechanisms and may provide a therapeutic approach to treat various proteinopathies, including Alzheimer's disease.

Keywords:  degradation; delivery; extracellular vesicle; proteasome; tau

DOI:  https://doi.org/10.1002/jev2.70086
Autophagy. 2025 May 20.

S-palmitoylation coordinates the trafficking of ATG9A to mediate autophagy initiation.

Fan Xia, Weining Li, Wenru Wang, Jiru Liu, Xiaolin Li, Jing Cai, Hao Shan, Zhe Cai, Jun Cui.

  Macroautophagy (hereafter autophagy), a major intracellular catabolic process, is evolutionarily conserved from yeasts to mammals, and is associated with a broad range of human diseases. Autophagy is morphologically characterized by the formation of double-membrane autophagosomes. ATG9A, a multi-spanning transmembrane protein and lipid scramblase, is a core component of the autophagy machinery that complements membrane sources and equilibrates lipids across membrane bilayers. Here, we report that palmitoyltransferase ZDHHC5 is indispensable for autophagosome nucleation and subsequent autophagosome formation. Upon autophagy induction, ZDHHC5 is internalized from the plasma membrane into intracellular compartments via clathrin-mediated endocytosis. This enzyme activates ATG9A S-palmitoylation at cysteine 155/156, which orchestrates the interaction of ATG9A with the heterotetrameric adaptor protein complex family member AP4E1/AP-4ε and subsequent trafficking from the trans-Golgi network to endosomal compartments. Functionally, impairment of ATG9A S-palmitoylation results in defects in autophagy initiation and autophagosome formation. These findings identify a regulatory mechanism that coordinates ATG9A-binding with AP4E1 and vesicular trafficking events through ATG9A S-palmitoylation by ZDHHC5, thereby ensuring the spatiotemporal fidelity of membrane trafficking and maintenance of autophagic homeostasis.

Keywords:  AP4E1; Trans-golgi network; ZDHHC5; autophagosome formation; clathrin-mediated endocytosis; membrane trafficking

DOI:  https://doi.org/10.1080/15548627.2025.2509376
Cell Rep. 2025 May 15. pii: S2211-1247(25)00499-1. [Epub ahead of print]44(5): 115728

An integrated proteomics approach identifies phosphorylation sites on viral and host proteins that regulate West Nile virus infection.

Zachary Walter, Minghua Li, Melissa Molho, Lauren Berish, Andrew Isopi, Mary O'Mara, Mark Dittmar, Chike Nwaezeapu, Alicia Richards, Martin McCullagh, Nevan J Krogan, Sara Cherry, Jeffrey R Johnson, Holly Ramage.

  Upon infection, viruses alter the proteome, creating a hospitable environment for infection. Cells respond to limit viral replication, including through protein regulation by post-translational modifications. We use mass spectrometry to define proteome alterations during West Nile virus (WNV) infection. Our studies identify upregulation of HERPUD1, which restricts WNV replication through a mechanism independent of its role in endoplasmic reticulum (ER)-associated degradation (ERAD). We also identify modifications on viral proteins, including a WNV NS3 phosphorylation site that impacts viral replication. Finally, we reveal activation of two host kinases with antiviral activity. We identify phosphorylation at S108 of AMPKβ1, a non-catalytic subunit that regulates activity of the AMPK complex. We also show activation of PAK2 by phosphorylation at S141, which restricts translation of the viral genome. This work contributes to our understanding of the interplay between host and virus while providing a resource to define the changes to the proteome that regulate viral infection.

Keywords:  CP: Microbiology; Orthoflaviviruses; West Nile virus; innate immunity; phosphorylation; post-translational modifications; viral helicase; viral translation; virus-host interactions

DOI:  https://doi.org/10.1016/j.celrep.2025.115728
Elife. 2025 May 23. pii: RP92757. [Epub ahead of print]13

Commissureless acts as a substrate adapter in a conserved Nedd4 E3 ubiquitin ligase pathway to promote axon growth across the midline.

Kelly G Sullivan, Greg J Bashaw.

  In both vertebrates and invertebrates, commissural neurons prevent premature responsiveness to the midline repellant Slit by downregulating surface levels of its receptor Roundabout1 (Robo1). In Drosophila, Commissureless (Comm) plays a critical role in this process; however, there is conflicting data on the underlying molecular mechanism. Here, we demonstrate that the conserved PY motifs in the cytoplasmic domain of Comm are required allow the ubiquitination and lysosomal degradation of Robo1. Disruption of these motifs prevents Comm from localizing to Lamp1 positive late endosomes and to promote axon growth across the midline in vivo. In addition, we conclusively demonstrate a role for Nedd4 in midline crossing. Genetic analysis shows that nedd4 mutations result in midline crossing defects in the Drosophila embryonic nerve cord, which can be rescued by introduction of exogenous Nedd4. Biochemical evidence shows that Nedd4 incorporates into a three-member complex with Comm and Robo1 in a PY motif-dependent manner. Finally, we present genetic evidence that Nedd4 acts with Comm in the embryonic nerve cord to downregulate Robo1 levels. Taken together, these findings demonstrate that Comm promotes midline crossing in the nerve cord by facilitating Robo1 ubiquitination by Nedd4, ultimately leading to its degradation.

Keywords:  D. melanogaster; Nedd4; Robo; Slit; axon guidance; commissureless; developmental biology; neuroscience; ubiquitin ligase

DOI:  https://doi.org/10.7554/eLife.92757
Mol Cell. 2025 May 15. pii: S1097-2765(25)00365-X. [Epub ahead of print]85(10): 1891-1893

Quality control of ribosome assembly: Kinetic discrimination at work.

Yunyang Zhang, Ning Gao.

Ribosome biogenesis is a complex and error-prone process, necessitating quality control mechanisms to degrade defective pre-ribosomal intermediates. In this issue of Molecular Cell, Akers et al.1 report the identification of a previously uncharacterized quality control pathway named ribosome assembly surveillance pathway (RASP), which functions to eliminate aberrant "dead-end" pre-60S assembly intermediates.

DOI: https://doi.org/10.1016/j.molcel.2025.04.023
Proc Natl Acad Sci U S A. 2025 May 27. 122(21): e2426644122

Macrophages release neuraminidase and cleaved calreticulin for programmed cell removal.

