bims-proteo 2025-10-05 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2025–10–05
forty-five papers selected by
Eric Chevet, INSERM

From serendipity to strategy: molecular glue degraders in cancer therapeutics.
Frustration in the protein-protein interface plays a central role in the cooperativity of PROTAC ternary complexes.
Hsp104-Hsp70-Hsp110 chaperones disintegrate kinase aggregates formed upon stress in Saccharomyces cerevisiae.
Nuclear and cytosolic J-domain proteins provide synergistic control of Hsf1 at distinct phases of the heat shock response.
The mechanistic basis of cargo selection during Golgi maturation.
RNF114 and RNF166 exemplify reader-writer E3 ligases that extend K11 polyubiquitin onto sites of MARUbylation.
Translation landscape of stress granules.
Towards a unified framework for the function of endoplasmic reticulum exit sites.
A FMRF-amide peptide that regulates cell non-autonomous protein homeostasis in C. elegans.
GCN1 couples GCN2 to ribosomal state to initiate amino acid response pathway signaling.
Proteostasis of immune checkpoint receptors.
Transcriptome-wide mapping of small ribosomal subunits elucidates scanning mechanisms of translation initiation in the mammalian brain.
ER stress tolerance is regulated by copper-dependent PERK kinase activity.
USP9X is a mechanosensitive deubiquitinase that controls tumor cell invasiveness and drug response through YAP stabilization.
Systematic mRNA interactome analysis reconceptualizes translational quiescence in bovine sperm.
Autophagy-dependent proteostasis suppresses breast cancer metastasis.
Expression of IMPACT Curtails Metabolic Plasticity and Augments NK Cell Killing to Abrogate Metastatic Growth.
CRISPR Screen Identifies BAP1 as a Deubiquitinase Regulating SPIN4 Stability.
Codon-specific ribosome stalling reshapes translational dynamics during branched-chain amino acid starvation.
Mitochondrial ROS triggers mitophagy through activating the DNA damage response signaling pathway.
Allosteric control of the bacterial ClpC/ClpP protease and its hijacking by antibacterial peptides.
Identification of the client-binding site on the Golgi membrane protein adaptor Vps74/yGOLPH3.
Expansion of lysosomal capacity in early adult neurons driven by TFEB/HLH-30 protects dendrite maintenance during aging in Caenorhabditis elegans.
Cytosolic fMet-Protein Synthesis as Source of Endogenous Ligands for Formyl Peptide Receptors.
Proteotoxic stress response drives T cell exhaustion and immune evasion.
Muscular dystrophy-associated lamin variants disrupt cellular organization through a nucleolar-ribosomal axis controlling cytoplasmic macromolecular crowding.
Synthetic protein degradation circuits using programmable cleavage and ligation by Sortase A.
Bacterial RNA promotes proteostasis through inter-tissue communication in C. elegans.
Mapping cryptic phosphorylation sites in the human proteome.
ER stress sensor PERK promotes T-cell pathogenicity in GVHD by regulating ER-associated degradation.
Interconnectivity of mitochondrial protein biogenesis and quality control.
Mitochondrial ROS trigger interorganellular signaling and prime ER processes to establish enhanced plant immunity.
All-at-once RNA folding with 3D motif prediction framed by evolutionary information.
Visualization of liquid-liquid phase transitions using a tiny G-quadruplex binding protein.
Mechanisms of Messenger RNA Packaging and Export.
Missense mutations in intrinsically disordered protein regions link pathogenicity and phase separation.
Nanoscale profiling of evolving intermolecular interactions in ageing FUS condensates.
Dual membrane receptor degradation via folate receptor targeting chimera.
Sequencing-free whole-genome spatial transcriptomics at single-molecule resolution.
Integrated multi-dimensional comparison of proteomic profiles across 54 cancer cell lines.
DrugDomain 2.0: Comprehensive database of protein domains-ligands/drugs interactions across the whole Protein Data Bank.
In situ secondary structure imaging of protein phase separation and aggregation by hyperspectral stimulated Raman scattering microscopy.
How SecB maintains clients in a translocation competent state.
Pioglitazone Reduces Hepatic Alpha-1 Antitrypsin Accumulation Through Autophagy and AMPK Activation in Alpha-1 Antitrypsin Deficient Mice.
Holding Out for a Model: Rhomboid Superfamily in Vertebrate Development and Disease.

Crit Rev Biochem Mol Biol. 2025 Oct 01. 1-31

From serendipity to strategy: molecular glue degraders in cancer therapeutics.

Sarah Darling, Ilirjana Bajrami, Stephen C West.

  Targeted protein degradation is an elegant therapeutic strategy that harnesses the cell's own degradation machinery to selectively eliminate target proteins. This approach marks a paradigm shift in drug discovery, moving beyond traditional occupancy-based inhibition toward target degradation, thereby silencing proteins that have historically resisted pharmacological intervention. Degrader molecules function by inducing proximity between target proteins and effectors, most commonly E3 ubiquitin ligases, triggering their ubiquitylation and proteasomal degradation. Molecular glue degraders - monovalent small molecules that promote these neo-interactions - have emerged as powerful tools in this space. Serendipity was once synonymous with molecular glue discovery, but increasing mechanistic understanding is now guiding their rational design. In this review, we trace their evolution from chance discovery, explore the biological mechanisms that underpin molecular glue activity, examine key examples that have advanced into the clinic, and discuss the challenges that remain in harnessing these compounds for broader therapeutic impact.

Keywords:  CRBN; E3 ligase; PROTACs; Targeted protein degradation; degraders; induced proximity; molecular glues; neo-substrates

DOI:  https://doi.org/10.1080/10409238.2025.2564068
Nat Commun. 2025 Sep 29. 16(1): 8595

Frustration in the protein-protein interface plays a central role in the cooperativity of PROTAC ternary complexes.

Ning Ma, Supriyo Bhattacharya, Sanychen Muk, Zuzana Jandova, Philipp S Schmalhorst, Soumadwip Ghosh, Keith Le, Emelyne Diers, Nicole Trainor, William Farnaby, Michael J Roy, Christiane Kofink, Peter Greb, Harald Weinstabl, Alessio Ciulli, Gerd Bader, Kyra Sankar, Andreas Bergner, Nagarajan Vaidehi.

