bims-proteo 2025-08-10 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2025–08–10
43 papers selected by
Eric Chevet, INSERM

NAC controls nascent chain fate through tunnel sensing and chaperone action.
UFMylation of BiP/GRP78 Is Crucial for Maintenance of Endoplasmic Reticulum Homeostasis.
NAC promotes co-translational protein folding at the ribosomal tunnel exit.
A molecular switch in NAC prevents mitochondrial protein mistargeting by SRP.
Functional integrity of the SEL1L-HRD1 complex is critical for ERAD and organismal viability.
Convergent activation of the integrated stress response and ER-mitochondria uncoupling in VAPB-associated ALS.
The EMC acts as a chaperone for membrane proteins.
Integral Synthesis and Clearance Analysis via DIA (ISDia) Reveals Coordinated Protein Dynamics Regulation during Endoplasmic Reticulum Stress.
Computationally Driven Top-Down Mass Spectrometry of Ubiquitinated Proteins.
IRE1 coordinates UPR and ERAD pathways to maintain protein homeostasis under ER stress in Arabidopsis.
Tuning ubiquitin transfer by RING E3 ubiquitin ligases through the linchpin residue.
Response of UBR-box E3 ubiquitin ligases and protein quality control pathways to perturbations in protein synthesis and skeletal muscle size.
SON-dependent nuclear speckle rehabilitation alleviates proteinopathies.
Time-resolved photocatalytic proximity labeling uncovers ER proteome dynamics underlying UPR-to-apoptosis transition.
The intrinsically disordered regions of organellophagy receptors are interchangeable and control organelle fragmentation, ER-phagy and mitophagy flux.
Pelota-mediated ribosome-associated quality control counteracts aging and age-associated pathologies across species.
Deubiquitinase-Targeting Chimeras Mediated Stabilization of Tumor Suppressive E3 Ligase Proteins as a Strategy for Cancer Therapy.
Expression of amyotrophic lateral sclerosis associated protein disulfide isomerase A3 D217N variant recapitulates early morphological alterations at the neuromuscular junction.
RNA N-glycosylation enables immune evasion and homeostatic efferocytosis.
Optogenetic Clustering of Human IRE1 Reveals Differential Regulation of Transcription and mRNA Splice Isoform Abundance by the UPR.
Post-Translational Modifications Remodel Proteome-Wide Ligandability.
Structures of dynamic interactors at native proteasomes by PhIX-MS and cryoelectron microscopy.
Autophagy activator AA-20 improves proteostasis and extends Caenorhabditis elegans lifespan.
BBSome-Mediated Clearance of Ubiquitinated IMPG2 Defines a Constitutive Ciliary Retrieval Pathway in Photoreceptors.
A microsporidial deubiquitinase blocks ubiquitin transfer from adenylated E1 to human UBE2K ubiquitin conjugating enzyme.
A light-resuming strategy as a screening method for selecting Sec61 inhibitors down-modulating PD-L1 expression.
Structural analyses define the molecular basis of clusterin chaperone function.
Non-native entanglement protein misfolding observed in all-atom simulations and supported by experimental structural ensembles.
Diabetic retinopathy and Alzheimer's disease: Convergence of the unfolded protein response in neurodegeneration.
Natural SEL1L variants rescue a model of NGLY1 deficiency and modify ERAD function and proteasome sensitivity.
Saturated lipid stress attenuates mitochondrial genome synthesis in human cells.
A Three-Stage Assembly Program Governing Pancreatic, Plasma, Pituitary, and Bone Secretory Cell Differentiation: A Strategy to Augment Protein Delivery.
Establishment of the phagophore-ERES membrane contact site initiates phagophore elongation.
A mechano-resistance mechanism in skin adapts to terrestrial locomotion.
PDZD8 links organelle crosstalk to synaptic remodeling via autophagy.
RNase L regulates the antiviral proteome by accelerating mRNA decay, inhibiting nuclear mRNA export, and repressing RNAPII-mediated transcription.
SOD1 is delivered to lysosomes via autophagy to maintain lysosomal function and integrity.
A Charge-Driven Strategy for Covalent Modification and Modulation of Biomolecular Condensates.
The E3 ligase HECTD4 regulates COX-2-dependent tumor progression and metastasis.
Lysosomal membrane homeostasis and its importance in physiology and disease.
Decoding Cellular Stress States for Toxicology Using Single-Cell Transcriptomics.
Structural Plasticity of the Membrane-Bound Protein Degradation Assembly Supports Bacterial Adaptation to Stress.
DirectContacts2: A network of direct physical protein interactions derived from high-throughput mass spectrometry experiments.

bioRxiv. 2025 Jul 31. pii: 2025.07.27.667080. [Epub ahead of print]

NAC controls nascent chain fate through tunnel sensing and chaperone action.

Jae Ho Lee, Laurenz Rabl, Martin Gamerdinger, Vaishali Goyal, Katrin Michaela Khakzar, Natalia Moreira Barbosa, Juliana Abramovich, Fabian Morales-Polanco, Ann-Kathrin Köhler, Ekaterina Samatova, Marina V Rodnina, Elke Deuerling, Judith Frydman.

The nascent polypeptide-associated complex (NAC) is a conserved ribosome-bound factor with essential yet incompletely understood roles in protein biogenesis. Here, we show that NAC is a multifaceted regulator that coordinates translation elongation, cotranslational folding, and organelle targeting through distinct interactions with nascent polypeptides both inside and outside the ribosome exit tunnel. Using NAC-selective ribosome profiling in C. elegans , we identify thousands of sequence-specific NAC binding events across the nascent proteome, revealing broad cotranslational engagement with hydrophobic and helical motifs in cytosolic, nuclear, ER, and mitochondrial proteins. Unexpectedly, we discover an intra-tunnel sensing mode, where NAC engages ribosomes with extremely short nascent polypeptides inside the exit tunnel in a sequence-specific manner. These early NAC interactions induce an early elongation slowdown that tunes ribosome flux and prevent ribosome collisions, linking NAC's chaperone activity to kinetic control of translation. We propose that NAC action protects aggregation-prone intermediates by shielding amphipathic helices thus promoting cytonuclear folding and supporting mitochondrial membrane protein biogenesis and ER targeting by early recognition of signal sequences and transmembrane domain. Our findings establish NAC as an early-acting, multifaceted orchestrator of cotranslational proteostasis, with distinct mechanisms of action on nascent chains depending on their sequence features and subcellular destinations.

DOI: https://doi.org/10.1101/2025.07.27.667080
FASEB J. 2025 Aug 15. 39(15): e70905

UFMylation of BiP/GRP78 Is Crucial for Maintenance of Endoplasmic Reticulum Homeostasis.

Zhu Li, Qian Liang, Yanmin Guo, Yaxiang Wang, Miao Wang, Yu-Sheng Cong.

  UFMylation is essential for the embryonic development of metazoans and is associated with several human diseases. Accumulating evidence indicates that the UFMylation pathway plays a critical role in the maintenance of endoplasmic reticulum (ER) homeostasis. However, the underlying mechanisms and relevant cellular targets remain largely unknown. Here, we report that the ER chaperone protein BiP can be covalently modified by UFM1 at lysine residues 294, 296, 352, 353, and 370. This modification destabilizes BiP by promoting its ubiquitination and subsequent proteasome degradation. Depletion of the UFM1 E3 ligase UFL1 or mutation of the BiP UFMylation sites impairs BiP's functions as an ER chaperone and ER stress sensor, thereby increasing apoptotic cell death under ER stress. Our findings suggest that the UFMylation of BiP is critical for maintaining ER homeostasis.

Keywords:  BiP; ER homeostasis; UFL1; UFMylation; UPR

DOI:  https://doi.org/10.1096/fj.202500976RR
bioRxiv. 2025 Aug 02. pii: 2025.08.02.668148. [Epub ahead of print]

NAC promotes co-translational protein folding at the ribosomal tunnel exit.

Jaime Santos, Manuel Günnigmann, Radoslaw J Gora, Marija Iljina, Masa Predin, Ilgin Eser Kotan, Pratiman De, Dhawal Choudhary, Juwon Jang, Frank Tippmann, Nenad Ban, Sander J Tans, Shu-Ou Shan, Günter Kramer, Bernd Bukau.

The nascent polypeptide-associated complex (NAC) coordinates enzymatic modifications and membrane targeting of nascent chains during translation. While NAC's function as a dynamic hub for other factors is well-established, its direct role in co-translational folding is unclear. By proteome-wide profiling NAC co-translational interactions in human cells, we found that NAC recognizes emerging segments enriched in hydrophobicity and α-helical propensity, within folded domains of cytonuclear proteins. Single-molecule and structural analyses reveal that NAC, via its β-barrel domain, dynamically interacts with nascent chains at the ribosomal tunnel exit and is capable of promoting on-pathway folding. Compartment-specific nascent chain interactions of NAC further elucidate its role in targeting to the endoplasmic reticulum and mitochondrial membrane protein biogenesis. Together, these findings show that NAC acts as a bona fide co-translational chaperone that facilitates early protein folding at the ribosomal tunnel exit, expanding its functional repertoire in protein biogenesis.

DOI: https://doi.org/10.1101/2025.08.02.668148
bioRxiv. 2025 Aug 01. pii: 2025.07.29.667405. [Epub ahead of print]

A molecular switch in NAC prevents mitochondrial protein mistargeting by SRP.

Emir Maldosevic, Radoslaw Gora, Liangguang Leo Lin, Linyao Elina Zhou, Zexin Jason Li, Yelena Peskova, Ling Qi, Shu-Ou Shan, Ahmad Jomaa.