Allison Banuelos, Michelle Baez, Allison Zhang, Leyla Yılmaz, William Kasberg, Regan Volk, Nardin Georgeos, Elle Koren-Sedova, Uyen Le, Andrew T Burden, Kristopher D Marjon, Jennifer Lippincott-Schwartz, Balyn W Zaro, Irving L Weissman.

  Calreticulin (CALR) is primarily an endoplasmic reticulum chaperone protein that also plays a key role in facilitating programmed cell removal (PrCR) by acting as an "eat-me" signal for macrophages, directing their recognition and engulfment of dying, diseased, or unwanted cells. Recent findings have demonstrated that macrophages can transfer their own CALR onto exposed asialoglycans on target cells, marking them for PrCR. Despite the critical role CALR plays in this process, the molecular mechanisms behind its secretion by macrophages and the formation of binding sites on target cells remain unclear. Our findings show that CALR undergoes C-terminal cleavage upon secretion, producing a truncated form that functions as the active eat-me signal detectable on target cells. We identify cathepsins as potential proteases involved in this cleavage process. Furthermore, we demonstrate that macrophages release neuraminidases, which modify the surface of target cells and facilitate CALR binding. These insights reveal a coordinated mechanism through which lipopolysaccharide (LPS)-activated macrophages regulate CALR cleavage and neuraminidase activity to mark target cells for PrCR. How they recognize the cells to be targeted remains unknown.

Keywords:  calreticulin; cancer; macrophages; phagocytosis

DOI:  https://doi.org/10.1073/pnas.2426644122
Autophagy Rep. 2024 ;3(1): 2392450

ATG5 is dispensable for ATG8ylation of cellular proteins.

Robin Ketteler, Koshiro Kiso, Lucas von Chamier, Alexander Agrotis.

  Protein ATG8ylation refers to a post-translational modification involving covalent attachment of ubiquitin-like autophagy-related protein ATG8 (LC3/GABARAP) to other cellular proteins, with reversal mediated by ATG4 proteases. While lipid ATG8ylation is important for autophagosome formation and mechanistically well-characterized, little is known about the mechanism of protein ATG8ylation. Here, we investigated the conjugation machinery of protein ATG8ylation in CRISPR/Cas9-engineered knockout human cell lines, utilizing a deconjugation-resistant (Q116P G120) form of MAP1LC3B. We report that protein ATG8ylation requires the E1-like activating enzyme ATG7 and E2-like conjugating enzyme ATG3, in common with ATG8 lipidation. However, in contrast, the E3-like ATG12-ATG5-ATG16L1 complex involved in lipidation is dispensable for protein ATG8ylation, since ATG5 knockout cells can form ATG8ylated protein conjugates. Further, we uncover that ATG7 itself is a target of ATG8ylation. Overall, our work provides crucial insight into the mechanism of protein ATG8ylation, distinguishing it from ATG8 lipidation, which will aid investigating its functional role.

Keywords:  ATG3; ATG4B; ATG7; LC3/GABARAP; LC3ylation; autophagy; conjugating; deconjugation; post-translational modification; ubiquitin-like

DOI:  https://doi.org/10.1080/27694127.2024.2392450
J Clin Invest. 2025 May 20. pii: e179845. [Epub ahead of print]

Trapα deficiency impairs the early events of insulin biosynthesis and glucose homeostasis.

Xin Li, Jingxin Hu, Yumeng Huang, Hai Zhang, Ning Xu, Yang Liu, Xuan Liu, Yuanyuan Ye, Xinxin Zhang, Xiaoxi Xu, Yuxin Fan, Ziyue Zhang, Weiping J Zhang, Shusen Wang, Wenli Feng, Peter Arvan, Ming Liu.

  Defects in the early events of insulin biosynthesis, including inefficient preproinsulin (PPI) translocation across the membrane of the endoplasmic reticulum (ER) and proinsulin (PI) misfolding in the ER, can cause diabetes. Cellular machineries involved in these events remain poorly defined. Gene encoding TRanslocon-Associated Protein alpha (TRAPα) shows linkage to glycemic control in humans, although their pathophysiological role remains unknown. Here we found that β-cell specific TRAPα knockout (TRAPα-βKO) mice fed with chow diet or high fat diet (HFD) exhibit decreased circulating insulin, with age- and diet-related glucose intolerance. Multiple independent approaches revealed that TRAPα-βKO not only causes inefficient PPI translocation, but also leads to PI misfolding and ER stress, selectively limiting PI ER export and β-cell compensatory potential. Importantly, decreased TRAPα expression was evident in islets of wild-type mice fed with high fat diet and in patients with type 2 diabetes (T2D). Furthermore, TRAPα expression was positively correlated with insulin content in human islet β cells, and decreased TRAPα was associated with PI maturation defects in T2D islets. Together, these data demonstrate that TRAPα deficiency in pancreatic β-cells impairs PPI translocation, PI folding, insulin production, and glucose homeostasis, contributing to its genetic linkage to T2D.

Keywords:  Beta cells; Endocrinology; Insulin; Metabolism; Mouse models

DOI:  https://doi.org/10.1172/JCI179845
Commun Biol. 2025 May 21. 8(1): 781

NOD1 deficiency promotes inflammation via autophagic degradation of ASK1.

Yoshitaka Kimura, Miyako Kimura, Noriko Miura, Yusuke Yoshino, Hajime Kono.

Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) is a pattern recognition receptor of bacterial peptidoglycans. NOD1 facilitates the elimination of invading intracellular bacteria via autophagy induction. Here, we demonstrate that NOD1 exerts an anti-inflammatory effect mediated via the selective autophagy of host cell protein. In our study of Candida albicans water-soluble fraction (CAWS)-induced coronary arteritis, which is a mouse model of Kawasaki disease, we observed an exacerbated disease phenotype in NOD1-deficient mice. NOD1 deficiency induced a higher expression of inflammatory cytokines via CAWS and CAWS-induced endoplasmic reticulum (ER) stress in bone marrow-derived dendritic cells. Furthermore, exaggerated inflammation was dependent on apoptosis signal-regulated kinase 1 (ASK1). Notably, NOD1 directly interacted with ASK1, inducing selective autophagy of ASK1, which was dependent on ATG16L1, and thus competitively inhibiting ER stress-dependent ASK1 activation. Altogether, these results show that NOD1 modulates excessive inflammatory responses through the upregulation of autophagy.