Targeted protein degradation using proteolysis-targeting chimeras (PROTACs) offers a promising strategy to eliminate previously undruggable proteins. PROTACs are bifunctional molecules that link a target protein with an E3 ubiquitin ligase, enabling the formation of a ternary complex that promotes ubiquitination and subsequent proteasomal degradation. Although many ternary complex structures are available, understanding how structural features relate to PROTAC function remains challenging due to the dynamic nature of these complexes. Here we show that the interface between the target protein SMARCA2 and the E3 ligase VHL is conformationally flexible and stabilized by interactions involving disordered loops. Using molecular dynamics simulations and X-ray crystallography of SMARCA2-VHL complexes bound to five different PROTACs, we find that interfacial residues often adopt energetically suboptimal, or 'frustrated,' configurations. We further show that the degree of frustration correlates with experimentally measured cooperativity for a set of 11 PROTACs. These findings suggest that quantifying interface frustration provides a rational, structure-based approach to guiding PROTAC design.

DOI: https://doi.org/10.1038/s41467-025-63713-7
FEBS Lett. 2025 Sep 29.

Hsp104-Hsp70-Hsp110 chaperones disintegrate kinase aggregates formed upon stress in Saccharomyces cerevisiae.

Pramit Bhattacharjee, Atin K Mandal.

  The cellular protein quality control (PQC) machinery maintains proteostasis. However, knowledge of PQC machinery-mediated handling of stress-induced misfolded proteins is still insufficient. We used the yeast kinase Ste11 to observe its fate upon heat stress or Hsp90 inhibition. We observed that while mild heat stress (37 °C) primarily resulted in proteasomal degradation of Ste11, severe heat stress (42 °C) resulted predominantly in aggregation. Ste11 aggregates sequestered with Hsp42 upon heat stress or Hsp90 inhibition. These aggregates associate with Hsp70 and Hsp104, the yeast disaggregase machinery. Notably, Ste11 aggregates disappear upon recovery from stress. This phenomenon is impaired in the absence of Hsp104 or Sse1, a co-chaperone recruited to the aggregates by Hsp70, suggesting the involvement of Hsp104, Hsp70 and Sse1 in aggregate mobilisation.

Keywords:  disintegration; kinase; molecular chaperones; protein quality control; spatial sequestration; ubiquitin proteasome system

DOI:  https://doi.org/10.1002/1873-3468.70174
Elife. 2025 Sep 30. pii: RP107157. [Epub ahead of print]14

Nuclear and cytosolic J-domain proteins provide synergistic control of Hsf1 at distinct phases of the heat shock response.

Carmen Ruger-Herreros, Lucia Svoboda, Gurranna Male, Aseem Shrivastava, Markus Höpfler, Katharina Jetzinger, Jiří Koubek, Günter Kramer, Fabian den Brave, Axel Mogk, David S Gross, Bernd Bukau.

  The heat shock response (HSR) is the major defense mechanism against proteotoxic stress in the cytosol and nucleus of eukaryotic cells. Initiation and attenuation of the response are mediated by stress-dependent regulation of heat shock transcription factors (HSFs). Saccharomyces cerevisiae encodes a single HSF (Hsf1), facilitating the analysis of HSR regulation. Hsf1 is repressed by Hsp70 chaperones under non-stress conditions and becomes activated under proteotoxic stress, directly linking protein damage and its repair to the HSR. J-domain proteins (JDPs) are essential for targeting of Hsp70s to their substrates, yet the specific JDP(s) regulating Hsf1 and connecting protein damage to HSR activation remain unclear. Here, we show that the yeast nuclear JDP Apj1 primarily controls the attenuation phase of the HSR by promoting Hsf1's displacement from heat shock elements in target DNA. In apj1Δ cells, HSR attenuation is significantly impaired. Additionally, yeast cells lacking both Apj1 and the major JDP Ydj1 exhibit increased HSR activation even in non-stress conditions, indicating their distinct regulatory roles. Apj1's role in both nuclear protein quality control and Hsf1 regulation underscores its role in directly linking nuclear proteostasis to HSR regulation. Together, these findings establish the nucleus as key stress-sensing signaling hub.

Keywords:  Hsf1; Hsp70; J-protein; S. cerevisiae; chaperone; chromosomes; gene expression; heat shock response

DOI:  https://doi.org/10.7554/eLife.107157
Sci Adv. 2025 Oct 03. 11(40): eaea0016

The mechanistic basis of cargo selection during Golgi maturation.

Rebecca J Taylor, Nikita Zubkov, Katarzyna A Ciazynska, Jonathan G G Kaufman, Grigory Tagiltsev, David J Owen, John A G Briggs, Sean Munro.

The multiple cisternae of the Golgi apparatus contain resident membrane proteins crucial for lipid and protein glycosylation. How Golgi residents remain in their designated compartments despite a constant flow of secretory cargo is incompletely understood. Here, we determine the structure of the COPI vesicle coat containing GOLPH3, an adaptor protein that binds the cytosolic tails of many Golgi residents. Analysis of this structure, together with structure-guided mutagenesis and functional assays, reveals how GOLPH3 uses coincidence detection of COPI and lipids to engage Golgi residents preferentially at late cisternae. Our findings rationalize the logic of cisternal maturation and explain how COPI can engage different types of substrates in different Golgi cisternae to retrieve some proteins back to the ER while retaining others within the Golgi apparatus.

DOI: https://doi.org/10.1126/sciadv.aea0016
EMBO J. 2025 Oct 02.

RNF114 and RNF166 exemplify reader-writer E3 ligases that extend K11 polyubiquitin onto sites of MARUbylation.

Rachel E Lacoursiere, Kapil Upadhyaya, Jasleen Kaur Sidhu, Ivan Rodriguez Siordia, Daniel S Bejan, Michael S Cohen, Jonathan N Pruneda.

  Ubiquitin (Ub) cooperates with other post-translational modifications to provide a tiered opportunity for protein regulation. Deltex E3 ligases were previously implicated in ubiquitylation of ADP-ribose (ADPr)-containing macromolecules in vitro, generating a noncanonical mono-ADPr-Ub ester (MARUbe). We previously identified mono-ADPr ubiquitylation (MARUbylation) on PARP7 in cells, which was extended with K11-linked polyUb, suggesting an intricately regulated, multilayered post-translational modification. Here, we show that the Deltex DTX2 ubiquitylates ADPr modifications on PARP7 in cells, which depends on PARP7 catalytic activity. We further identify RNF114 as the E3 ligase responsible for K11-linked polyUb extension on sites of PARP7 MARUbylation. Using a chemoenzymatic approach, we developed a fluorescent Ub-ADPr probe and find that RNF114 explicitly recognizes MARUbylated species. We used AlphaFold3 to examine the mechanisms of Ub-ADPr recognition and K11-linked polyUb extension by RNF114. We identify a tandem Di19-UIM module in RNF114 as a MARUbe-binding domain (M-UBD), thus providing a reader function that interfaces with K11-specific writer activity. Finally, we describe a small family of M-UBD-containing E3 ligases that demonstrate preference for Ub-ADPr, which we call MARUbe-Targeted Ligases (M-UTLs).