The nascent polypeptide-associated complex (NAC) co-translationally screens all nascent proteins and regulates their access to the signal recognition particle (SRP) to ensure the fidelity of protein targeting to the endoplasmic reticulum (ER). However, the mechanism by which NAC prevents the mistargeting of nascent mitochondrial proteins remains unclear. Here, we identified a molecular switch in NAC that allows its central barrel domain to adopt a stabilized conformation on ribosomes exposing a mitochondrial targeting sequence (MTS). Mutations of the MTS on the nascent chain or in the NAC switch region increases NAC barrel dynamics and reduces its binding to the ribosome. This leads to an impaired ability of NAC to prevent mistargeting by SRP and causes ER stress in human cells. Our work reveals how NAC detects nascent mitochondrial proteins early in translation and prevents their promiscuous access to SRP, elucidating the structural basis that underlies this role and providing novel insights into protein targeting fidelity with broader implications for cellular proteostasis.

DOI: https://doi.org/10.1101/2025.07.29.667405
bioRxiv. 2025 Aug 02. pii: 2025.08.01.668162. [Epub ahead of print]

Functional integrity of the SEL1L-HRD1 complex is critical for ERAD and organismal viability.

Xiawei Zhang, Liangguang Leo Lin, Linxiu Pan, Xiaoqiong Wei, Huilun Helen Wang, Zexin Jason Li, Ling Qi.

The SEL1L-HRD1 complex represents the most evolutionarily conserved branch of endoplasmic reticulum-associated degradation (ERAD), with SEL1L acting as a key cofactor for the E3 ubiquitin ligase HRD1. While the physiological relevance of this complex has been increasingly recognized, whether SEL1L is strictly required for HRD1 function in mammals has remained unclear. Here, using complementary in vivo and in vitro approaches, we define the architecture and physiological significance of the mammalian SEL1L-HRD1 ERAD complex. Our data demonstrate that direct binding between SEL1L and HRD1 is essential for ERAD function and neonatal survival in mice. In three knock-in mouse models harboring targeted mutations at the SEL1L-HRD1 interface, we show that the L709P variant - unlike the benign P699T mutation - results in complete neonatal lethality within 30 hours of birth, a phenotype more severe than that of the partially lethal S658P variant. Mechanistically, the L709P mutation abolishes SEL1L-HRD1 interaction, disrupting substrate engagement and impairing recruitment of the E2 enzyme UBE2J1, leading to the accumulation and aggregation of misfolded proteins in the ER. Notably, these defects can be partially rescued by HRD1 overexpression, echoing findings from yeast. Together, our results provide definitive evidence that the SEL1L-HRD1 interaction is essential for ERAD activity and neonatal viability in mammals, resolving a long-standing question in ERAD biology and identifying a new therapeutic strategy for modulating ERAD activity in humans.

DOI: https://doi.org/10.1101/2025.08.01.668162
EMBO Mol Med. 2025 Aug 05.

Convergent activation of the integrated stress response and ER-mitochondria uncoupling in VAPB-associated ALS.

Curran Landry, James P Costanzo, Miguel Mitne-Neto, Mayana Zatz, Ashleigh E Schaffer, Maria Hatzoglou, Alysson R Muotri, Helen C Miranda.

  Vesicle-associated membrane protein-associated protein-B (VAPB) is an endoplasmic reticulum (ER) membrane-bound protein. The P56S mutation in VAPB causes a dominant, familial form of amyotrophic lateral sclerosis (ALS). However, the mechanism by which this mutation leads to motor neuron (MN) degeneration remains unclear. Utilizing inducible pluripotent stem cell (iPSC)-derived MNs expressing either wild-type (WT) or P56S VAPB, we demonstrate that the mutant protein reduces neuronal firing and disrupts ER-mitochondria-associated membranes (ER MAMs), with a time-dependent decline in mitochondrial membrane potential (MMP), hallmarks of MN pathology. These findings were validated in patient-derived iPSC-MNs. Additionally, VAPB P56S MNs show increased susceptibility to ER stress, elevated expression of the Integrated Stress Response (ISR) regulator ATF4 under stress, and reduced global protein synthesis. Notably, pharmacological ISR inhibition using ISRIB rescued ALS-associated phenotypes in both VAPB P56S and patient-derived iPSC-MNs. We present the first evidence that the VAPB P56S mutation activates ISR signaling via mitochondrial dysfunction in human MNs. These findings support ISR modulation as a strategy for ALS intervention and highlight the need for patient stratification in clinical trials.

Keywords:  ALS (Amyotrophic Lateral Sclerosis); ER-MAM (Endoplasmic Reticulum Mitochondria Associated Membrane); ISR (Integrated Stress Response); Neurodegeneration; VAPB ((Vesicle Associated Membrane Protein Associated Protein B)

DOI:  https://doi.org/10.1038/s44321-025-00279-3
Nat Commun. 2025 Aug 02. 16(1): 7097

The EMC acts as a chaperone for membrane proteins.

Carolin J Klose, Kevin M Meighen-Berger, Martin Kulke, Marina Parr, Barbara Steigenberger, Martin Zacharias, Dmitrij Frishman, Matthias J Feige.

Structure formation of membrane proteins is error-prone and thus requires chaperones that oversee this essential process in cell biology. The ER membrane protein complex (EMC) is well-defined as a transmembrane domain (TMD) insertase. In this study, we characterize an additional chaperone function of the EMC. We use interactomics and systematic studies with model proteins to comprehensively define client features for this EMC chaperone mode. Based on this data, we develop a machine learning-based tool for client prediction. Mechanistically, our study reveals that the EMC engages TMDs via its EMC1 subunit and modulates their orientation within the lipid bilayer. Productive TMD assembly reduces binding to the EMC chaperone site. Taken together, our study provides detailed insights into an EMC chaperone function, further establishing the role of the EMC as a multifunctional molecular machine in membrane protein biogenesis.

DOI: https://doi.org/10.1038/s41467-025-62109-x
bioRxiv. 2025 Jul 30. pii: 2025.07.23.666381. [Epub ahead of print]

Integral Synthesis and Clearance Analysis via DIA (ISDia) Reveals Coordinated Protein Dynamics Regulation during Endoplasmic Reticulum Stress.

Yue Dou, Danny Qiu, Vivien Li, Maja E Wierzbińska, Gregory R Keele, Wenpeng Liu, Jie Yang, Joao A Paulo, Ling Qi, Tian Zhang.

Endoplasmic Reticulum (ER) stress disrupts protein homeostasis and impacts protein dynamics, driving cellular responses critical for survival, development and disease. However, no current proteome-wide technology enables simultaneous identification of proteins undergoing altered synthesis and clearance and distinguish their relative contribution during ER stress. To fill this gap, we developed Integral Synthesis and clearance analysis via DIA (ISDia), a robust mass spectrometry-based platform that integrates pulsed-SILAC labeling with data-independent acquisition (DIA) to quantify heavy and light peptide changes and determine the drivers of protein dynamics with high proteome coverage under non-steady-state conditions. Using ISDia, we uncover diverse regulatory mechanisms by which protein synthesis and clearance are modulated to control protein abundances during ER stress, revealing PERK dependent and independent regulatory mechanisms across subcellular compartments, complexes and isoforms. These findings highlight the potential of ISDia as a powerful and widely applicable platform for elucidating protein dynamic regulatory mechanisms.

DOI: https://doi.org/10.1101/2025.07.23.666381
bioRxiv. 2025 Jul 29. pii: 2025.07.24.666707. [Epub ahead of print]

Computationally Driven Top-Down Mass Spectrometry of Ubiquitinated Proteins.

Elizaveta I Shestoperova, Daniil G Ivanov, Joyce Zhong, Liam Chien, Eric R Strieter.

Ubiquitination regulates numerous cellular processes through the attachment of polyubiquitin (Ub) chains that vary in linkage type, length, and branching topology. However, current mass spectrometry approaches cannot simultaneously define both the site of ubiquitination and the topology of the attached Ub chain on intact protein substrates. Here, we present the first integrated strategy that enables simultaneous determination of ubiquitin site and chain architecture using top-down mass spectrometry (TD-MS). Central to this approach is UbqTop , a custom computational platform that predicts Ub chain topology from tandem MS (MS²) fragmentation data by utilizing Bayesian-like scoring algorithm. To address the challenge of analyzing complex substrates, we combine this with selective Asp-N proteolysis, which digests the substrate while preserving intact Ub chains. This enables direct, site-resolved mapping of Ub chain topology on proteins. We demonstrate the broad utility of this method on both free Ub chains and multiply ubiquitinated protein substrates, including the resolution of isomeric chains and branched architectures. Together, this work establishes a powerful new framework for proteoform-level analysis of ubiquitin signaling with unprecedented structural resolution.

DOI: https://doi.org/10.1101/2025.07.24.666707
Plant Physiol Biochem. 2025 Jul 31. pii: S0981-9428(25)00849-6. [Epub ahead of print]228 110321

IRE1 coordinates UPR and ERAD pathways to maintain protein homeostasis under ER stress in Arabidopsis.

Jae Yong Yoo, Ha Na Choi, Ki Seong Ko, Wahyu Indra Duwi Fanata, Rikno Harmoko, Sun Ho Kim, Jong Chan Hong, Sang-Kyu Lee, Woo Sik Chung, Kyun Oh Lee.