DOI: https://doi.org/10.1038/s42003-025-08213-6
Nat Commun. 2025 May 21. 16(1): 4720

H/ACA snR30 snoRNP guides independent 18S rRNA subdomain formation.

Paulina Fischer, Matthias Thoms, Benjamin Lau, Timo Denk, Maria Kuvshinova, Otto Berninghausen, Dirk Flemming, Ed Hurt, Roland Beckmann.

Ribosome biogenesis follows a cascade of pre-rRNA folding and processing steps, coordinated with ribosomal protein incorporation. Nucleolar 90S pre-ribosomes are well-described stable intermediates, composed of pre-18S rRNA, ribosomal S-proteins, U3 snoRNA, and ~70 assembly factors. However, how numerous snoRNAs control pre-rRNA modification and folding during early maturation events remains unclear. We identify snR30 (human U17), the only essential H/ACA snoRNA in yeast, which binds with Cbf5-Gar1-Nop10-Nhp2 to a pre-18S rRNA subdomain containing platform helices and ES6 of the 40S central domain. Integration into the 90S is blocked by RNA hybridization with snR30. The snoRNP complex coordinates the recruitment of early assembly factors Krr1-Utp23-Kri1 and ribosomal proteins uS11-uS15, enabling isolated subdomain assembly. Krr1-dependent release of snR30 culminates in integration of the platform into the 90S. Our study reveals the essential role of snR30 in chaperoning central domain formation as a discrete assembly unit externalized from the pre-ribosomal core.

DOI: https://doi.org/10.1038/s41467-025-59656-8
Nat Commun. 2025 May 23. 16(1): 4794

Linker-free PROTACs efficiently induce the degradation of oncoproteins.

Jianchao Zhang, Congli Chen, Xiao Chen, Kefan Liao, Fengming Li, Xiaoxiao Song, Chaowei Liu, Ming-Yuan Su, Huiyong Sun, Tingjun Hou, Chris Soon Heng Tan, Lijing Fang, Hai Rao.

Proteolysis-targeting chimeras (PROTACs) present a potentially effective strategy against various diseases via selective proteolysis. How to increase the efficacy of PROTACs remains challenging. Here, we explore the necessity of the linker, which has been deemed as an integral part of heterobifunctional PROTACs. Adopting single amino acid-based degradation signals, we find that the linker is not a required feature of the PROTACs. Notably, the linker-free PROTAC, Pro-BA, exhibits superior efficacy over its linker-bearing counterparts in degrading EML4-ALK and inhibiting lung cancer cell growth, as Pro-BA induces a stronger interaction between the target and the E3 ubiquitin ligase. Pro-BA is a water-soluble, orally administered degrader that significantly inhibits the tumor growth in a xenograft mouse model. The broad applicability of this linker-free PROTAC strategy is further validated through the development of BCR-ABL degrader. Our study introduces a design paradigm for PROTACs, potentially facilitating the advancement of more efficient therapeutic degraders.

DOI: https://doi.org/10.1038/s41467-025-60107-7
Proc Natl Acad Sci U S A. 2025 May 27. 122(21): e2504077122

CD83 suppresses endogenous March-I-dependent MHC class II ubiquitination, endocytosis, and degradation.

Sunil Kaul, Nelson B Cole, Joanna Bandola-Simon, Ying Lu, Elena Tondini, Even Walseng, Paul A Roche.

  MHC class II glycoproteins (MHC-II) bind peptides derived from exogenous antigens and dendritic cells (DCs) present these peptide MHC-II (pMHC-II) complexes to antigen-specific CD4 T cells during immune responses. The turnover of surface pMHC-II on antigen-presenting cells (APCs) is controlled by ubiquitin-mediated degradation of pMHC-II by the E3 ubiquitin ligase March-I. To study March-I protein expression, we have generated a mouse in which a V5 epitope-tag was knocked-in to the endogenous March-I gene, thereby allowing us to follow the fate of March-I using high-affinity anti-V5 antibodies. Quantitative analysis revealed that resting spleen DCs and B cells express only ~500 and 125 March-I molecules/cell, respectively. Endogenous March-I protein has a very short half-life in DCs and March-I mRNA, March-I protein, and MHC-II ubiquitination are rapidly terminated upon activation of both DCs and B cells. Like March-I, CD83 is a known regulator of MHC-II expression in APCs and we also show that CD83 suppresses endogenous March-I-dependent MHC-II ubiquitination, endocytosis, and degradation in mouse spleen DCs. Thus, our study reveals molecular mechanisms for both March-I- and CD83-dependent regulation of MHC-II expression in APCs.

Keywords:  MHC class II; antigen presentation; ubiquitination

DOI:  https://doi.org/10.1073/pnas.2504077122
Sci Adv. 2025 May 23. 11(21): eadt7013

Single-molecule analysis reveals the mechanism of chromatin ubiquitylation by variant PRC1 complexes.

Alexandra Teslenko, Beat Fierz.

Chromatin regulation relies on "writer" enzymes that add posttranslational modifications to histone proteins. Variant polycomb repressive complex 1 (PRC1) exists as several subtypes, which are "writers" of ubiquitylation on histone H2A K118 and K119, crucial for transcriptional repression during development and cell identity determination. The mechanism by which dynamic chromatin exploration by variant PRC1 complexes couples to ubiquitin writing is unknown. Here, we developed a single-molecule approach to directly observe chromatin interactions and ubiquitylation by PRC1. We find that variant PRC1 transiently samples chromatin until it reaches a catalytically competent nucleosome-bound state, resulting in E2 recruitment and ubiquitin transfer. Variant PRC1 is weakly processive in ubiquitylating neighboring nucleosomes. Moreover, activity differences between PRC1 subtypes, containing either a PCGF1 or PCGF4 subunit, result from distinct probabilities of achieving a catalytically competent state. Our results thus demonstrate that the dynamic formation of an active complex between variant PRC1, E2, and chromatin is the critical determinant of subtype-specific variant PRC1 activity.

DOI: https://doi.org/10.1126/sciadv.adt7013
J Cell Biol. 2025 Jul 07. pii: e202405162. [Epub ahead of print]224(7):

Phosphatidic acid drives spatiotemporal distribution of Pex30 at ER-LD contact sites.