Keywords:  ADP-ribose Ubiquitylation; K11 Polyubiquitylation; PARP/Ubiquitin Crosstalk; Post-translational Modifications

DOI:  https://doi.org/10.1038/s44318-025-00577-z
Sci Adv. 2025 Oct 03. 11(40): eady6859

Translation landscape of stress granules.

Yichun Wu, Xing Wang, Lingyu Meng, Zhizhao Liao, Wei Ji, Peipei Zhang, Jie Lin, Qiang Guo.

Stress granules, cytoplasmic assemblies of RNA binding proteins and messenger RNAs formed during cellular stress, are implicated in translational control. However, their exact functions remain elusive. Here, we used cryogenic correlative light and electron microscopy to visualize stress granules in their native environment and reconstructed them in three dimensions using tomography. This approach provided the first quantitative and spatial analysis of the translational machinery within stress granules. Our findings suggest that stress granules have a limited impact on global translation regulation but serve to protect small ribosomal subunits and preinitiation complexes from degradation. Numerical simulations based on a phase-field model accurately reproduced the spatial distribution of ribosomal components inside and outside the stress granules, shedding light on the thermodynamic principles governing this process.

DOI: https://doi.org/10.1126/sciadv.ady6859
Nat Rev Mol Cell Biol. 2025 Sep 29.

Towards a unified framework for the function of endoplasmic reticulum exit sites.

Hesso Farhan, Ishier Raote, Felix Campelo, Liang Ge, Koret Hirschberg, Alison Forrester, Giulia Zanetti, Jennifer Lippincott-Schwartz, José Carlos Pastor-Pareja, Franck Perez, Kota Saito, Vivek Malhotra.

Endoplasmic reticulum exit sites (ERES) are specialized, ribosome-free ER subdomains that serve as dynamic portals for COPII-mediated export of proteins from the ER. Beyond their role in the secretory pathway, ERES are implicated in diverse processes, including autophagy and the maturation of lipid droplets, highlighting their functional plasticity. ERES integrate cargo load, membrane tension and spatial cues to remodel their architecture and function in real time. This Roadmap synthesizes our current knowledge on the biogenesis, structural diversity and regulatory logic of ERES. We highlight key unanswered questions in the field, particularly concerning how ERES integrate signals to coordinate protein trafficking under varying cellular states. Finally, we propose a multidisciplinary framework - leveraging advances in high-resolution imaging, synthetic reconstitution and computational modelling - to delineate the principles governing the function and plasticity of ERES. Understanding these mechanisms holds significant potential for developing targeted therapeutic strategies in diseases linked to trafficking dysfunction.

DOI: https://doi.org/10.1038/s41580-025-00899-0
bioRxiv. 2025 Mar 15. pii: 2025.03.14.643343. [Epub ahead of print]

A FMRF-amide peptide that regulates cell non-autonomous protein homeostasis in C. elegans.

Carrie A Sheeler, Jacqueline Y Lo, Daviana Menendez Escalera, Emmett Griffin-Baldwin, Karmen Mah, Adam Roszczyk, Antoine E Roux, Cynthia Kenyon, Jennifer L Garrison, Ashley E Frakes.

The coordination of protein homeostasis from the brain to periphery is essential for the health and survival of all animals. In C. elegans , glia serve a central role in coordinating organismal protein homeostasis and longevity via the unfolded protein response of the endoplasmic reticulum (UPR ER ). However, the full extent of the cell non-autonomous response and the identity of the signaling molecules required remained unknown. Here, we show that glial UPR ER activation induces robust transcriptomic changes in specific tissue types across the animal, particularly in pathways related to neuropeptide signaling. We performed neuropeptidomics and loss and gain-of-function genetic screens and identified a single neuropeptide, FLP-17, that is sufficient but not necessary to induce cell non-autonomous activation of the UPR ER . FLP-17 is sufficient to protect against chronic ER stress and age-dependent protein aggregation. We determined that FLP-17 acts through the receptor, EGL-6, to activate cell non-autonomous UPR ER . This work reveals a complex peptidergic signaling network initiated by glial activation of the UPR ER to regulate organismal protein homeostasis.

DOI: https://doi.org/10.1101/2025.03.14.643343
Science. 2025 Oct 02. 390(6768): eads8728

GCN1 couples GCN2 to ribosomal state to initiate amino acid response pathway signaling.

Changqian Zhou, Miao Zhang, Jason Murray, Joao Paulo, Steve Gygi, Sichen Shao, Malcolm Whitman, Tracy Keller.

During nutrient deprivation, activation of the protein kinase GCN2 regulates cell survival and metabolic homeostasis. In addition to amino acid stress, GCN2 is activated by a variety of cellular stresses. GCN2 activation has been linked to its association with uncharged tRNAs, specific ribosomal proteins, and conditions of translational arrest, but their relative contribution to activation is unclear. Here, we used in vitro translation to reconstitute GCN2 activation by amino acid stress and compared collided ribosome populations induced by diverse translational stressors. Initiation of GCN2 signaling required the di-ribosome sensor GCN1, which recruits GCN2 to ribosomes in a collision-dependent manner, where GCN2 becomes activated by key ribosomal interactions and stably associated with collided ribosomes. Our findings define the molecular requirements and dynamics of GCN2 activation.

DOI: https://doi.org/10.1126/science.ads8728
Biochem J. 2025 Oct 01. 482(19): 1489-1516

Proteostasis of immune checkpoint receptors.

Pei Yee Tey, Sylvie Urbé, Michael J Clague.

  Immunotherapy relies on the targeting of immune checkpoint receptors and their respective ligands by specific antibodies that bind to the cell surface proteins. The pace of this highly successful clinical advancement has outstripped our cell biological understanding of these receptors. Here, we discuss what is known about their intracellular trafficking itineraries, which determine the bioavailability of these proteins for clinical targeting. Some of them are amongst the shortest-lived membrane proteins (CTLA-4), whilst others can be very stable (PD-L1). We highlight the ubiquitin system, which is key to determining their turnover, as it plays a key role in disposing of misfolded newly synthesised proteins via the ERAD pathway and generating a key signal for endosomal sorting towards lysosomes. In some cases, ubiquitylation can modulate the signalling function of the immune checkpoint receptor, as seen for LAG-3. Immune checkpoint proteins can evade lysosomal degradation by effective recycling to the plasma membrane using highly specialised factors, including CMTM6 (for PD-L1) and LRBA (for CTLA-4). Lastly, we consider how reprogramming the ubiquitin system emerges as an alternative modality in targeting immune checkpoint receptors.