  Environmental stress in plants often leads to the accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER), triggering the unfolded protein response (UPR) and ER-associated degradation (ERAD) to restore protein homeostasis. In Arabidopsis, the ER stress sensor IRE1 catalyzes the unconventional splicing of bZIP60 mRNA, producing a transcription factor that regulates UPR-related genes. However, the role of IRE1 in ERAD regulation remains unclear. Here, we demonstrate that ire1a&b double mutants show delayed induction of key ERAD components, including HRD1B, EBS5/HRD3, and EBS6/OS9, under tunicamycin-induced ER stress. In a genetic background carrying the misfolded brassinosteroid receptor Bri1-5, loss of IRE1 exacerbates dwarfism and increases the stability of Bri1-5 protein, indicating impaired ERAD activity. Overexpression of Bri1-5 in the ire1a&b background phenocopied the dwarf and stress-sensitive traits, confirming the role of IRE1 in mitigating ERAD substrate accumulation. Chemical inhibition experiments further revealed that proper glycoprotein processing and degradation are essential for normal growth and stress tolerance, and that IRE1 function is critical for coordinating these processes. Our findings uncover a key regulatory function of IRE1 in linking the UPR and ERAD pathways, thereby maintaining ER homeostasis and supporting plant growth under stress conditions.

Keywords:  Arabidopsis; Bri1-5; Dwarfism; Endoplasmic reticulum stress; IRE1

DOI:  https://doi.org/10.1016/j.plaphy.2025.110321
Life Sci Alliance. 2025 Oct;pii: e202503394. [Epub ahead of print]8(10):

Tuning ubiquitin transfer by RING E3 ubiquitin ligases through the linchpin residue.

Mark A Nakasone, Lori Buetow, Mads Gabrielsen, Syed F Ahmed, Karolina A Majorek, Gary J Sibbet, Brian O Smith, Danny T Huang.

RING family ubiquitin ligases (E3s) employ the RING domain to recruit the E2 thioester ubiquitin (E2∼Ub) intermediate to catalyze the transfer of ubiquitin (Ub) to substrates. A cationic Arg linchpin (LP) residue in the RING domain plays a key role in stabilizing the interface with E2∼Ub, but the identity of the LP residue varies across E3s. Here, we investigate how the LP residue contributes to ubiquitination. Using the model RNF38 system, we demonstrate that substitution of LPArg to the other 19 available amino acids modulates ubiquitination, ranging from minor reduction to complete abolition. The identity of the LP residue influences E2∼Ub binding but does not correlate with E3 activity. NMR and X-ray crystallography analyses reveal that RNF38 LPArg variants stabilize E2∼Ub in a catalytically competent conformation to varying degrees. By altering the LP residue in XIAP, we show that the XIAPY485R variant promotes E2∼Ub stabilization and enhances substrate ubiquitination in cells. Our work demonstrates the importance of the LP residue in modulating E2∼Ub conformation to control ubiquitination.

DOI: https://doi.org/10.26508/lsa.202503394
bioRxiv. 2025 Jul 27. pii: 2025.07.23.666188. [Epub ahead of print]

Response of UBR-box E3 ubiquitin ligases and protein quality control pathways to perturbations in protein synthesis and skeletal muscle size.

Leslie M Baehr, Luis Gustavo Oliveira de Sousa, Craig A Goodman, Adam P Sharples, David S Waddell, Sue C Bodine, David C Hughes.

The N-degron pathway contributes to proteolysis by targeting N-terminal residues of destabilized proteins via E3 ligases that contain a UBR-box domain. Emerging evidence suggests the UBR-box family of E3 ubiquitin ligases (UBR1-7) are involved in the positive regulation of skeletal muscle mass. The purpose of this study was to explore the role of UBR-box E3 ubiquitin ligases under enhanced protein synthesis and skeletal muscle growth conditions. Cohorts of adult male mice were electroporated with constitutively active Akt (Akt-CA) or UBR5 RNAi constructs with a rapamycin diet intervention for 7 and 30 days, respectively. In addition, the UBR-box family was studied during the regrowth phase post nerve crush induced inactivity. Skeletal muscle growth with Akt-CA or regrowth following inactivity increased protein abundance of UBR1, UBR2, UBR4, UBR5 and UBR7. This occurred with corresponding increases in Akt-mTORC1/S6K and MAPK/p90RSK signaling and protein synthesis. The increases in UBR-box E3s, ubiquitination, and proteasomal activity occurred independently of mTORC1 activity and were associated with increases in markers related to autophagy, ER-stress, and protein quality control pathways. Finally, while UBR5 knockdown (KD) evokes atrophy, it occurs together with hyperactivation of mTORC1 and protein synthesis. In UBR5 KD muscles, we identified an increase in protein abundance for UBR2, UBR4 and UBR7, which may highlight a compensatory response to maintain proteome integrity. Future studies will seek to understand the role of UBR-box E3s towards protein quality control in skeletal muscle plasticity.
New and Noteworthy: Novel UBR-box E3 ubiquitin ligases are responsive to heightened protein synthesis and alterations in skeletal muscle mass and fiber size, in order to maintain proteome integrity.

DOI: https://doi.org/10.1101/2025.07.23.666188
Nat Commun. 2025 Aug 05. 16(1): 7065

SON-dependent nuclear speckle rehabilitation alleviates proteinopathies.

William Dion, Yuren Tao, Maci Chambers, Shanshan Zhao, Riley K Arbuckle, Michelle Sun, Syeda Kubra, Matthew A Schaich, Yuhang Nie, Megan Ye, Imran Jamal, Mads B Larsen, Daniel Camarco, Eleanor Ickes, Haokun H Wang, C DuPont, Bingjie Wang, Silvia Liu, Shaohua Pi, Bennett Van Houten, Bill B Chen, Yuanyuan Chen, Xu Chen, Bokai Zhu.

Current treatments targeting individual protein quality control pathways have limited efficacy in alleviating proteinopathies, highlighting the prerequisite for a common druggable target capable of global proteostasis modulation. Building upon our prior research establishing nuclear speckles as pivotal membrane-less organelles for transcriptional control of proteostasis, we aim to alleviate proteinopathies through nuclear speckle rehabilitation. We identify pyrvinium pamoate as a nuclear speckle rehabilitator that enhances protein quality control gene expression and suppresses YAP1 transcriptional activity via decreasing the surface/interfacial tension of nuclear speckle condensates through interaction with the intrinsically disordered region of nuclear speckle scaffold protein SON. In pre-clinical models, nanomolar pyrvinium pamoate protects against retinal degeneration and tauopathy mainly by promoting autophagy and ubiquitin-proteasome activity in a SON-dependent manner without causing stress. Aberrant nuclear speckle morphology, reduced protein quality control and increased YAP1 activity are observed in human tauopathies. Our study provides proof-of-principle of targeting nuclear speckles to ameliorate proteinopathies.

DOI: https://doi.org/10.1038/s41467-025-62242-7
Proc Natl Acad Sci U S A. 2025 Aug 12. 122(32): e2503115122

Time-resolved photocatalytic proximity labeling uncovers ER proteome dynamics underlying UPR-to-apoptosis transition.

Nan Zhou, Yan Zhang, Peng R Chen, Xinyuan Fan.

  Apoptosis is a critical outcome of stress-induced processes, with the endoplasmic reticulum (ER) playing a central role in apoptotic protein processing and stress signal transduction. Profiling the ER proteome during stress to cell death offers valuable insights into these processes, but existing methods often suffer from a loss of in situ information or requirement of genetic manipulation. In this study, we introduce CAT-ER, a nongenetic ER proteomics system that provides in situ labeling, spatiotemporal resolution, and compatibility across diverse cell types. By combining an ER-targeted iridium photocatalyst with a thio-quinone methide (thioQM) probe, CAT-ER achieves high specificity in enriching ER proteins, comparable to traditional enzymatic methods. Importantly, CAT-ER is free of genetic manipulation, allowing its use in hard-to-transfect cell types like HeLa and immune cells (e.g., Raji, Jurkat, and RAW264.7). Given the high spatiotemporal resolution of CAT-ER, we revealed dynamic ER proteome changes during thapsigargin (Tg)-induced unfolded protein response (UPR) to apoptosis. Notably, NFIP2 mitigated ER stress by halting translation when UPR initiated, while compromised EMC2 delayed apoptosis during prolonged stress. These findings provide insights into the molecular dynamics linking the UPR and apoptosis. Collectively, CAT-ER serves as a versatile tool for spatiotemporal proteomic analysis without the need for genetic manipulation, offering a powerful approach to study ER dynamics in various biological contexts.

Keywords:  apoptosis; endoplasmic reticulum; proximity labeling; subcellular proteomics; unfolded protein response

DOI:  https://doi.org/10.1073/pnas.2503115122
Nat Cell Biol. 2025 Aug 04.

The intrinsically disordered regions of organellophagy receptors are interchangeable and control organelle fragmentation, ER-phagy and mitophagy flux.

Mikhail Rudinskiy, Carmela Galli, Andrea Raimondi, Maurizio Molinari.

Organellophagy receptors control the generation and delivery of portions of their homing organelle to acidic degradative compartments to recycle nutrients, remove toxic or aged macromolecules and remodel the organelle upon physiologic or pathologic cues. How they operate is not understood. Here we show that organellophagy receptors are composed of a membrane-tethering module that controls organellar and suborganellar distribution and by a cytoplasmic intrinsically disordered region (IDR) with net cumulative negative charge that controls organelle fragmentation and displays an LC3-interacting region (LIR). The LIR is required for lysosomal delivery but is dispensable for organelle fragmentation. Endoplasmic reticulum (ER)-phagy receptors' IDRs trigger DRP1-assisted mitochondrial fragmentation and mitophagy when transplanted at the outer mitochondrial membrane. Mitophagy receptors' IDRs trigger ER fragmentation and ER-phagy when transplanted at the ER membrane. This offers an interesting example of function conservation on sequence divergency. Our results imply the possibility to control the integrity and activity of intracellular organelles by surface expression of organelle-targeted chimeras composed of an organelle-targeting module and an IDR module with net cumulative negative charge that, if it contains a LIR, eventually tags the organelle portions for lysosomal clearance.