Morgan House, Karan Khadayat, Thomas N Trybala, Nikhil Nambiar, Elizabeth Jones, Steven M Abel, Joshua Baccile, Amit S Joshi.

Lipid droplets (LDs) are ubiquitous neutral lipid storage organelles that form at discrete subdomains in the ER bilayer. The assembly of these ER subdomains and the mechanism by which proteins are recruited to them is poorly understood. Here, we investigate the spatiotemporal distribution of Pex30 at the ER-LD membrane contact sites (MCSs). Pex30, an ER membrane-shaping protein, has a reticulon homology domain, a dysferlin (DysF) domain, and a Duf4196 domain. Deletion of SEI1, which codes for seipin, a highly conserved protein required for LD biogenesis, results in accumulation of Pex30 and phosphatidic acid (PA) at ER-LD contact sites. We show that PA recruits Pex30 at ER subdomains by binding to the DysF domain. The distribution of Pex30 as well as PA is also affected by phosphatidylcholine (PC) levels. We propose that PA regulates the spatiotemporal distribution of Pex30 at ER subdomains that plays a critical role in driving the formation of LDs in the ER membrane.

DOI: https://doi.org/10.1083/jcb.202405162
Autophagy Rep. 2025 ;4(1): 2501365

A new role of RAB21 and VARP in autophagy and autophagic exocytosis of ATP.

María Carolina Barbosa, Pablo Reta, Sébastien Nola, Milton Osmar Aguilera, Thierry Galli, María Isabel Colombo, Claudio Marcelo Fader.

  Autophagy has been implicated in various cellular processes, including non-conventional secretion. Our previous findings suggest that ATP is loaded into amphisomes and secreted upon autophagy stimulation at focal adhesion sites in a VAMP7-dependent manner. Here, we demonstrate that the knockout (KO) of VAMP7, along with its partners RAB21 and its guanine nucleotide exchange factor (GEF) VARP, inhibits ATP release, indicating a key role for this pathway in amphisome secretion. Constitutively inactive RAB21 also inhibited ATP secretion. RAB21 overexpression rescued starvation-induced ATP secretion in RAB21 KO, but not in VAMP7 or VARP KO cells. RAB21-LC3-positive vesicles redistributed to the cell periphery upon starvation. KO cells and overexpression experiments showed that RAB21 plays a positive role in autophagosome biogenesis, particularly in controlling the number of LC3-II- and DFCP1-positive structures upon starvation, suggesting a role in the early steps of autophagosome formation. Accordingly, VARP partially colocalized with LC3 upon starvation. Together, these findings identify a novel role for RAB21 in regulating autophagic ATP secretion likely in amphisome biogenesis and their localization in the cell periphery.

Keywords:  ATP release; LC3; RAB proteins; VAMP7; macroautophagy; secretory autophagy

DOI:  https://doi.org/10.1080/27694127.2025.2501365
Sci Adv. 2025 May 23. 11(21): eadt2724

Metabolic traits shape responses to LSD1-directed therapy in glioblastoma tumor-initiating cells.

Giulia Marotta, Daniela Osti, Elena Zaccheroni, Brunella Costanza, Stefania Faletti, Adriana Marinaro, Cristina Richichi, Deborah Mesa, Simona Rodighiero, Chiara Soriani, Enrica Migliaccio, Federica Ruscitto, Chiara Priami, Sara Sigismund, Francesco Manetti, Dario Polli, Galina V Beznusenko, Mara-Camelia Rusu, Francesco Favero, Davide Corà, Domenico A Silvestris, Angela Gallo, Valentina Gambino, Fabio Alfieri, Sara Gandini, Matthias J Schmitt, Gaetano Gargiulo, Roberta Noberini, Tiziana Bonaldi, Giuliana Pelicci.

Lysine-specific histone demethylase 1A (LSD1) is an epigenetic regulator involved in various biological processes, including metabolic pathways. We demonstrated the therapeutic potential of its pharmacological inhibition in glioblastoma using DDP_38003 (LSD1i), which selectively targets tumor-initiating cells (TICs) by hampering their adaptability to stress. Through biological, metabolic, and omic approaches, we now show that LSD1i acts as an endoplasmic reticulum (ER) stressor, activating the integrated stress response and altering mitochondrial structure and function. These effects impair TICs' oxidative metabolism and generate reactive oxygen species, further amplifying cellular stress. LSD1i also impairs TICs' glycolytic activity, causing their metabolic decline. TICs with enhanced glycolysis benefit from LSD1-directed therapy. Conversely, metabolically silent TICs mantain ER and mitochondrial homeostasis, adapting to stress conditions, including LSD1i treatment. A dropout short hairpin RNA screening identifies postglycosylphosphatidylinositol attachment to proteins inositol deacylase 1 (PGAP1) as a mediator of resistance to LSD1i. Disruptions in ER and mitochondrial balance holds promise for improving LSD1-targeted therapy efficacy and overcoming treatment resistance.

DOI: https://doi.org/10.1126/sciadv.adt2724
Proc Natl Acad Sci U S A. 2025 May 27. 122(21): e2421258122

Induction of the ISR by AB5 subtilase cytotoxin drives type-I IFN expression in pDCs via STING activation.

Daniela Barros, Beatriz H Ferreira, Paulina Garcia-Gonzalez, Francesco Carbone, Marine Luka, Fátima Leite-Pinheiro, Mariana D Machado, Theopisti Nikolaou, Angelo Pilotti, Eliot Goguet, Paulo Antas, Andreia Mendes, Lichen Zhang, Marina Cresci, Lou Galliot, Julien P Gigan, Marisa Reverendo, Bing Su, Miwako Narita, Adrienne W Paton, James C Paton, Stéphane Rocchi, Frédéric Rieux-Laucat, Rafael J Argüello, Béatrice Nal, Yinming Liang, Mickaël Ménager, Evelina Gatti, Catarina R Almeida, Philippe Pierre.