Keywords:  Protein turnover; immune checkpoint; membrane trafficking; proteostasis; ubiquitin

DOI:  https://doi.org/10.1042/BCJ20253299
Commun Biol. 2025 Sep 30. 8(1): 1399

Transcriptome-wide mapping of small ribosomal subunits elucidates scanning mechanisms of translation initiation in the mammalian brain.

Preeti Madhav Kute, Francois P Pauzin, Kornel Labun, Clive R Bramham, Eivind Valen.

Neuronal protein synthesis is highly compartmentalised and regulated, with key roles for translation initiation and elongation factors. Ribosome profiling, the most widely used transcriptome-wide method for measuring translation, captures translation elongation, but not the initiation phase involving small ribosomal subunit (SSU) scanning. Here, we adapted ribosome complex profiling (RCP-seq) for mouse dentate gyrus and cerebral cortex, to characterize translation initiation. In both tissues, SSUs accumulate near the start codon on synaptically localised RNAs, and this 'poised' SSU configuration correlates with enhanced translational efficiency. Upstream open reading frames (uORFs) are associated with less poised SSUs, potentially by disassociating the SSUs. We further find that neuron-specific transcripts recruit more ribosomes and are more efficiently translated than glia-specific transcripts. For neuronal transcripts, monosome-preferring mRNAs show less SSU occupancy relative to polysome-preferring mRNAs, suggesting reduced recruitment of ribosomes. In summary, RCP-seq elucidates translation initiation dynamics and cell-type- and transcript-specific regulation in the mammalian brain.

DOI: https://doi.org/10.1038/s42003-025-08804-3
Cell Rep. 2025 Sep 25. pii: S2211-1247(25)01089-7. [Epub ahead of print]44(10): 116318

ER stress tolerance is regulated by copper-dependent PERK kinase activity.

Sarah E Bond Newton, Xinglong Shi, Noah R Beratan, Julia Perhacs, Jitendra K Arya, Margaret K Bond, Tali Gidalevitz, Cagla Akay-Espinoza, Donita C Brady, Kelly L Jordan-Sciutto.

  Pancreatic/PKR-like endoplasmic reticulum (ER) kinase (PERK) is a kinase that, in response to ER stress, mediates dual homeostatic and pro-apoptotic signaling. Thus, intricate regulation is required for physiological function. Attempts to modulate PERK activity have shown that the determinants of adaptive vs. maladaptive signaling remain ambiguous. Here, with purified protein, we provide evidence that PERK binds copper, identifies residues required for interaction, and demonstrates that copper is necessary for kinase activity. Furthermore, cellular PERK activity can be modulated via copper availability, and this regulatory relationship can be manipulated to dictate ER stress tolerance. Critically, these phenomena translate to phenotypes in vivo, as C. elegans harboring a "PERK-copper mutant" exhibit exacerbated ER-stress sensitivity. The copper-PERK paradigm suggests that copper homeostasis, as a regulator of PERK, may constitute a critical factor in resolving the long-standing ambiguity in endeavors to therapeutically target PERK.

Keywords:  CP: Cell biology; ER stress; ISR; PERK; UPR; copper; kinase regulation; stress tolerance

DOI:  https://doi.org/10.1016/j.celrep.2025.116318
Cell Rep. 2025 Oct 01. pii: S2211-1247(25)01143-X. [Epub ahead of print]44(10): 116372

USP9X is a mechanosensitive deubiquitinase that controls tumor cell invasiveness and drug response through YAP stabilization.

Pierric Biber, Alexandrine Carminati, Walaa Mohager, Mickael Ohanna, Christophe A Girard, Margaux Lecacheur, Mira Kahil, Océane Bouvet, Marie Irondelle, Stéphane Audebert, Mehdi Khaled, Sophie Tartare-Deckert, Marcel Deckert.

  Ubiquitin removal by deubiquitinases (DUBs) is crucial for protein activity and homeostasis. While tumor cells adapt to treatment and environmental stress, the role of DUBs in sensing mechanical signals from the extracellular matrix (ECM) remains an unexplored area. Using melanoma cells cultured on collagen matrices of varying stiffness and activity-based ubiquitin probe profiling combined with quantitative proteomics, we identify ubiquitin specific peptidase 9 X-linked (USP9X) as a stiffness-sensitive DUB acting through the discoidin domain receptor (DDR)/actomyosin signaling pathway. USP9X regulates levels of the mechanosensor YAP by preventing its proteasomal degradation via deubiquitination. Inhibition or knockdown of USP9X reduced YAP expression, impaired tumor cell migration, invasion, and ECM contraction, and decreased metastatic potential in vivo. Targeting USP9X also enhanced the effectiveness of BRAF-targeted therapies by limiting YAP-mediated mechanosensing, drug resistance, and tumor relapse. These findings establish USP9X as a mechanoresponsive DUB essential for cancer cell adaptation to mechanical cues, proposing it as a targetable mechanosensitive therapeutic target in cancer.

Keywords:  CP: Cancer; CP: Cell biology; DUB; USP9X; YAP; extracellular matrix stiffness; invasion; mechanotransduction; therapy resistance; ubiquitin proteasome system

DOI:  https://doi.org/10.1016/j.celrep.2025.116372
Commun Biol. 2025 Oct 03. 8(1): 1419

Systematic mRNA interactome analysis reconceptualizes translational quiescence in bovine sperm.

Saurabh Tiwari, Nehal Thakor, Jacob Thundathil.

Ejaculated mammalian sperm must undergo a series of biochemical changes called capacitation to gain fertilizing competence. Mounting evidence on protein synthesis during capacitation contradicts the widely accepted dogma of translational quiescence in sperm. However, mechanisms regulating mRNA translation in sperm is ambiguous, necessitating elucidation and understanding its role in enabling fertilizing competence. Here, we perform proteome analysis from bovine sperm and identify proteins involved in translation, encompassing initiation and elongation factors, ribosomal proteins, tRNA synthetase, ligase, and RNA-binding proteins (RBPs) involved in mRNA export, degradation, and binding. We further explore the mRNA-binding activity of RBPs during capacitation, identifying 48 RBPs; 13 and 8 RBPs were exclusive to fresh-uncapacitated and capacitation groups, respectively, with an overlap of 27 RBPs present in both groups. Interestingly, cytoskeletal proteins and metabolic enzymes associate differentially with mRNAs during capacitation. Since phosphorylation is a known regulatory mechanism dynamically modulating RBPs' interactions with mRNAs, we performed sperm phosphoproteome analysis, revealing few RBPs to phosphorylate during capacitation. These observations suggest that RNA-binding functions of these proteins are coupled with capacitation-associated phosphorylation events, enabling concomitant protein synthesis and fertilizing competence in sperm. These findings will assist in elucidating translational regulation of mRNA in sperm and advancing our knowledge in regulation of male fertility.