DOI: https://doi.org/10.1038/s41556-025-01728-4
Proc Natl Acad Sci U S A. 2025 Aug 12. 122(32): e2505217122

Pelota-mediated ribosome-associated quality control counteracts aging and age-associated pathologies across species.

Jongsun Lee, Bora Lee, Hyein Lee, Eun Ji E Kim, Sieun S Kim, Hyunwoo C Kwon, Hanseul Lee, Gee-Yoon Lee, Woojin Hong, Seokjin Ham, Jae Won Yang, Heeju Park, Tatiana M Moreno, Michelle E Brown, Hae-Eun H Park, Eunah Kim, Yoonji Jung, Eunseok Kang, Sangsoon Park, Yeo Jin Shin, Seokjun G Ha, Sujeong Kwon, Seungjae Hwang, Hyemin Min, Caroline Kumsta, Chunghun Lim, Chanhee Kang, Jinsoo Seo, Kwang-Pyo Lee, Seung-Jae V Lee.

  Ribosome-associated quality control (RQC) is a pivotal biological process that governs the fidelity of messenger RNA (mRNA) homeostasis and protein synthesis. Defects in RQC are implicated in cellular dysfunction and proteotoxicity, but their impact on aging remains elusive. Here, we show that Pelota, the ribosome rescue factor, promotes longevity and protects against age-related pathological phenotypes in multiple metazoan species. By performing a targeted genetic screen, we find that Pelota is indispensable for longevity in the nematode Caenorhabditis elegans. We show that Pelota mitigates premature senescence in cultured human cells, muscle aging in mice, and neuropathology in cellular and organoid models of Alzheimer's disease. Mechanistically, we demonstrate that Pelota maintains autophagy-mediated proteostasis, by preventing the hyperactivation of mechanistic target of rapamycin signaling. Overall, our work highlights the conserved functional significance of RQC, regulated by Pelota, in extending lifespan and protecting diverse species against age-associated disease phenotypes.

Keywords:  C. elegans; RNA quality control (RQC); aging

DOI:  https://doi.org/10.1073/pnas.2505217122
J Am Chem Soc. 2025 Aug 06.

Deubiquitinase-Targeting Chimeras Mediated Stabilization of Tumor Suppressive E3 Ligase Proteins as a Strategy for Cancer Therapy.

Li Chen, Zhijie Deng, Yan Xiong, Jing Liu, Daoyuan Huang, Jingchao Wang, Yong Chen, Hiroyuki Inuzuka, Ling Xie, Xian Chen, Jian Jin, Wenyi Wei.

Targeted protein stabilization has emerged as a promising therapeutic strategy to combat various human diseases linked to aberrant protein degradation. However, the deubiquitinase-targeting chimera (DUBTAC) technology is still in its infancy, with only a few proteins being successfully stabilized. To this end, the stabilization of tumor suppressor proteins represents a critical therapeutic strategy to combat cancer, as their loss-of-function mutations and reduced expression are frequently implicated in the pathogenesis of diverse types of human cancer. In this study, we present an innovative PRO-DUBTAC platform that, for the first time, stabilizes tumor-suppressive E3 ubiquitin ligases as a novel anticancer therapeutic approach. Through the conjugation of E3 ligase ligands with a small-molecule ligand of the deubiquitinase OTUB1 via a linker, we developed two series of PRO-DUBTACs─VHL-DUBTAC and KEAP1-DUBTAC─that effectively stabilize the tumor-suppressive E3 ligases VHL and KEAP1 in cells, respectively, in an OTUB1-dependent manner to retard tumor cell growth. PRO-DUBTAC could be a versatile and generalizable platform for the selective stabilization of tumor-suppressor E3 ligases, thereby opening new therapeutic avenues for targeted cancer therapies by harnessing the tumor-suppressive potential of E3 ligases.

DOI: https://doi.org/10.1021/jacs.5c06306
Neurobiol Dis. 2025 Aug 01. pii: S0969-9961(25)00261-X. [Epub ahead of print] 107045

Expression of amyotrophic lateral sclerosis associated protein disulfide isomerase A3 D217N variant recapitulates early morphological alterations at the neuromuscular junction.

Martin Sepulveda, Francisca MartinezTraub, Patricia Ojeda, Jessica Mella, Jorge Ojeda, Cristina Pinto, Rodrigo Diaz, Pablo Rozas, Claudia Sepulveda, Bredford Kerr, Vania Morales, Mirva Saaranen, Lloyd Ruddock, Danilo B Medinas, Juan Pablo Henriquez, Claudio Hetz.

  Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by neuromuscular connectivity decline followed by motoneuron loss. Altered proteostasis is suggested as a transversal pathogenic mechanism, notably involving dysfunction at the level of the endoplasmic reticulum (ER). Protein disulfide isomerases (PDIs) are key enzymes that catalyze protein folding and disulfide bond formation in the ER. Importantly, PDIs function is disrupted in ALS. We previously identified mutations in the gene encoding PDIA3 (also known as Grp58 or ERp57) as risk factors for ALS, which were associated with altered neuromuscular junction (NMJ) organization when expressed in zebrafish, a phenotype recapitulated in PDIA3-null mice. Here, we generated a transgenic mouse line overexpressing the ALS-linked PDIA3 variant D217N and performed a comprehensive characterization of ALS-like features. The transgenic line exhibited moderate overexpression of mutant PDIA3D217N, which led to morphological alterations at the NMJ resembling those observed in ALS models and patients, along with abnormal distribution of oxidative and glycolytic muscle fibers. However, mutant PDIA3D217N expression did not result in motor impairment, coordination deficits, or motoneuron loss. At the molecular level, we observed reduced expression of SV2 in the spinal cord, an important synaptic protein involved in NMJ function. Our findings further support the involvement of PDIA3 dysfunction as a risk factor in the emergence of early features of ALS.

Keywords:  Amyotrophic lateral sclerosis; Endoplasmic reticulum; Neuromuscular junction; Protein disulfide isomerase; Proteostasis

DOI:  https://doi.org/10.1016/j.nbd.2025.107045
Nature. 2025 Aug 06.

RNA N-glycosylation enables immune evasion and homeostatic efferocytosis.

Vincent R Graziano, Jennifer Porat, Marie Dominique Ah Kioon, Ivana Mejdrová, Alyssa J Matz, Charlotta G Lebedenko, Peiyuan Chai, John V Pluvinage, Rafael Ricci-Azevedo, Andrew G Harrison, Skylar S Wright, Xinzheng Wang, Madison S Strine, Penghua Wang, Michael R Wilson, Sivapriya Kailasan Vanaja, Beiyan Zhou, Franck J Barrat, Thomas Carell, Ryan A Flynn, Vijay A Rathinam.

Glycosylation is central to the localization and function of biomolecules1. We recently discovered that small RNAs undergo N-glycosylation2 at the modified RNA base 3-(3-amino-3-carboxypropyl) uridine (acp3U)3. However, the functional significance of N-glycosylation of RNAs is unknown. Here we show that the N-glycans on glycoRNAs prevent innate immune sensing of endogenous small RNAs. We found that de-N-glycosylation of cell-culture-derived and circulating human and mouse glycoRNA elicited potent inflammatory responses including the production of type I interferons in a Toll-like receptor 3- and Toll-like receptor 7-dependent manner. Furthermore, we show that N-glycans on cell surface RNAs prevent apoptotic cells from triggering endosomal RNA sensors in efferocytes, thus facilitating the non-inflammatory clearance of dead cells. Mechanistically, N-glycans conceal the hypermodified uracil base acp3U, which we identified as immunostimulatory when exposed in RNA. Consistent with this, genetic deletion of an enzyme (DTWD2) that synthesizes acp3U abrogated innate immune activation by de-N-glycosylated small RNAs and apoptotic cells. Furthermore, synthetic acp3U-containing RNAs are sufficient to trigger innate immune responses. Thus, our study has uncovered a natural mechanism by which N-glycans block RNAs from inducing acp3U-dependent innate immune activation, demonstrating how glycoRNAs exist on the cell surface and in the endosomal network without inducing autoinflammatory responses.

DOI: https://doi.org/10.1038/s41586-025-09310-6
bioRxiv. 2025 Jul 21. pii: 2025.07.16.665212. [Epub ahead of print]

Optogenetic Clustering of Human IRE1 Reveals Differential Regulation of Transcription and mRNA Splice Isoform Abundance by the UPR.

Jacob W Smith, Damien B Wilburn, Vladislav Belyy.

Inositol-requiring enzyme 1 (IRE1) is one of three known sensor proteins that respond to homeostatic perturbations in the metazoan endoplasmic reticulum. The three sensors collectively initiate an intertwined signaling network called the Unfolded Protein Response (UPR). Although IRE1 plays pivotal roles in human health and development, understanding its specific contributions to the UPR remains a challenge due to signaling crosstalk from the other two stress sensors. To overcome this problem, we engineered a light-activatable version of IRE1 and probed the transcriptomic effects of IRE1 activity in isolation from the other branches of the UPR. We demonstrate that 1) oligomerization alone is sufficient to activate IRE1 in human cells, 2) IRE1's transcriptional response evolves substantially under prolonged activation, and 3) the UPR induces major changes in mRNA splice isoform abundance in an IRE1-independent manner. Our data reveal previously unknown targets of IRE1 transcriptional regulation and direct degradation. Additionally, the tools developed here will be broadly applicable for precise dissection of signaling networks in diverse cell types, tissues, and organisms.