  We demonstrate that exposure to the AB5 subtilase cytotoxin (SubAB) induces the unfolded protein response (UPR) in human peripheral blood mononuclear cells, concomitant with a proinflammatory response across distinct cell subsets. Notably, SubAB selectively induces type-I interferon (IFN) expression in plasmacytoid dendritic cells, acting synergistically with Toll-like receptor 7 stimulation. The induction of type-I IFN in response to SubAB relies on stimulator of interferon genes (STING) activation, coupled with protein synthesis inhibition mediated by protein kinase R-like endoplasmic reticulum kinase (PERK) and phosphorylation of the eukaryotic translation initiation factor 2 subunit-alpha. By impeding mRNA translation through the integrated stress response, SubAB precipitates the downregulation of the negative innate signaling feedback regulator Tax1-binding protein 1. This downregulation is necessary to unleash TANK-binding kinase 1 signaling associated with STING activation. These findings shed light on how UPR-inducing conditions may regulate the immune system during infection or pathogenesis.

Keywords:  innate immunity; pDC; subtilase cytotoxin; unfolded protein stress response

DOI:  https://doi.org/10.1073/pnas.2421258122
Elife. 2025 May 21. pii: RP100747. [Epub ahead of print]13

Glutamine catabolism supports amino acid biosynthesis and suppresses the integrated stress response to promote photoreceptor survival.

Moloy T Goswami, Eric Weh, Shubha Subramanya, Katherine M Weh, Hima Bindu Durumutla, Heather Hager, Nicholas Miller, Sraboni Chaudhury, Anthony Andren, Peter Sajjakulnukit, Li Zhang, Cagri Besirli, Costas A Lyssiotis, Thomas J Wubben.

  Photoreceptor loss results in vision loss in many blinding diseases, and metabolic dysfunction underlies photoreceptor degeneration. So, exploiting photoreceptor metabolism is an attractive strategy to prevent vision loss. Yet, the metabolic pathways that maintain photoreceptor health remain largely unknown. Here, we investigated the dependence of photoreceptors on glutamine (Gln) catabolism. Gln is converted to glutamate via glutaminase (GLS), so mice lacking GLS in rod photoreceptors were generated to inhibit Gln catabolism. Loss of GLS produced rapid rod photoreceptor degeneration. In vivo metabolomic methodologies and metabolic supplementation identified Gln catabolism as critical for glutamate and aspartate biosynthesis. Concordant with this amino acid deprivation, the integrated stress response (ISR) was activated with protein synthesis attenuation, and inhibiting the ISR delayed photoreceptor loss. Furthermore, supplementing asparagine, which is synthesized from aspartate, delayed photoreceptor degeneration. Hence, Gln catabolism is integral to photoreceptor health, and these data reveal a novel metabolic axis in these metabolically demanding neurons.

Keywords:  cell biology; glutaminase; metabolism; mouse; neurodegeneration; neuroscience; photoreceptor

DOI:  https://doi.org/10.7554/eLife.100747
Dev Dyn. 2025 May 21.

Protein turnover downstream of the Nipbl/CRL4 axis contributes to abnormal development in zebrafish embryos.

Annie C Sanchez, Niusha Banoukh, Fiona Mensching, Robert V Skibbens, M Kathryn Iovine.

   BACKGROUND: Mutations in cohesins cause cohesinopathies such as Cornelia de Lange Syndrome (CdLS) and Roberts Syndrome (RBS). Prior findings demonstrate that Esco2 (a cohesin activator) and Smc3 (a core cohesin subunit) regulate the CRL4 E3 ubiquitin ligase. SMC3 mutations, however, account for a small percentage of CdLS. Here, we test whether NIPBL, which when mutated is responsible for 65% of CdLS cases, also regulates CRL4.
RESULTS: We report that Nipbl knockdown in zebrafish embryos produces developmental abnormalities and reduces the transcription of ddb1, which encodes a key component of CRL4 E3 ligase. The severity of phenotypes in Nipbl knockdown embryos is partially rescued by exogenous ddb1 mRNA, demonstrating that CRL4 ligase function is downstream of Nipbl. These findings suggest that aberrant accumulation of CRL4 ligase substrates contributes to developmental abnormalities. To test this model, we identified candidate CRL4 substrates in zebrafish embryos by LC-MS. The results reveal that elevated expression of one of these candidates, pparαa, is sufficient to produce developmental defects in zebrafish embryos.
CONCLUSIONS: Nipbl impacts CRL4 ligase activity via regulation of ddb1 expression. We provide evidence that the aberrant accumulation of substrates is sufficient to produce developmental abnormalities consistent with those observed in RBS and CdLS models.

Keywords:  CRL4 ubiquitin ligase; Cohesinopathies; Cornelia de Lange Syndrome (CdLS); Ddb1; Roberts Syndrome (RBS); birth defects; thalidomide

DOI:  https://doi.org/10.1002/dvdy.70037
J Biol Chem. 2025 May 21. pii: S0021-9258(25)02112-X. [Epub ahead of print] 110262

Symmetric stimulation of Hsp90 catalyzed ATP hydrolysis through enhanced active site gate dynamics.

Breanna Magnan, Thomas Dumont, Suad Rashid, Paul LaPointe, Leo Spyracopoulos.

  Heat shock protein 90 (Hsp90) is a vital molecular chaperone that is essential for activating a diverse array of regulatory proteins through an ATP-dependent clamping cycle. The Hsp90 clamping cycle is driven by large-amplitude conformational changes within the N-terminal ATPase domain, including the release of an autoinhibitory N-terminal β-strap followed by a less well-characterized ATP gate rearrangement involving N-terminal helix 1. Here, we employed a combination of 19F nuclear magnetic resonance (NMR) spectroscopy, molecular dynamics (MD) simulations, and ATPase assays to examine the effects of targeted β-strap and helix 1 mutations. Our findings reveal that targeted disruption of helix 1 packing against the ATPase domain accelerates clamp closure, symmetrically enhancing ATP hydrolysis for both subunits of the Hsp90 dimer, whereas activation by the Aha1 cochaperone is disrupted. Decreasing the energy barrier associated with helix 1 release is a key step in modulating the energy landscape that governs the dynamics of the Hsp90 clamping cycle.

Keywords:  (19)F NMR; ATPase; chaperone; enzyme kinetics; heat shock protein 90 (Hsp90); molecular chaperone; molecular dynamics; protein dynamics

DOI:  https://doi.org/10.1016/j.jbc.2025.110262
Cell Rep. 2025 May 21. pii: S2211-1247(25)00506-6. [Epub ahead of print]44(6): 115735

PUS10-induced tRNA fragmentation impacts retrotransposon-driven inflammation.