DOI: https://doi.org/10.1038/s42003-025-08919-7
Autophagy. 2025 Oct 02.

Autophagy-dependent proteostasis suppresses breast cancer metastasis.

Jayanta Debnath, Gourish Mondal.

  In breast cancer, macroautophagy/autophagy suppresses key steps of the metastatic cascade, including colonization and outgrowth at distant sites. However, the molecular mechanisms behind this suppression have remained unclear. Our recent study shows that increased metastasis observed in the setting of autophagy deficiency is driven by the accumulation of phase-separated biomolecular condensates containing the autophagy cargo receptors NBR1 and SQSTM1. These NBR1-SQSTM1 condensates sequester ITCH, an E3 ubiquitin ligase responsible for degrading TP63, a transcription factor that promotes basal differentiation. Hence, ITCH sequestration stabilizes and activates TP63 in breast cancer cells, hence promoting an aggressive, pro-metastatic basal-like differentiation state. Overall, our findings suggest that the potential benefits of targeting autophagy in cancer therapy are accompanied by defects in proteostasis, which disrupts epithelial lineage fidelity and enhances metastatic potential. We propose that targeting NBR1-SQSTM1 condensates may offer new therapeutic avenues to prevent metastasis, particularly in the context of autophagy deficiency.

Keywords:  Autophagy; autophagy cargo receptors; biomolecular condensates; lineage infidelity; metastasis

DOI:  https://doi.org/10.1080/15548627.2025.2569677
Cancer Discov. 2025 Sep 30. OF1-OF30

Expression of IMPACT Curtails Metabolic Plasticity and Augments NK Cell Killing to Abrogate Metastatic Growth.

Surajit Sinha, Abir Kumar Panda, Rodrigo Xavier das Neves, Zeribe C Nwosu, Ke Xu, Elke van Beek, Priyanka P Desai, Sivasish Sindiri, Sruthi Chempati, Kirsten Remmert, Billel Gasmi, Linda Bojmar, Constantinos Zambirinis, Alexander J Rossi, Reed I Ayabe, Michael M Wach, James D McDonald, Samantha M Ruff, Emily A Verbus, Areeba Saif, Alyssa V Eade, Carolina M Larrain, Lindsay R Friedman, Shreya Gupta, Alok Ranjan, Martha E Teke, Tahsin M Khan, Tracey Pu, Amber Leila Sarvestani, Carrie E Ryan, Jacob T Lambdin, Kenneth Luberice, Stephanie N Gregory, Stephanie C Lux, Hanna Hong, Allen J Luna, Imani A Alexander, Sarfraz R Akmal, Shahyan U Rehman, Ashley Rainey, Todd D Prickett, Vishal N Koparde, Samantha Sevilla, Skyler A Kuhn, King Chan, Zhonghe Sun, Nina Bubunenko, Eileen Li, Cathleen Hannah, Geneti Gaga, Thorkell Andresson, Margaret C Cam, Xiaolin Wu, Lisa M Jenkins, Andrew M Blakely, Jeremy L Davis, Giorgio Trinchieri, Pankaj K Singh, James C Yang, Marina Pasca di Magliano, Costas A Lyssiotis, Michael B Yaffe, Ethan M Shevach, Jonathan M Hernandez.

Given the propensity of aggressive epithelial tumors to form hepatic metastases, we performed an in vivo cDNA screen using the mouse liver and KRASG12D/TP53R273H pancreatic cells that identified the RNA-binding protein GCN1 as an integral component of hepatic outgrowth. RNAi experiments reveal that GCN1 triggers the integrated stress response (ISR) to activate serine, folate, and methionine biosynthetic pathways together with amino acid transporters, which act in concert to facilitate acquisition of metabolites and to restore redox homeostasis. Alongside the activation of the ISR, we found that GCN1 also functions in the nucleus where it interacts with HNRNPK to suppress the expression of MHC-I molecules and NK ligands. Intriguingly, we identified IMPACT as an endogenous competitive inhibitor of GCN1 that blocks both ISR-dependent metabolic control and disrupts HNRNPK interaction. In doing so, IMPACT enhances tumor immunogenicity to unleash NK cell killing, in addition to sensitizing metastatic tumor cells to immune checkpoint blockade.
SIGNIFICANCE: Metastatic tumor cells display profound immunometabolic plasticity to colonize distant organs. We identify IMPACT, an inhibitor of GCN1-stress signaling, expression of which curtailed metabolic plasticity and augmented tumor immunogenicity, sensitizing metastatic tumor cells to NK cell-mediated destruction.

DOI: https://doi.org/10.1158/2159-8290.CD-24-1055
Biochemistry. 2025 Sep 30.

CRISPR Screen Identifies BAP1 as a Deubiquitinase Regulating SPIN4 Stability.

Alondra Sanchez, Chen Zhou, Rima Tulaiha, Francisco Ramirez, Lu Wang, Xiaoyu Zhang.

Protein homeostasis is tightly controlled by the coordinated actions of E3 ubiquitin ligases and deubiquitinases (DUBs). We previously identified Spindlin-4 (SPIN4), a histone H3K4me3 reader, as a degradation substrate of DCAF16. In this study, we confirmed this degradation pathway using an E3 ligase-focused CRISPR-Cas9 knockout screen. Furthermore, through a DUB-focused CRISPR-Cas9 knockout screen and biochemical analyses, we demonstrated that the deubiquitinase BAP1 interacts with and stabilizes SPIN4 via its deubiquitination activity. Inhibition or loss of BAP1 reduces SPIN4 levels, highlighting its critical role in maintaining SPIN4 homeostasis. Proteomics and interactome analyses further support this regulatory axis. These findings reveal a dynamic balance controlling SPIN4 stability, with potential implications for epigenetic regulation and disease processes.

DOI: https://doi.org/10.1021/acs.biochem.5c00493
Genome Biol. 2025 Sep 27. 26(1): 315

Codon-specific ribosome stalling reshapes translational dynamics during branched-chain amino acid starvation.

Lina Worpenberg, Cédric Gobet, Felix Naef.