DOI: https://doi.org/10.1101/2025.07.16.665212
bioRxiv. 2025 Aug 02. pii: 2025.07.31.667978. [Epub ahead of print]

Post-Translational Modifications Remodel Proteome-Wide Ligandability.

Weichao Li, Qijia Wei, Paolo Governa, Manuel Llanos, Tzu-Yuan Chiu, Jacob M Wozniak, Appasso M Jadhav, Clara Gathmann, Jacob Cravatt, Ashok Dongre, Mia L Huang, Stefano Forli, Christopher G Parker.

Post-translational modifications (PTMs) vastly expand the diversity of human proteome, dynamically reshaping protein activity, interactions, and localization in response to environmental, pharmacologic, and disease-associated cues. While it is well established that PTMs modulate protein function, structure, and biomolecular interactions, their proteome-wide impact on small-molecule recognition-and thus druggability-remains largely unexplored. Here, we introduce a chemical proteomic strategy to delineate how PTM states remodel protein ligandability in human cells. By deploying broad profiling photoaffinity probes, we identified over 400 functionally diverse proteins whose ability to engage small molecules is impacted by phosphorylation or N-linked glycosylation status. Integration of binding site mapping with structural analyses revealed a diverse array of PTM-dependent pockets. Among these targets, we discovered that the phosphorylation status of common oncogenic KRAS mutants impact the action of small molecules, including clinically approved inhibitors. These findings illuminate an underappreciated, PTM-governed layer of proteome plasticity and uncover opportunities for the development of chemical probes to selectively target proteins in defined modification states.

DOI: https://doi.org/10.1101/2025.07.31.667978
bioRxiv. 2025 Aug 02. pii: 2025.07.31.667872. [Epub ahead of print]

Structures of dynamic interactors at native proteasomes by PhIX-MS and cryoelectron microscopy.

Kitaik Lee, Hitendra Negi, Xiang Chen, Katerina Atallah-Yunes, Sunny Truslow, Rithik E Castelino, Mary R Guest, Anthony M Ciancone, Sergey G Tarasov, Raj Chari, Kylie J Walters, Francis J O'Reilly.

  Proteasome function depends on a network of transient interactions that remain structurally and functionally unresolved. We developed PhIX-MS (Photo-induced In situ Crosslinking-Mass Spectrometry), a structural proteomics workflow that stabilizes transient interactions in cells by UV-activated crosslinking to capture topological information. Applying PhIX-MS with cryo-electron microscopy (cryo-EM), we mapped redox sensor TXNL1 at the proteasome regulatory particle (RP), placing its PITH domain above deubiquitinase RPN11 and resolving its dynamic thioredoxin domain near RPN2/PSMD1 and RPN13/ADRM1, ideally located to reduce substrates prior to proteolysis. We also resolved chaperone PSMD5 bound to RP without the proteolytic core particle (CP) where its C-terminus inserts into the ATPase pore blocking CP binding. PhIX-MS and AlphaFold modeling tether ubiquitin ligase UBE3C/Hul5 along the RP placing its catalytic site above the RPN11 active site, enabling their coupled activities. Our integrative approach enables the localization of native, low-affinity protein interactions and is broadly applicable to dynamic macromolecular assemblies.

Keywords:  PSMD5; TXNL1; UBE3C; in situ mass spectrometry; proteasome

DOI:  https://doi.org/10.1101/2025.07.31.667872
Proc Natl Acad Sci U S A. 2025 Aug 12. 122(32): e2423455122

Autophagy activator AA-20 improves proteostasis and extends Caenorhabditis elegans lifespan.

Ee Phie Tan, Nora Lyang, Saam Doroodian, Pablo Sanz-Martinez, Jin Xu, Sviatlana Zaretski, José L Nieto-Torres, Hiroshi Ebata, Shaun H Y Lim, William C Hou, Khalyd J Clay, Leonard Yoon, Lynée A Massey, Derek Rhoades, Danny Garza, Kristen A Johnson, Alan To, Lydia Ambaye, Emily P Bentley, Michael Petrascheck, Alexandra Stolz, Jeffery W Kelly, Malene Hansen.

  The degradation of cellular components through autophagy is essential for longevity and healthy aging. However, autophagy function decreases with aging, contributing to age-related diseases. In this study, we characterized a small-molecule activator of autophagy called AA-20 that enhances autophagy and lipid droplet clearance in human cells and in the nematode Caenorhabditis elegans. AA-20 reduces polyglutamine aggregation in an autophagy-dependent manner in both human cells and C. elegans, where it also promotes fitness. Consistently, we found that AA-20 extends lifespan in WT C. elegans, but not in autophagy-deficient mutants. Interestingly, our findings suggest that AA-20 acts, at least in part, through a mechanism involving the transcription factor EB, but without inhibiting the protein kinase mammalian target of rapamycin complex 1. Collectively, our results identify an autophagy activator AA-20, which may have potential therapeutic implications for aging-related proteinopathies and lipid storage disorders.

Keywords:  C. elegans; autophagy activator; healthspan; lifespan; lipophagy

DOI:  https://doi.org/10.1073/pnas.2423455122
bioRxiv. 2025 Aug 01. pii: 2025.07.29.667331. [Epub ahead of print]

BBSome-Mediated Clearance of Ubiquitinated IMPG2 Defines a Constitutive Ciliary Retrieval Pathway in Photoreceptors.

Tirthasree Das, Gary A Bradshaw, Jeanette Hyer, Markus Masek, Yien-Ming Kuo, Ruxandra Bachmann-Gagescu, Marian Kalocsay, Maxence V Nachury.

The BBSome mediates the retrieval of ubiquitinated membrane proteins from cilia, but its physiological cargoes in photoreceptors remain largely unidentified. Here, we find that K63-linked ubiquitin (UbK63) chains accumulate in the outer segment (OS, equivalent of cilia) of Bbs4 -/- photoreceptors from the onset of OS formation. Through quantitative profiling of the UbK63-associated OS proteome, we identify the transmembrane fragment of interphotoreceptor matrix proteoglycan 2 (IMPG2 m ) as a principal cargo of the BBSome. In Bbs4 -/- mice, ubiquitinated IMPG2 m aberrantly accumulates in OSs, and disruption of IMPG2 m ubiquitination impairs its retrieval and clearance. Because full-length IMPG2 traffics to the OS to deliver its extracellular domain to the matrix, our data support a model in which IMPG2 m undergoes constitutive cycling between the inner and outer segments. These findings redefine the BBSome's role in photoreceptors from quality control to constitutive membrane protein turnover.

DOI: https://doi.org/10.1101/2025.07.29.667331
bioRxiv. 2025 Aug 01. pii: 2025.07.31.666823. [Epub ahead of print]

A microsporidial deubiquitinase blocks ubiquitin transfer from adenylated E1 to human UBE2K ubiquitin conjugating enzyme.

Tomiwa Lawal, Alana H Chang, Lauren G Carnley, Nehry Y Celoge, Taylor A Blount, Cynthia Vied, Antonia A Nemec, Robert J Tomko.

Intracellular pathogens frequently subjugate the ubiquitin system to evade host immune defenses and establish intracellular replication niches. Microsporidia are obligate intracellular animal parasites that typically cause self-limiting infections in humans, but can sometimes cause fatal disseminated disease. At present, the ubiquitin system of microsporidia is virtually unexplored. Here, we discover a likely effector deubiquitinase (DUB) of the otubain subgroup from the human pathogenic microsporidian Encephalitozoon hellem, which we designate ehOTUB1. We find that ehOTUB1 selectively binds the human ubiquitin conjugating (E2) enzyme UBE2K and inhibits its ubiquitin conjugation activity independent of ehOTUB1 DUB activity. We show that ehOTUB1 obstructs docking of UBE2K onto ubiquitin E1 enzyme via steric conflict with ubiquitin in the E1 adenylation site to prevent ubiquitin transfer to UBE2K. This unconventional mechanism of E2 inhibition expands the known repertoire by which pathogens manipulate ubiquitin signaling, and suggests that direct inhibition of E2 enzymes may be a broader function of otubain subfamily DUBs than originally appreciated.

DOI: https://doi.org/10.1101/2025.07.31.666823
Nat Commun. 2025 Aug 06. 16(1): 7243

A light-resuming strategy as a screening method for selecting Sec61 inhibitors down-modulating PD-L1 expression.

Fulvia Vitale, Gianluca Scerra, Laura Marrone, Anna Di Micco, Magda Cannata Serio, Amarawan Intasiri, Giuseppina Amodio, Vittorio Cirillo, Paolo Remondelli, Antonietta Luongo, Raffaella Bonavita, Maria Gabriella Caporaso, Franck Perez, Maurizio Renna, Thomas W Bell, Simona Romano, Massimo D'Agostino.

The perturbation of protein translocation into the secretory pathway using Sec61 translocon inhibitors is a novel and promising strategy for tackling many pathological situations, including cancer and viral infections. However, a highly sensitive and direct screening platform for selecting Sec61 inhibitors is unavailable. Here, we develop a "resuming luminescence upon translocation interference" (RELITE) assay capable of selecting Sec61 inhibitors in a single round of screening. This assay exploits the inactivation of firefly luciferase, once translocated into the endoplasmic reticulum (ER), and the possibility of diverting and "re-lighting" luciferase into the cytosol by a Sec61 inhibitor. Using this method, we select small molecules capable of hampering the protein expression of the PD-L1 immune checkpoint by interfering with its ER translocation and delivering it for degradation. In conclusion, our screening method will greatly facilitate the selection of Sec61 inhibitors for down-modulating the expression of many disease-relevant proteins.