Magdalena Madej, Phuong Cao Thi Ngoc, Sowndarya Muthukumar, Anna Konturek-Cieśla, Silvia Tucciarone, Alexandre Germanos, Christian Ashworth, Knut Kotarsky, Sudip Ghosh, Zhimeng Fan, Helena Fritz, Izei Pascual-Gonzalez, Alain Huerta, Nicola Guzzi, Anita Colazzo, Giulia Beneventi, Hang-Mao Lee, Maciej Cieśla, Christopher Douse, Hiroki Kato, Vinay Swaminathan, William W Agace, Ainara Castellanos-Rubio, Paolo Salomoni, David Bryder, Cristian Bellodi.

  Pseudouridine synthases (PUSs) catalyze the isomerization of uridine (U)-to-pseudouridine (Ψ) and have emerging roles in development and disease. How PUSs adapt gene expression under stress remains mostly unexplored. We identify an unconventional role for the Ψ "writer" PUS10 impacting intracellular innate immunity. Using Pus10 knockout mice, we uncover cell-intrinsic upregulation of interferon (IFN) signaling, conferring resistance to inflammation in vivo. Pus10 loss alters tRNA-derived small RNAs (tdRs) abundance, perturbing translation and endogenous retroelements expression. These alterations promote proinflammatory RNA-DNA hybrids accumulation, potentially activating cyclic GMP-AMP synthase (cGAS)-stimulator of interferon gene (STING). Supplementation with selected tdR pools partly rescues these effects through interactions with RNA processing factors that modulate immune responses, revealing a regulatory circuit that counteracts cell-intrinsic inflammation. By extension, we define a PUS10-specific molecular fingerprint linking its dysregulation to human autoimmune disorders, including inflammatory bowel diseases. Collectively, these findings establish PUS10 as a viral mimicry modulator, with broad implications for innate immune homeostasis and autoimmunity.

Keywords:  CP: Molecular biology; PUS10; RNA-DNA hybrids; cGAS-STING; hematopoietic stem cell; inflammation; inflammatory bowel disease; interferon; pseudouridine; tRNA-derived small RNAs; transposable elements; viral mimicry

DOI:  https://doi.org/10.1016/j.celrep.2025.115735
J Mol Biol. 2025 May 21. pii: S0022-2836(25)00292-X. [Epub ahead of print] 169226

Disease-associated Kv1.3 variants are energy compromised with impaired nascent chain folding.

Aaron Sykes, Lannawill Caruth, Sophia Gross, Shefali Setia Verma, Toshinori Hoshi, Carol Deutsch.

  Nascent proteins fold in a stepwise manner during all stages of biogenesis. This progression is particularly complex for ion channels composed of multiple biogenic and functional domains and subunits. The human Kv1.3 ion channel, encoded by KCNA3, is expressed in neuronal and immune cells. Its dysregulation produces chronic inflammatory disease and autoimmune disorders, which affect many in the US population, especially women. Using the unbiased 'genome-first' approach with integrated patient biobank databases, we identified KCNA3 gene variants associated with human disease and examined their impact on Kv1.3 channel biogenesis. Our tertiary and quaternary folding assays and all-atom molecular dynamics simulations show that KCNA3 gene variants in T1, the channel's intersubunit recognition domain, manifest early-stage T1 folding defects, energetic instabilities, and conformational distortion of subunits concomitant with tertiary unwinding. These findings identify molecular mechanisms by which patient-associated variants influence channel assembly, potentially contributing to diverse clinical phenotypes underlying human disease.

Keywords:  Kv channels; Kv1.3; genetic analysis; molecular dynamic simulations; protein biogenesis; protein folding

DOI:  https://doi.org/10.1016/j.jmb.2025.169226
Elife. 2025 May 20. pii: RP104979. [Epub ahead of print]14

mTOR inhibition in Q175 Huntington's disease model mice facilitates neuronal autophagy and mutant huntingtin clearance.

Philip Stavrides, Chris N Goulbourne, James Peddy, Chunfeng Huo, Mala Rao, Vinod Khetarpal, Deanna M Marchionini, Ralph A Nixon, Dun-Sheng Yang.

  Huntington's disease (HD) is caused by the expansion of the polyglutamine stretch in huntingtin protein (HTT) resulting in hallmark aggresomes/inclusion bodies (IBs) composed of mutant huntingtin protein (mHTT) and its fragments. Stimulating autophagy to enhance mHTT clearance is considered a potential therapeutic strategy for HD. Our recent evaluation of the autophagic-lysosomal pathway (ALP) in human HD brain reveals upregulated lysosomal biogenesis and relatively normal autophagy flux in early Vonsattel grade brains, but impaired autolysosome clearance in late grade brains, suggesting that autophagy stimulation could have therapeutic benefits as an early clinical intervention. Here, we tested this hypothesis by crossing the Q175 HD knock-in model with our autophagy reporter mouse TRGL (Thy-1-RFP-GFP-LC3) to investigate in vivo neuronal ALP dynamics. In the Q175 and/or TRGL/Q175 mice, mHTT was detected in autophagic vacuoles and also exhibited a high level of colocalization with autophagy receptors p62/SQSTM1 and ubiquitin in the IBs. Compared to the robust lysosomal pathology in late-stage human HD striatum, ALP alterations in Q175 models are also late-onset but milder, that included a lowered phospho-p70S6K level, lysosome depletion, and autolysosome elevation including more poorly acidified autolysosomes and larger-sized lipofuscin granules, reflecting impaired autophagic flux. Administration of a mTOR inhibitor to 6-mo-old TRGL/Q175 normalized lysosome number, ameliorated aggresome pathology while reducing mHTT-, p62-, and ubiquitin-immunoreactivities, suggesting the beneficial potential of autophagy modulation at early stages of disease progression.

Keywords:  Huntington's disease; Q175 mouse model; TRGL mice; autophagy; autophagy modulation; huntingtin; medicine; mouse; neuroscience

DOI:  https://doi.org/10.7554/eLife.104979
Nat Commun. 2025 May 16. 16(1): 4575

USP37 prevents unscheduled replisome unloading through MCM complex deubiquitination.