   BACKGROUND: Cells regulate protein synthesis in response to fluctuating nutrient availability through mechanisms that affect both translation initiation and elongation. Branched-chain amino acids, leucine, isoleucine, and valine, are essential nutrients. However, how their depletion affects translation remains largely unclear. Here, we investigate the immediate effects of single, double, and triple branched-chain amino acid deprivation on translational dynamics in NIH3T3 cells using RNA-seq and ribosome profiling.
RESULTS: All starvation conditions increased ribosome dwell times, with pronounced stalling at all valine codons during valine and triple starvation, whereas leucine and isoleucine starvation produced milder, codon-specific effects. Notably, stalling under isoleucine deprivation largely decreased under triple starvation. Positional enrichment of valine codons near the 5' end and downstream isoleucine codons potentially contributes to these patterns, suggesting a possible elongation bottleneck that influences translational responses under branched-chain amino acid starvation. The presence of multiple valine stalling sites was associated with decreased protein levels. Finally, codon-specific dwell time changes correlated strongly with patterns of tRNA isoacceptor charging.
CONCLUSIONS: Together, these findings suggest that differential ribosome stalling under branched-chain amino acid starvation reflects a balance between amino acid supply, tRNA charging dynamics, codon position, and stress-response signaling.

Keywords:  Amino acid starvation; Codon-specific stalling; Elongation bottleneck; Nutrient stress response; Ribosome profiling; Translation regulation; tRNA charging

DOI:  https://doi.org/10.1186/s13059-025-03800-6
Proc Natl Acad Sci U S A. 2025 Oct 07. 122(40): e2502841122

Mitochondrial ROS triggers mitophagy through activating the DNA damage response signaling pathway.

Qi-Qiang Guo, Shan-Shan Wang, Xiao-You Jiang, Xiao-Chen Xie, Yu Zou, Jing-Wei Liu, Yang Guo, Yu-Han Li, Xi-Yan Liu, Shuang Hao, Xin-Yue Zhang, Xiao-Xu Wu, Song-Ming Lu, Hong-De Xu, Wen-Dong Guo, Yan-Ling Feng, Chuan-Gui Wang, Sheng-Ping Zhang, Jia-Bin Li, Chen Liu, Xiao-Yu Song, Toren Finkel, Liu Cao.

  The homeostatic link between the production of mitochondrial ROS (mtROS) and mitophagy plays a significant role in how cells respond to various physiological and pathological conditions. However, it remains unclear how cells translate oxidative stress signals into adaptive mitophagy responses. Here, we show that mtROS act as signaling molecules that activate the ataxia-telangiectasia mutated (ATM)-cell cycle checkpoint kinase 2 (CHK2), a DNA damage response (DDR) pathway. When activated, CHK2 regulates three critical steps in mitophagy. First, CHK2 phosphorylates mitochondrial membrane protein ATAD3A at Ser371, which inhibits the transport of PINK1 to the inner mitochondrial membrane and leads to the accumulation of PINK1 and the commencement of mitophagy. Second, activated CHK2 targets the autophagy adaptor OPTN at Ser177 and Ser473, thereby enhancing the targeting of ubiquitinated mitochondria to autophagosomes. Finally, CHK2 phosphorylates Beclin 1 at Ser90 and Ser93, hence promoting the formation of autophagosomal membranes. Consistent with these effects, Chk2-/- mice show impaired mitophagic induction and impaired recovery in a ROS-dependent model of renal ischemia-reperfusion. Our study reveals a mtROS-triggered adaptive pathway that coordinates mitophagic induction, in order to protect cells and tissues exposed to pathophysiological stress-induced damage.

Keywords:  ATM; CHK2; PINK1; mitophagy; mtROS

DOI:  https://doi.org/10.1073/pnas.2502841122
EMBO J. 2025 Sep 29.

Allosteric control of the bacterial ClpC/ClpP protease and its hijacking by antibacterial peptides.

Timo Jenne, Lisa Engelhardt, Ieva Baronaite, Dorit Levy, Inbal Riven, Maciej Malolepszy, Stavros Azinas, Taras Sych, Erdinc Sezgin, Dirk Flemming, Irmgard Sinning, Gilad Haran, Marta Carroni, Axel Mogk.

  The hexameric AAA+ protein ClpC, combined with peptidase ClpP, forms a critical ATP-dependent protease in bacteria, essential for virulence. ClpC is usually repressed in an inactive resting state, where two ClpC spirals interact via coiled-coil M-domains. Antibacterial peptides and partner proteins trigger ClpC activation by binding to its N-terminal domain (NTD). This study reveals that the NTD stabilizes the resting state through multiple anchoring points to M-domains and ATPase domains. The same NTD sites also serve as binding sites for adaptor proteins and substrates carrying phosphorylated arginines (pArg), disrupting resting state interactions and promoting active ClpC hexamer formation. This coupling ensures that ClpC activation aligns with substrate and partner protein availability. Toxic peptides exploit this regulatory mechanism, leading to continuous ClpC activation and harmful, uncontrolled proteolysis. These findings highlight the dual role of the NTD in maintaining resting state stability and mediating activation, emphasizing its critical role in bacterial protease regulation and its potential as a drug target.

Keywords:  ATPase Associated with Diverse Cellular Activities (AAA); Chaperone; Hsp100; Protein Degradation; Protein Quality Control

DOI:  https://doi.org/10.1038/s44318-025-00575-1
iScience. 2025 Oct 17. 28(10): 113494

Identification of the client-binding site on the Golgi membrane protein adaptor Vps74/yGOLPH3.

Agnieszka M Lesniak, Ziyun Ye, David K Banfield.

  Vps74 and its mammalian counterpart GOLPH3 are COPI associated protein sorting adaptors that function to maintain the cisternal distributions of a diversity of Golgi integral membrane protein clients by binding to their short cytoplasmically exposed N-termini. Here, we identify the client-binding site on yeast GOLPH3 (Vps74) which maps to two evolutionarily conserved loops on the membrane-facing surface, and includes residues mediating binding of GOLPH3s to PI4P, as well as the membrane-binding β hairpin. As an orthogonal approach, we isolated an inhibitory anti-Vps74 nanobody (which also binds to GOLPH3s) with which we corroborate the client-binding site and reveal that Sac1 and Arf1 binding to Vps74 blocks client access. We also identify an additional mode for the recruitment of Vps74 to Golgi membranes whereby the adaptor binds directly to its client N-termini. This study elucidates the molecular mechanism of Vps74 and identifies an inhibitory GOLPH3 nanobody with potential therapeutic applications.

Keywords:  Biochemistry; cell biology; molecular biology

DOI:  https://doi.org/10.1016/j.isci.2025.113494
PLoS Biol. 2025 Sep 30. 23(9): e3002957

Expansion of lysosomal capacity in early adult neurons driven by TFEB/HLH-30 protects dendrite maintenance during aging in Caenorhabditis elegans.

Ruiling Zhong, Claire E Richardson.