DOI: https://doi.org/10.1038/s41467-025-62439-w
Nat Struct Mol Biol. 2025 Aug 08.

Structural analyses define the molecular basis of clusterin chaperone function.

Patricia Yuste-Checa, Alonso I Carvajal, Chenchen Mi, Sarah Paatz, F Ulrich Hartl, Andreas Bracher.

Clusterin (apolipoprotein J), a conserved glycoprotein abundant in blood and cerebrospinal fluid, functions as a molecular chaperone and apolipoprotein. Dysregulation of clusterin is linked to late-onset Alzheimer disease. Despite its prominent role in extracellular proteostasis, the mechanism of clusterin function remained unclear. Here, we present crystal structures of human clusterin, revealing a discontinuous three-domain architecture. Structure-based mutational analysis demonstrated that two disordered, hydrophobic peptide tails enable diverse activities. Resembling the substrate-binding regions of small heat-shock proteins, these sequences mediate clusterin's chaperone function in suppressing amyloid-β, tau and α-synuclein aggregation. In conjunction with conserved surface areas, the tail segments also participate in clusterin binding to cell surface receptors and cellular uptake. While contributing to lipoprotein formation, the hydrophobic tails remain accessible for chaperone function in the lipoprotein complex. The remarkable versatility of these sequences allows clusterin to function alone or bound to lipids in maintaining the solubility of aberrant extracellular proteins and facilitating their clearance by endocytosis and lysosomal degradation.

DOI: https://doi.org/10.1038/s41594-025-01631-4
Sci Adv. 2025 Aug 08. 11(32): eadt8974

Non-native entanglement protein misfolding observed in all-atom simulations and supported by experimental structural ensembles.

Quyen V Vu, Ian Sitarik, Yang Jiang, Yingzi Xia, Piyoosh Sharma, Divya Yadav, Hyebin Song, Mai Suan Li, Stephen D Fried, Edward P O'Brien.

Several mechanisms are known to cause monomeric protein misfolding. Coarse-grained simulations have predicted an additional mechanism exists involving off-pathway, noncovalent lasso entanglements, which are long-lived kinetic traps and structurally resemble the native state. Here, we examine whether such misfolded states occur in long-timescale, all-atom folding simulations of ubiquitin and λ-repressor. We find that these entangled misfolded states are populated in higher-resolution models. However, because of the small size of ubiquitin and λ-repressor, these states are short-lived. In contrast, coarse-grained simulations of a larger protein, IspE, predict that it populates long-lived misfolded states. Using an Arrhenius extrapolation applied to all-atom simulations, we estimate that these IspE misfolded states have lifetimes similar to the native state while remaining soluble. We further show that these misfolded states are consistent with the structural changes inferred from limited proteolysis and cross-linking mass spectrometry experiments. Our results indicate that misfolded states composed of non-native entanglements can persist for long timescales in both all-atom simulations and experiments.

DOI: https://doi.org/10.1126/sciadv.adt8974
Alzheimers Dement. 2025 Aug;21(8): e70497

Diabetic retinopathy and Alzheimer's disease: Convergence of the unfolded protein response in neurodegeneration.

Adrián G Palacios, Sarah X Zhang, Mónica L Acosta.

  Diabetic retinopathy (DR) and Alzheimer's disease (AD) are progressive neurodegenerative disorders affecting the eye and the brain, respectively. Despite targeting different organs, they share common molecular mechanisms. A central process connecting these conditions is the unfolded protein response (UPR), which maintains protein homeostasis in the endoplasmic reticulum (ER). Dysregulation of UPR pathways, particularly the IRE1-XBP1 and PERK-eIF2α pathways, can lead to inflammation, oxidative stress, and neurodegeneration. While the IRE1-XBP1 pathway regulates protein folding and inflammatory signaling, the PERK-eIF2α pathway reduces protein synthesis but may trigger apoptosis if persistently activated. Emerging therapies targeting UPR pathways and ER chaperones show promise in mitigating neurodegenerative damage in DR and AD. This review highlights shared pathophysiological mechanisms, explore retinal biomarkers for early AD detection, and emphasizes UPR modulation as a therapeutic strategy for neurodegeneration in aging-related diseases. HIGHLIGHTS: Diabetic retinopathy (DR, ocular disorder) and Alzheimer's disease (AD, cerebral disorder) share common molecular mechanisms, including oxidative stress, inflammation, and proteostasis dysfunction. UPR is a critical pathway linking both diseases through endoplasmic reticulum (ER) stress and neurodegeneration and targeting unfolded protein response (UPR) pathways, ER chaperones (e.g., P58IPK), and anti-inflammatory treatments show promise. The IRE1-XBP1 pathway regulates protein homeostasis and inflammation; XBP1s protects against ER stress in both retinal and brain neurons. The PERK-eIF2α pathway suppresses protein synthesis under stress but may induce apoptosis via ATF4 and CHOP if chronically activated. Age-related decline in metabolism, proteostasis, and neurovascular function intensifies disease progression and exacerbates molecular and cellular damage in both DR and AD.

Keywords:  Alzheimer's disease; aging; diabetes; retina; unfolded protein response

DOI:  https://doi.org/10.1002/alz.70497
PLoS Genet. 2025 Aug 07. 21(8): e1011823

Natural SEL1L variants rescue a model of NGLY1 deficiency and modify ERAD function and proteasome sensitivity.

Travis K Tu'ifua, Clement Y Chow.

N-glycanase 1 (NGLY1) deficiency is an ultra-rare disease caused by autosomal recessive loss-of-function mutations in the NGLY1 gene. NGLY1 removes N-linked glycans from glycoproteins in the cytoplasm and is thought to help clear misfolded proteins from the endoplasmic reticulum (ER) through the ER associated degradation (ERAD) pathway. Despite this, the physiological significance of NGLY1 in ERAD is not understood. The best characterized substrate of NGLY1 is NRF1, a transcription factor that upregulates proteasome expression and the proteasome bounce-back response. We previously performed a genetic modifier screen using a Drosophila model of NGLY1 deficiency and identified potential modifiers that alter the lethality of the model. We identified two protein-coding variants in Hrd3/SEL1L: S780P and Δ806-809. Both variants are localized to the SEL1L cytoplasmic tail, an uncharacterized domain. SEL1L is a component of the ERAD complex that retrotranslocates misfolded proteins from the ER to the cytoplasm for degradation. We used CRISPR to generate fly lines carrying these SEL1L variants in a common genetic background and tested them with our model of NGLY1 deficiency. Validating our previous screen, the SEL1LS780P and SEL1LΔ806-809 variants increased the survival of the NGLY1 deficiency model, compared to the SEL1LS780 variant. To determine how these SEL1L variants were modifying lethality in NGLY1 deficiency, we interrogated the ERAD and NRF1 signaling pathways. We found that the SEL1LS780P and SEL1LΔ806-809 variants improve resistance to ER stress, with enhanced ERAD function as a likely contributing mechanism. This effect depends on NGLY1 activity, further implicating NGLY1 in general ERAD function. We also found that, in heterozygous NGLY1 null flies, these variants protect against some defects like increased lethality caused by proteasome inhibition. These results provide new insights into the role of SEL1L in the disease pathogenesis of NGLY1 deficiency. SEL1L is a strong candidate modifier gene in patients, where variability in presentation is common.

DOI: https://doi.org/10.1371/journal.pgen.1011823
bioRxiv. 2025 Jul 29. pii: 2025.07.28.667051. [Epub ahead of print]

Saturated lipid stress attenuates mitochondrial genome synthesis in human cells.

Casadora Boone, Sophie Judge, Ahmad Shami, Bezawit Danna, Andrea B Ball, Tejashree Pradip Waingankar, Hera Saqub, Ajit S Divakaruni, Samantha C Lewis.

Fatty acids are trafficked between organelles to support membrane biogenesis and act as signaling molecules to rewire cellular metabolism in response to starvation, overnutrition, and environmental cues. Mitochondria are key cellular energy converters that harbor their own multi-copy genome critical to metabolic control. In homeostasis, mitochondrial DNA (mtDNA) synthesis is coupled to mitochondrial membrane expansion and division at sites of contact with the endoplasmic reticulum (ER). Here, we provide evidence from cultured hepatocytes that mtDNA synthesis and lipid droplet biogenesis occur at spatially and functionally distinct ER-mitochondria membrane contact sites. We find that, during saturated lipid stress, cells pause mtDNA synthesis and mitochondrial network expansion secondary to rerouted fatty acid trafficking through the ER and lipid droplet biogenesis, coincident with a defect in soluble protein import to the ER lumen. The relative composition of fatty acid pools available to cells is critical, as monounsaturated fatty acid supplementation rescued both ER proteostasis and mtDNA synthesis, even in the presence of excess saturated fat. We propose that shutoff of mtDNA synthesis conserves mtDNA-to-mitochondrial network scaling until cells can regain ER homeostasis.
Summary: Overnutrition of cultured human cells causes endoplasmic reticulum dysfunction, which downregulates mitobiogenesis in turn by constraining mtDNA synthesis.

DOI: https://doi.org/10.1101/2025.07.28.667051
J Biol Chem. 2025 Aug 05. pii: S0021-9258(25)02413-5. [Epub ahead of print] 110562

A Three-Stage Assembly Program Governing Pancreatic, Plasma, Pituitary, and Bone Secretory Cell Differentiation: A Strategy to Augment Protein Delivery.

Joseph P Bidwell, Alexander G Robling, Ronald C Wek.