Derek L Bolhuis, Dalia Fleifel, Thomas Bonacci, Xianxi Wang, Brandon L Mouery, Jeanette Gowen Cook, Nicholas G Brown, Michael J Emanuele.

The CMG helicase (CDC45-MCM2-7-GINS) unwinds DNA as a component of eukaryotic replisomes. Replisome (dis)assembly is tightly coordinated with cell cycle progression to ensure genome stability. However, factors that prevent premature CMG unloading and replisome disassembly are poorly described. Since disassembly is catalyzed by ubiquitination, deubiquitinases (DUBs) represent attractive candidates for safeguarding against untimely and deleterious CMG unloading. We combined a targeted loss-of-function screen with quantitative, single-cell analysis to identify human USP37 as a key DUB preventing replisome disassembly. We demonstrate that USP37 maintains active replisomes on S phase chromatin and promotes normal cell cycle progression. Proteomics and biochemical assays revealed USP37 interacts with the CMG complex to deubiquitinate MCM7, antagonizing replisome disassembly. Significantly, USP37 protects normal epithelial cells from oncoprotein-induced replication stress. Our findings reveal USP37 to be critical to the maintenance of replisomes in S phase and suggest USP37-targeting as a potential strategy for treating malignancies with defective DNA replication control.

DOI: https://doi.org/10.1038/s41467-025-59770-7
Nat Biotechnol. 2025 May 23.

Deep-learning-based single-domain and multidomain protein structure prediction with D-I-TASSER.

Wei Zheng, Qiqige Wuyun, Yang Li, Quancheng Liu, Xiaogen Zhou, Chunxiang Peng, Yiheng Zhu, Lydia Freddolino, Yang Zhang.

The dominant success of deep learning techniques on protein structure prediction has challenged the necessity and usefulness of traditional force field-based folding simulations. We proposed a hybrid approach, deep-learning-based iterative threading assembly refinement (D-I-TASSER), which constructs atomic-level protein structural models by integrating multisource deep learning potentials with iterative threading fragment assembly simulations. D-I-TASSER introduces a domain splitting and assembly protocol for the automated modeling of large multidomain protein structures. Benchmark tests and the most recent critical assessment of protein structure prediction, 15 experiments demonstrate that D-I-TASSER outperforms AlphaFold2 and AlphaFold3 on both single-domain and multidomain proteins. Large-scale folding experiments further show that D-I-TASSER could fold 81% of protein domains and 73% of full-chain sequences in the human proteome with results highly complementary to recently released models by AlphaFold2. These results highlight a new avenue to integrate deep learning with classical physics-based folding simulations for high-accuracy protein structure and function predictions that are usable in genome-wide applications.

DOI: https://doi.org/10.1038/s41587-025-02654-4
Nat Commun. 2025 May 19. 16(1): 4652

Dual Ribosome Profiling reveals metabolic limitations of cancer and stromal cells in the tumor microenvironment.

Daniela Aviles-Huerta, Rossella Del Pizzo, Alexander Kowar, Ali Hyder Baig, Giuliana Palazzo, Ekaterina Stepanova, Cinthia Claudia Amaya Ramirez, Sara D'Agostino, Edoardo Ratto, Catarina Pechincha, Nora Siefert, Helena Engel, Shangce Du, Silvia Cadenas-De Miguel, Beiping Miao, Victor M Cruz-Vilchez, Karin Müller-Decker, Ilaria Elia, Chong Sun, Wilhelm Palm, Fabricio Loayza-Puch.

The tumor microenvironment (TME) influences cancer cell metabolism and survival. However, how immune and stromal cells respond to metabolic stress in vivo, and how nutrient limitations affect therapy, remains poorly understood. Here, we introduce Dual Ribosome Profiling (DualRP) to simultaneously monitor translation and ribosome stalling in multiple tumor cell populations. DualRP reveals that cancer-fibroblast interactions trigger an inflammatory program that reduces amino acid shortages during glucose starvation. In immunocompetent mice, we show that serine and glycine are essential for optimal T cell function and that their deficiency impairs T cell fitness. Importantly, immune checkpoint blockade therapy imposes amino acid restrictions specifically in T cells, demonstrating that therapies create distinct metabolic demands across TME cell types. By mapping codon-resolved ribosome stalling in a cell‑type‑specific manner, DualRP uncovers metabolic crosstalk that shapes translational programs. DualRP thus offers a powerful, innovative approach for dissecting tumor cell metabolic interplay and guiding combined metabolic-immunotherapeutic strategies.

DOI: https://doi.org/10.1038/s41467-025-59986-7
Nat Chem Biol. 2025 May 16.

Nuclear ubiquitination permits Hippo-YAP signal for liver development and tumorigenesis.

Jinsong Wei, Zhifa Cao, Qing Li, Xiaoyu Li, Qingzhe Wang, Yiming Zhang, Run Zhang, Xingru Wu, Quanhui Dai, Xinyang Li, Zhaocai Zhou, Fenyong Sun, Shi Jiao, Bing Zhao.

Hippo-YAP signaling is crucial to organ development and tumorigenesis. VGLL4, which occupies TEAD to prevent YAP binding, is the main transcriptional repressor of Hippo-YAP activity. Here we identified the nuclear E3 ligase ubiquitin protein ligase E3 component n-recognin 5 (UBR5) poly-ubiquitinated VGLL4 at Lys61 for its degradation, which permits Hippo-YAP signaling for the development of the liver biliary system in mice and multiple cancers in humans. In mouse liver development, Ubr5 and Vgll4 exhibited reciprocal expression patterns spatiotemporally. Ubr5 deletion impaired cholangiocyte development and hepatocyte reprogramming, which could be efficiently rescued by restoring Hippo-YAP through ablating Vgll4. We also found that the UBR5-VGLL4-YAP axis is associated with the progression of human pan-cancers. Targeting nuclear E3 ligases in multiple types of patient-derived tumor organoids suppressed their expansion. Our identification of UBR5 as the bona fide E3 ligase of VGLL4 offers a molecular framework of nuclear Hippo-YAP regulation and suggests nuclear ubiquitination as a potential therapeutic target for YAP-dependent malignancies.