Lysosomes are essential for neuronal homeostasis, providing degradation and recycling functions necessary to support neurons' complex operations and long lifespans. However, the regulation of lysosomal degradative capacity in healthy neurons is poorly understood. Here, we investigate the role of HLH-30, the sole Caenorhabditis elegans homolog of Transcription Factor EB (TFEB), a master regulator of lysosome biogenesis and autophagy that is thought to predominantly function in the context of starvation or stress. We demonstrate that HLH-30 is dispensable for neuronal development but acts cell-intrinsically to expand lysosomal degradative capacity during early adulthood. Loss of HLH-30 leads to lysosomal dysfunction and delayed turnover of synaptic vesicle proteins from the synapse. Notably, we show that basal HLH-30 activity is sufficient to expand neuronal lysosomal capacity without nuclear enrichment, in contrast to the nuclear translocation associated with starvation- and stress-induced activation of TFEB and HLH-30. Furthermore, we show that neuronal lysosomal function declines with age in wild-type animals, and this corresponds to a decrease in basal HLH-30-mediated transcription. We further demonstrate that basal HLH-30 activity is crucial for neuron maintenance: lysosomal dysfunction due to inadequate HLH-30 activity leads to dendrite degeneration and aberrant outgrowths. In summary, our study establishes a critical role for HLH-30/TFEB in promoting lysosomal capacity to preserve neuronal homeostasis and structural integrity of mature neurons in vivo.

DOI: https://doi.org/10.1371/journal.pbio.3002957
Bioessays. 2025 Sep 28. e70074

Cytosolic fMet-Protein Synthesis as Source of Endogenous Ligands for Formyl Peptide Receptors.

Chang-Seok Lee, Cheol-Sang Hwang.

  Formyl peptides, exemplified by the synthetic tripeptide formyl-Met-Leu-Phe (fMLF), are well-established ligands for formyl peptide receptors (FPRs), central to neutrophil chemotaxis, and innate immune signaling. Traditionally attributed to bacterial and mitochondrial origins, these peptides are now proposed to arise from an additional, stress-inducible source within the eukaryotic cytosol. Recent findings suggest that under specific stress conditions, eukaryotic translation can initiate with formylmethionine (fMet), producing fMet-bearing nascent chains that are processed by the fMet/N-degron and fMet-mediated ribosome quality control (fMet-RQC) pathways. These proteostatic mechanisms may generate short, structurally diverse formyl peptides with the potential to function as endogenous FPR ligands. By introducing cytosolic proteostasis as a hypothetical source of formyl peptides, this perspective expands the landscape of formyl peptide biology and opens new directions for investigating their roles in immune regulation under stress and disease.

Keywords:  FPR; fMLF; fMet/N‐degron; fMet‐RQC pathway; formylmethionine; proteostasis

DOI:  https://doi.org/10.1002/bies.70074
Nature. 2025 Oct 01.

Proteotoxic stress response drives T cell exhaustion and immune evasion.

Yi Wang, Anjun Ma, No-Joon Song, Ariana E Shannon, Yaa S Amankwah, Xingyu Chen, Weidong Wu, Ziyu Wang, Abbey A Saadey, Amir Yousif, Gautam Ghosh, Jay K Mandula, Maria Velegraki, Tong Xiao, Haitao Wen, Stanley Ching-Cheng Huang, Ruoning Wang, Christian M Beusch, Abdelhameed S Dawood, David E Gordon, Mohamed S Abdel-Hakeem, Hazem E Ghoneim, Gang Xin, Brian C Searle, Zihai Li.

Chronic infections and cancer cause T cell dysfunction known as exhaustion. This cell state is caused by persistent antigen exposure, suboptimal co-stimulation and a plethora of hostile factors that dampen protective immunity and limit the efficacy of immunotherapies1-4. The mechanisms that underlie T cell exhaustion remain poorly understood. Here we analyse the proteome of CD8+ exhausted T (Tex) cells across multiple states of exhaustion in the context of both chronic viral infections and cancer. We show that there is a non-stochastic pathway-specific discordance between mRNA and protein dynamics between T effector (Teff) and Tex cells. We identify a distinct proteotoxic stress response (PSR) in Tex cells, which we term Tex-PSR. Contrary to canonical stress responses that induce a reduction in protein synthesis5,6, Tex-PSR involves an increase in global translation activity and an upregulation of specialized chaperone proteins. Tex-PSR is further characterized by the accumulation of protein aggregates and stress granules and an increase in autophagy-dominant protein catabolism. We establish that disruption of proteostasis alone can convert Teff cells to Tex cells, and we link Tex-PSR mechanistically to persistent AKT signalling. Finally, disruption of Tex-PSR-associated chaperones in CD8+ T cells improves cancer immunotherapy in preclinical models. Moreover, a high Tex-PSR in T cells from patients with cancer confers poor responses to clinical immunotherapy. Collectively, our findings indicate that Tex-PSR is a hallmark and a mechanistic driver of T cell exhaustion, which raises the possibility of targeting proteostasis pathways as an approach for cancer immunotherapy.

DOI: https://doi.org/10.1038/s41586-025-09539-1
bioRxiv. 2025 Sep 23. pii: 2025.09.22.677926. [Epub ahead of print]

Muscular dystrophy-associated lamin variants disrupt cellular organization through a nucleolar-ribosomal axis controlling cytoplasmic macromolecular crowding.

Xiangyi Ding, Sweta Kumari, Ellen Gregory, Daniel A Starr, G W Gant Luxton.

Emery-Dreifuss muscular dystrophy (EDMD) arises from mutations in nuclear lamins or emerin. Current pathological models emphasize defects in nuclear mechanics and transcription regulation. Yet these models do not fully explain the complexity of EDMD or other laminopathies. Here, we uncover an emerging pathway linking nuclear lamina defects to the reorganization of cytoplasmic biophysics, revealing how nuclear dysfunction cascades throughout the cell. Using Caenorhabditis elegans EDMD models, we demonstrate that lamin mutations dramatically alter cytoplasmic organization, reducing macromolecular crowding and increasing diffusivity of 40 nm G enetically E ncoded M ultimeric (GEM) nanoparticles. These striking biophysical changes coincide with nuclear positioning defects and collapsed endoplasmic reticulum architecture, mirroring phenotypes associated with ribosome depletion. We propose a mechanism where mutations in the C. elegans lamin lmn-1 disrupt nucleolar density and ribosome biogenesis, creating a nucleolar-ribosomal axis that propagates defects from the nucleus to the cytoplasm. Genetic interactions between lmn-1 and ribosomes support this regulatory relationship. While individual depletion of other nuclear envelope proteins produces minimal effects, combined loss of the functionally redundant emerin ortholog emr-1 and LEM-domain protein lem-2 phenocopied lmn-1 mutants, demonstrating that cytoplasmic biophysical disruption lies at EDMD's pathogenic core. Our findings establish a paradigm where nuclear lamina defects fundamentally rewire cellular biophysics through nucleolar-ribosomal dysfunction, opening transformative therapeutic avenues for treating laminopathies.