  Defective secretory cell function underlies many diseases, and recent therapeutic strategies have focused on enhancing protein synthesis and delivery by targeting the secretory machinery of mature cells. However, mature differentiated cells appear to have intrinsic limits to their secretory capacity. In this review, we propose new strategies for engineering these cells to overcome these limits on secretion. The integrated stress response (ISR) and the related unfolded protein response (UPR) are stress adaptation systems that modulate transcriptional and translational programs of gene expression. These programs drive remodeling of cellular architecture to boost protein production and trafficking but also play critical roles in the differentiation of secretory cells. This dual function suggests that the limits of the secretory capacity of mature cells are pre-programmed during development. A potentially more effective therapeutic approach to expand protein secretion may lie in reprogramming the secretory capacity early in differentiation. Two additional transcriptional programs work in concert with the ISR and UPR to shape differentiated cell identities, their secretory outputs, and production capacity. The first involves lineage-determining transcription factors that define both cell type and secretory products. The second involves 'scaling factors' that set the magnitude of the cell's protein synthesis and secretion capacity. We explore the mechanisms by which these three programs-lineage specification, scaling, and stress adaptation-interact to define and potentially enhance secretory capacity. We will illustrate this integrated model across several secretory cell types, including pancreatic, plasma, pituitary, and bone secretory cells, with emphasis on applications to improve therapeutic outcomes in osteoporosis.

Keywords:  Integrated stress response; lineage-determining factor; scaling factor; unfolded protein response

DOI:  https://doi.org/10.1016/j.jbc.2025.110562
Nat Struct Mol Biol. 2025 Aug 07.

Establishment of the phagophore-ERES membrane contact site initiates phagophore elongation.

Rubén Gómez-Sánchez, Sabrina Chumpen Ramirez, Prado Vargas Duarte, Yan Hu, Muriel Mari, Katharina Olschewski, Ralph Hardenberg, J Christopher Fromme, Christian Ungermann, Fulvio Reggiori.

The de novo generation of membrane contact sites (MCSs) between the phagophore and the endoplasmic reticulum exit sites (ERES) is important for the acquisition of the lipids necessary for phagophore elongation and autophagosome formation during autophagy. However, it is currently unclear how these MCSs are established. Here, we show that the TRAPPIII complex, the guanine nucleotide exchange factor of the Rab GTPase Ypt1, localizes to and regulates the formation of the MCS between the phagophore and the ERES. In particular, TRAPPIII and the lipid transfer protein Atg2 appear equally essential for the association of the phagophore with the ERES, TRAPPIII activation and Ypt1 activation onto the phagophore. Ypt1 redistributes over the entire surface of the phagophore and promotes its elongation through both stimulation of the local biosynthesis of phosphatidylinositol-3-phosphate and recruitment of the downstream effectors Atg18 and Atg21. Our data suggest that de novo generation of the phagophore-ER MCSs and subsequent Ypt1 activation initiates phagophore elongation.

DOI: https://doi.org/10.1038/s41594-025-01621-6
Cell. 2025 Aug 01. pii: S0092-8674(25)00801-3. [Epub ahead of print]

A mechano-resistance mechanism in skin adapts to terrestrial locomotion.

Ruonan Di, Qianqian Du, Yuhua Xie, Yanhua Lu, Wenxuan Gao, Lei Zhang, Xiaoli Qi, Yanyan Fan, Jiao Li, Fengchao Wang, She Chen, Ting Chen.

  The transition from water to land required animals to evolve specialized paw skin to support body weight and enable locomotion. We identify an evolutionarily emerged mechanism in skin epithelial cells that adapts to this mechanical demand. We show that the Slurp1 gene, conserved across tetrapods, is specifically expressed in palmoplantar skin. In humans, mutations in SLURP1 cause palmoplantar keratoderma (PPK), a condition marked by pathologically thickened skin epidermis on the soles and palms. Remarkably, reducing mechanical pressure on Slurp1 knockout paw skin fully rescues the PPK phenotype. Mechanistically, SLURP1 localizes to the endoplasmic reticulum (ER) membrane, where it binds the calcium pump SERCA2b. By preserving SERCA2b activity under mechanical pressure, SLURP1 maintains low cytoplasmic calcium levels and inhibits pressure-induced activation of the pPERK-NRF2 signaling-a pathway that can be genetically targeted to reverse PPK. These findings reveal an ER-based mechano-resistance mechanism that enhances cellular defense against prolonged mechanical pressure.

Keywords:  SERCA2b; SLURP1; calcium; endoplasmic reticulum; mechanical pressure; palmoplantar keratoderma

DOI:  https://doi.org/10.1016/j.cell.2025.07.012
Autophagy. 2025 Aug 03. 1-2

PDZD8 links organelle crosstalk to synaptic remodeling via autophagy.

Rajan S Thakur, Kate M O'Connor-Giles.

  Synapse formation and plasticity require coordinating cellular processes from signaling to protein turnover over long distances, placing high demands on intracellular communication. Membrane contact sites (MCSs) between organelles are specialized compartments for coordinating cellular processes, yet their functions in the developing nervous system remain poorly understood. Through an in vivo CRISPR screen in Drosophila, we identified the conserved endoplasmic reticulum (ER) MCS tethering protein Pdzd8 as a regulator of activity-dependent synapse development. Our in vivo studies demonstrate that Pdzd8 functions at ER-late endosome/lysosome MCSs to promote lysosomal maturation and increase autophagic flux during periods of high demand such as prolonged neuronal activity.

Keywords:  Autophagy; PDZD8; lipid transfer; lysosomes; membrane contact sites; synapse

DOI:  https://doi.org/10.1080/15548627.2025.2537983
bioRxiv. 2025 Jul 21. pii: 2025.07.18.665572. [Epub ahead of print]

RNase L regulates the antiviral proteome by accelerating mRNA decay, inhibiting nuclear mRNA export, and repressing RNAPII-mediated transcription.

J Monty Watkins, Cameron J Douglas, Renee Cusic, Ciaran P Seath, James M Burke.

Ribonuclease L (RNase L) is an antiviral endoribonuclease that triggers widespread degradation of cellular mRNAs. Here, we show that the degradation of cellular mRNA by RNase L is a conserved response to flaviviruses, including Zika virus (ZIKV), dengue virus serotype 2 (DENV-2), and West Nile virus (WNV). Quantitative mass spectrometry in response to dsRNA or ZIKV infection shows that RNase L broadly downregulates the cellular proteome, reducing proteins with short half-lives involved in cell cycle progression, cellular metabolism, and protein synthesis. The mRNAs encoded by interferon-stimulated genes (ISGs) evade mRNA decay by RNase L, allowing for protein synthesis of ISG-encoding mRNAs. However, RNase L dampens ISG protein synthesis by triggering a block in nuclear mRNA export and repressing RNAPII-mediated transcription at later times during the antiviral response. These findings implicate reprograming of the cellular proteome as primary means by which RNase L combats viral infection, tumorigenesis, and immune dysregulation.
HIGHLIGHTS: RNase L initiates widespread decay of cellular mRNA in response to flavivirusesRNase L-mediated mRNA decay broadly downregulates the homoeostatic proteomeAntiviral mRNAs are translated due to their ability to evade RNase L-mediated decayRNase L reduces antiviral proteins by inhibiting mRNA export and RNAPII-mediated transcription.

DOI: https://doi.org/10.1101/2025.07.18.665572
J Cell Biol. 2025 Oct 06. pii: e202501007. [Epub ahead of print]224(10):

SOD1 is delivered to lysosomes via autophagy to maintain lysosomal function and integrity.

Yanzhe Zheng, Meng Li, Xuelin Chen, Ze Zheng, Zixuan Chen, Ruilin Tian, Yan G Zhao.

The gene encoding superoxide dismutase 1 (SOD1) is often mutated in familial amyotrophic lateral sclerosis (ALS), affecting motor neurons. Compared with ALS-associated mutant SOD1, the function of WT SOD1 is less explored. We demonstrate that during starvation, WT and mutant SOD1 are transported into lysosomes. Genome-wide CRISPR interference (CRISPRi) screening identified autophagy-related proteins and the autophagic receptor TP53INP1 as key mediators. TP53INP1 binds ATG8 family proteins, preferentially LC3C, and directly interacts with SOD1. Within lysosomes, SOD1 retains its enzymatic activity. Starvation induces elevated levels of lysosomal reactive oxygen species (ROS), which are further increased by knocking down SOD1 or TP53INP1. Lysosomal degradation activities and membrane integrity are also compromised in the absence of SOD1 or TP53INP1. We reveal a novel function of SOD1 in maintaining lysosomal activity and integrity, and a previously unrecognized role of autophagy in delivering cytosolic enzymes into lysosomes for catalytic purposes, rather than for degradation.

DOI: https://doi.org/10.1083/jcb.202501007
J Am Chem Soc. 2025 Aug 02.

A Charge-Driven Strategy for Covalent Modification and Modulation of Biomolecular Condensates.

Zhewei Chen, Lu Liu, Jerome Cattin, Tuomas P J Knowles, Gonçalo J L Bernardes.

Small-molecule modulation of biomolecular condensates has emerged as a novel and attractive therapeutic modality. Increasing evidence implicates dysregulated condensate formation in neurodegenerative diseases and cancer. However, the proteins that mediate condensate formation are typically difficult to drug directly with small molecules. Here, we present a charge-driven strategy and demonstrate its implementation on Ras GTPase-activating protein-binding protein 1 (G3BP1) to inhibit G3BP1-mediated stress granule (SG) formation. Small-molecule SG inhibitors were developed from the carbonylacrylic amide covalent functionality and were used to modify the folded domain of G3BP1 with surface charges, leading to an alteration of the conformational dynamics of intrinsically disordered regions. Cellular experiments using HeLa cells expressing cysteine-mutated G3BP1, together with structure-activity relationship studies, support the proposed charge-driven mechanism of action. Molecular dynamics simulations further suggest that the small-molecule G3BP1 modification promotes a shift toward more compact conformations, comparable to that induced by an ∼26% increase in IDR1-IDR3 interaction. Together, our findings establish a new strategy for the rational modulation of biomolecular condensates.