DOI: https://doi.org/10.1038/s41589-025-01901-8
Cell Mol Gastroenterol Hepatol. 2025 May 14. pii: S2352-345X(25)00074-8. [Epub ahead of print] 101533

Goblet cell loss linked to NOD2 and secondary resection in Crohn's disease is induced by dysbiosis and epithelial MyD88.

Serre-Yu Wong, Maria Manuela Estevinho, Thomas Heaney, Allison A Marshall, Elisabeth Giselbrecht, Scott G Daniel, Chaoting Zhou, Adriana Rosas-Villegas, Kyung Ku Jang, Hairu Yang, Huaibin Mabel Ko, John D Paulson, Yi Ding, Kyle Bittinger, Judy H Cho, James D Lewis, Deepshika Ramanan, Ken Cadwell.

   BACKGROUND & AIMS: The role of goblet cells in small intestinal inflammation in Crohn's disease is unknown. Polymorphisms of NOD2 confer risk for Crohn's disease (CD) and associate with small intestinal disease location. We previously showed in mice that Nod2 deficiency leads to overexpansion of Phocaeicola vulgatus in the gut and downstream goblet cell defects, which preceded small intestinal inflammation. In this study, we ask whether goblet cell defects occur in CD patients with NOD2 polymorphisms and investigate in mice how P. vulgatus signals through the intestinal epithelium.
METHODS: We performed a retrospective study of patients with CD to assess clinical outcomes and goblet cell histology by NOD2 status. We evaluated the contribution of microbiota and MyD88 signaling in the intestinal epithelium to goblet cell defects in the setting of Nod2 deficiency using genetic mouse models and germ-free mice.
RESULTS: In patients with CD who have undergone ileocolic resection, NOD2 risk alleles confer a risk for re-operation (OR 8.12, P = .047) and for increased pERK and goblet cell defects in uninflamed ileal tissue. We show that patients with CD with ileal involvement harbor P. vulgatus regardless of NOD2 risk allele status. We show that intestinal epithelial MyD88 and TLR4 are required for goblet cell defects in Nod2-/- mice harboring P. vulgatus. Finally, we show that P. vulgatus requires complex microbiota to exert its effects in Nod2-deficient mice.
CONCLUSIONS: Goblet cell defects may be a harbinger of small intestinal inflammation in CD patients, particularly in the postoperative setting. Our findings in mice show that small intestinal goblet cell loss associated with Nod2 mutation is induced by microbiome dysbiosis and epithelial MyD88, in part due to TLR4 signaling.

Keywords:  Crohn’s disease; NOD2; TLR4; goblet cells; intestinal epithelium; microbiota dysbiosis

DOI:  https://doi.org/10.1016/j.jcmgh.2025.101533
Blood. 2025 May 22. pii: blood.2024026940. [Epub ahead of print]

Type 2 calreticulin mutations activate ATF6 to promote BCL-xL-mediated survival in myeloproliferative neoplasms.

Nicole S Arellano, William L Heaton, Mirielle C Nauman, Abigail E Runnels, Jacky Gomez-Villa, Daniele Vanni, Melissa Gaviria, Maihi Fujita, Nathan M Krah, Michele Ciboddo, Saveg Yadav, Callie T Brown, Parker D Bowden, Amy K Chen, Christopher Henning, Silvia Catricalà, Ilaria Carola Casetti, Oscar Borsani, Elisa Rumi, Daniela Pietra, Isabelle Plo, Caroline Marty, Marco Marchetti, Caner Saygin, Ami B Patel, Shannon E Elf.

The majority of calreticulin (CALR) mutations in myeloproliferative neoplasms (MPNs) are classified as either type 1, a 52 base-pair deletion (CALRdel52), or type 2, a 5 base-pair insertion (CALRins5). Both are gain-of-function (GOF) mutations that generate an identical mutant C-terminal tail, which mediates the binding to and activation of the thrombopoietin receptor MPL. We recently reported that despite this shared GOF, CALRdel52 but not CALRins5 mutations cause loss of calcium binding function, leading to activation of and dependency on the IRE1a/XBP1 pathway of the unfolded protein response (UPR). This led us to ask whether CALRins5 mutations activate and depend on a different UPR pathway, and whether this is likewise mediated by a mutation type-specific loss-of-function (LOF). Here, we show that CALRins5 mutations lead to activation of the ATF6 pathway of the UPR due to loss of CALR chaperone function. This LOF is caused by interference of the CALRins5 mutant C-terminus with key chaperone residue H170. Further, we show that CALRins5 cells are partially dependent on ATF6 for cytokine-independent growth, and identify BCL-xL as a transcriptional target of ATF6 that promotes type 2 CALR mutant cell survival.

DOI: https://doi.org/10.1182/blood.2024026940
Angew Chem Int Ed Engl. 2025 May 19. e202507092

Reengineering of a Proteomimetic Pan-Ras Inhibitor into a Ras Degrader.

Joseph F Ongkingco, Seong Ho Hong, Eugene D Toth, Thu Nguyen, Paramjit S Arora.

  Bifunctional ligands that can coax protein-protein interactions have become attractive therapeutic modalities. Herein, we describe conformationally defined helix dimers as proteomimetic molecular glues. The helix dimers can be rationally designed toengage helical protein interfaces. We previously described a synthetic Sos protein mimic, CHDSOS, as a Ras ligand that inhibits wild-type and oncogenic Ras signaling. This Sos proteomimetic consisted of a crosslinked helix dimer (CHD) that reproduced two helical domains, termed aH and aI, from Sos. The native aH helix of Sos constitutes the primary contact surface for Sos while aI has minimal engagement. We conjectured that the aI domain of CHDSOS could be reengineered to preserve Ras binding while engaging another protein to fully leverage the contact residues available in a proteomimetic. Herein, we incorporate a second distinct binding epitope into CHDSOS, thereby generating a bispecific proteomimetic. This secondary epitope was designed based on the p53 activation domain to engage the E3 ligase MDM2 and induce complexation with Ras. The resulting lead proteomimetic, CHDBI4, associates with both MDM2 and Ras and demonstrates reduction of cellular Ras levels. Overall, the study offers a proof-of-concept for the development of a bispecific proteomimetic scaffold to target multiple protein interfaces.

Keywords:  Molecular glues; Protein-protein interactions; Ras; constrained peptides; proteomimetics

DOI:  https://doi.org/10.1002/anie.202507092