DOI: https://doi.org/10.1021/jacs.5c06625
Proc Natl Acad Sci U S A. 2025 Aug 12. 122(32): e2425621122

The E3 ligase HECTD4 regulates COX-2-dependent tumor progression and metastasis.

Joanna A Vuille, Cem Tanriover, Ezgi Antmen, Douglas S Micalizzi, Richard Y Ebright, Sambhavi Animesh, Robert Morris, Soroush Hajizadeh, Zachary J Nicholson, Hunter C Russell, Eric F Zaniewski, Ben S Wittner, Ben K Wesley, Ji Eun Kwak, Julian Grünewald, Regan N Szalay, Douglas B Fox, Min Yang, J Keith Joung, Doga C Gulhan, Andrew E H Elia, Wilhelm Haas, Eugene Oh, Shyamala Maheswaran, Daniel A Haber.

  E3 ubiquitin ligases mediating turnover of proteins engaged in cancer progression point to key regulatory nodes. To uncover modifiers of metastatic competency, we conducted an in vivo genome-wide CRISPR-inactivation screen using cultured breast circulating tumor cells, following intravascular seeding and lung colonization. We identified HECTD4, a previously uncharacterized gene encoding a conserved potential homologous to E6AP C-terminus domain-containing ubiquitin transferase, as a potent tumor and metastasis suppressor. We show that purified HECTD4 mediates ubiquitin conjugation in vitro, and proteomic studies combined with ubiquitin remnant profiling identify a major degradation target as the prostaglandin synthetic enzyme cyclooxygenase-2 (COX-2; PTGS2). In addition to COX-2 itself, HECTD4 targets its regulatory kinase MKK7. In breast cancer models, HECTD4 expression is induced as cells lose adherence to the matrix, and its depletion massively increases COX-2 expression, enhancing anchorage-independent proliferation and tumorigenesis. Genetic or pharmacologic suppression of COX-2 reverses the protumorigenic and prometastatic phenotype of HECTD4-depleted cells. Thus, HECTD4 encodes an E3 ubiquitin ligase that downregulates COX-2 suppressing anchorage independence in epithelial cancer cells.

Keywords:  anchorage independence; breast cancer; metastasis suppressor genes; tumor suppressor gene; ubiquitination

DOI:  https://doi.org/10.1073/pnas.2425621122
Nat Rev Mol Cell Biol. 2025 Aug 04.

Lysosomal membrane homeostasis and its importance in physiology and disease.

Maja Radulovic, Chonglin Yang, Harald Stenmark.

Lysosomes are membranous organelles that are crucial for cell function and organ physiology. Serving as the terminal stations of the endocytic pathway, lysosomes have fundamental roles in the degradation of endogenous and exogenous macromolecules and particles as well as damaged or superfluous organelles. Moreover, the lysosomal membrane is a docking and activation platform for several signalling components, including mTOR complex 1 (mTORC1), which orchestrates metabolic signalling in the cell. The integrity of their membrane is crucial for lysosomes to function as hubs for the regulation of cell metabolism. Various agents, including pathogens, nanoparticles and drugs, can compromise lysosomal membrane integrity. Membrane permeabilization causes leakage of proteases and cations into the cytosol, which can induce cell death pathways and innate immunity signalling. Multiple pathways repair damaged lysosomes, and severely damaged lysosomes are degraded by an autophagic process, lysophagy. Moreover, lysosome damage activates transcriptional programmes that orchestrate lysosome biogenesis to replenish the cellular lysosome pool. In this Review, we discuss recent insights into the mechanisms that ensure the maintenance of lysosomal membrane homeostasis, including novel mechanisms of lysosomal membrane repair and the interplay between lysosome damage, repair, lysophagy and lysosome biogenesis. We highlight the importance of lysosomal membrane homeostasis in cell function, physiology, disease and ageing, and discuss the potential for therapeutic exploitation of lysosomal membrane permeabilization.

DOI: https://doi.org/10.1038/s41580-025-00873-w
bioRxiv. 2025 Aug 02. pii: 2025.06.10.657506. [Epub ahead of print]

Decoding Cellular Stress States for Toxicology Using Single-Cell Transcriptomics.

Imran Shah, David Gallegos, Brian Robinette, Bryant A Chambers, Dennis Eastburn, Douglas A Bell, Michelle R Campbell, Suzanne N Martos, Salvatore Camiolo, Kevin White, Nicole Martin, Gioele Montis, Joel McComb, Bruce Seligmann, Brian N Chorley.

We applied the TempO-LINC® platform to generate single-cell transcriptomic (SCTr) profiles of ~40,000 HepaRG cells exposed to etoposide, brefeldin A, cycloheximide, rotenone, tBHQ, troglitazone, and tunicamycin at three concentrations for 24 hours. SCTr enabled a detailed analysis of adaptive stress response pathways (SRPs), including the unfolded protein response (UPR), oxidative stress response (OSR), heat shock response (HSR), and DNA damage response (DDR). Troglitazone upregulated lipid metabolism genes (PLIN2, ACOX1) along with HSR and UPR activation, with co-expression of DNAJA1, HSP90AA1, and DDIT3 in subsets of cells. Brefeldin A and tunicamycin strongly induced UPR markers (HSPA5, SYVN1, LMF2, PDIA4) in subsets of cells, with some also expressing apoptotic (DDIT3, CASP8) and autophagic (SQSTM1) genes, indicating diverse stress responses. Rotenone activated GDF15, TRIB3, and DDIT3 in a fraction of cells, accompanied by PLIN2 and mild UPR induction, reflecting heterogeneous mitochondrial stress responses. We scored individual cells using literature-derived SRP gene signatures to characterize overall stress phenotypes and clustered them using a generalized Jaccard metric. The clustering revealed five phenotypic groups spanning cell states associated with homeostasis, adaptive responses, terminal outcomes, autophagy, and apoptosis. By systematically analyzing the distributions of cells in different states across treatments, we visualized dynamic shifts in cellular subpopulations responding to chemicals, revealing early stress responses and potential transitions to cell death. Our findings suggest the utility of SCTr in decoding stress states that could provide possible insights into transitions between cellular adaptive and terminal transitions involved in toxicity.

DOI: https://doi.org/10.1101/2025.06.10.657506
bioRxiv. 2025 Jul 25. pii: 2025.07.21.662073. [Epub ahead of print]

Structural Plasticity of the Membrane-Bound Protein Degradation Assembly Supports Bacterial Adaptation to Stress.

Naseer Iqbal, Sandro Keller, Alireza Ghanbarpour.

Protein degradation by AAA+ proteases is essential for bacterial adaptation to environmental stress. The membrane-bound AAA+ protease FtsH forms a large inner-membrane complex with the SPFH (Stomatin, Prohibitin, Flotillin, HflK/C) family transmembrane proteins HflK and HflC, playing a key role in bacterial recovery from aminoglycoside antibiotic stress. Recent structural studies have revealed both open, asymmetric and closed, symmetric conformations of the HflK/C assembly under different sample-preparation conditions, suggesting two distinct models for how this complex modulates FtsH proteolysis. To determine which conformation reflects the biologically active state, we engineered a disulfide-crosslinked HflK/C variant to stabilize the closed conformation and resolved its structure using high-resolution cryo-EM. Phenotypic assays showed that cells expressing either this stabilized, closed HflK/C variant or an HflK/C mutant that disrupts interactions with FtsH exhibit significantly impaired growth under aminoglycoside stress. Surprisingly, the cryo-EM structure of the FtsH•HflK/C complex from cells challenged with the aminoglycoside antibiotic tobramycin revealed a novel HflK/C arrangement, characterized by two openings on opposite sides that may facilitate substrate access to FtsH during proteotoxic stress. Together, our results suggest that both the dynamic open conformation of HflK/C and its specific interactions with FtsH are critical for adaptation to aminoglycoside-induced stress. Given the conserved structural and functional features of SPFH family members, our findings may offer a broader framework for understanding how this protein family operates under both basal and stress conditions.

DOI: https://doi.org/10.1101/2025.07.21.662073
bioRxiv. 2025 Jul 28. pii: 2025.07.17.665435. [Epub ahead of print]

DirectContacts2: A network of direct physical protein interactions derived from high-throughput mass spectrometry experiments.

Erin R Claussen, Miles D Woodcock-Girard, Samantha N Fischer, Kevin Drew.

Cellular function is driven by the activity proteins in stable complexes. Protein complex assembly depends on the direct physical association of component proteins. Advances in macromolecular structure prediction with tools like AlphaFold and RoseTTAFold have greatly improved our ability to model these interactions in silico, but an all-by-all analysis of the human proteome's ∼200M possible pairs remains computationally intractable. A comprehensive cellular map of direct protein interactions will therefore be an invaluable resource to direct screening efforts. Here, we present DirectContacts2 , a machine learning model that distinguishes direct from indirect protein interactions using features derived from over 25,000 mass spectrometry experiments. Applied to ∼26 million human protein pairs, our model outperforms previous resources in identifying direct physical interactions and enriches for accurate structural models including ∼2,500 new AlphaFold3 models. Our framework enables structural modeling of disease-relevant complexes (e.g. orofacial digital syndrome (OFDS) complex) offering insights into the molecular consequences of pathogenic mutations (OFD1) and broadly, establishes a highly accurate protein wiring diagram of the cell.

DOI: https://doi.org/10.1101/2025.07.17.665435