DOI: https://doi.org/10.1101/2025.09.22.677926
Nat Commun. 2025 Sep 30. 16(1): 8682

Synthetic protein degradation circuits using programmable cleavage and ligation by Sortase A.

Hopen K Yang, Pragati K Muthukumar, Wilfred Chen.

BioPROTACs are heterobifunctional proteins designed for targeted protein degradation (TPD). They are useful not only for probing protein functions but also offer a therapeutic avenue for modulating disease-related proteins. To extend the use of TPD beyond just protein attenuation, we introduce a synthetic framework for logic-gated, switchable TPD to achieve conditional control of protein content. By exploiting both the cleavage and ligation functionalities of Sortase A (SrtA), we present a new strategy utilizing SrtA as the control input to direct bioPROTAC activity for switchable TPD. Furthermore, by layering the SrtA input with protease gating, conditional degradation phenotypes can be readily adapted with minimal modifications to the design. This Logic-gated AdPROM deploying SrtA-mediated Element Recombination (LASER) platform allows us to expand the possible protein degradation outcomes in mammalian cells using Boolean logic operations depending on the input combinations. The flexibility to modulate the level of multiple native intracellular proteins can potentially lead to applications from therapy to diagnostics and biotechnology.

DOI: https://doi.org/10.1038/s41467-025-63819-y
Nat Commun. 2025 Oct 01. 16(1): 8650

Bacterial RNA promotes proteostasis through inter-tissue communication in C. elegans.

Emmanouil Kyriakakis, Chiara Medde, Danilo Ritz, Geoffrey Fucile, Alexander Schmidt, Anne Spang.

Life expectancy has been increasing over the last decades, which is not matched by an increase in healthspan. Besides genetic composition, environmental and nutritional factors influence both health- and lifespan. Diet is thought to be a major factor for healthy ageing. Here, we show that dietary RNA species improve proteostasis in C. elegans. Inherent bacterial-derived double stranded RNA reduces protein aggregation in a C. elegans muscle proteostasis model. This beneficial effect depends on low levels of systemic selective autophagy, the RNAi machinery in the germline, even when the RNA is delivered through ingestion in the intestine and the integrity of muscle cells. Our data suggest a requirement of inter-organ communication between the intestine, the germline and muscles. Our results demonstrate that bacterial-derived RNAs elicit a systemic response in C. elegans, which protects the animal from protein aggregation during ageing, which might extend healthspan.

DOI: https://doi.org/10.1038/s41467-025-63987-x
EMBO J. 2025 Oct 03.

Mapping cryptic phosphorylation sites in the human proteome.

Dino Gasparotto, Annarita Zanon, Valerio Bonaldo, Elisa Marchiori, Massimo Casagranda, Erika Di Domenico, Laura Copat, Tommaso Fortunato Asquini, Marta Rigoli, Sirio Vittorio Feltrin, Nuria Lopez Lorenzo, Graziano Lolli, Maria Pennuto, Jesùs R Requena, Omar Rota Stabelli, Giovanni Minervini, Cristian Micheletti, Giovanni Spagnolli, Pietro Faccioli, Emiliano Biasini.

  Advances in computational and experimental methods have revealed the existence of transient, non-native protein folding intermediates that could play roles in disparate biological processes, from regulation of protein expression to disease-relevant misfolding mechanisms. Here, we tested the possibility that specific post-translational modifications may involve residues exposed during the folding process by assessing the solvent accessibility of 87,138 post-translationally modified amino acids in the human proteome. Unexpectedly, we found that one-third of phosphorylated proteins present at least one phosphosite completely buried within the protein's inner core. Computational and experimental analyses suggest that these cryptic phosphosites may become exposed during the folding process, where their modification could destabilize native structures and trigger protein degradation. Phylogenetic investigation also reveals that cryptic phosphosites are more conserved than surface-exposed phosphorylated residues. Finally, cross-referencing with cancer mutation databases suggests that phosphomimetic mutations in cryptic phosphosites can increase tumor fitness by inactivating specific onco-suppressors. These findings define a novel role for co-translational phosphorylation in shaping protein folding and expression, laying the groundwork for exploring the implications of cryptic phosphorylation in health and disease.

Keywords:  Co-translational Phosphorylation; Cryptic Phosphosites; Post-translation Modification; Protein Folding; Protein Phosphorylation

DOI:  https://doi.org/10.1038/s44318-025-00567-1
J Clin Invest. 2025 Sep 30. pii: e190958. [Epub ahead of print]

ER stress sensor PERK promotes T-cell pathogenicity in GVHD by regulating ER-associated degradation.

Qiao Cheng, Hee-Jin Choi, Yongxia Wu, Xiaohong Yuan, Allison Pugel, Linlu Tian, Michael Hendrix, Denggang Fu, Reza Alimohammadi, Chen Liu, Xue-Zhong Yu.

  Endoplasmic reticulum (ER) stress through IRE1/XBP-1 is implicated in the onset and progression of graft-versus-host disease (GVHD), but the role of ER stress sensor PERK in T-cell allogeneic responses and GVHD remains unexplored. Here, we report that PERK is a key regulator in T-cell allogeneic response and GVHD induction. PERK augments GVHD through increasing Th1 and Th17 population, while reducing Treg differentiation by activating Nrf2 pathway. Genetical deletion or selective inhibition of PERK pharmacologically reduces GVHD while preserving graft-versus-leukemia (GVL) activity. At cellular level, PERK positively regulates CD4+ T-cell pathogenicity, while negatively regulating CD8+ T-cell pathogenicity in the induction of GVHD. At molecular level, PERK interacts with SEL1L and regulates SEL1L expression, leading to augmented T-cell allogeneic responses and GVHD development. In vivo, PERK deficiency in donor T cells alleviate GVHD through ER-associated degradation (ERAD). Furthermore, pharmacological inhibition of PERK with AMG44 significantly suppresses the severity of GVHD induced by murine or human T cells. In summary, our findings validate PERK as a potential therapeutic target for the prevention of GVHD while preserving GVL responses, and uncover the mechanism by which PERK differentially regulates CD4+ versus CD8+ T-cell allogeneic and anti-tumor responses.

Keywords:  Bone marrow transplantation; Immunology; Inflammation

DOI:  https://doi.org/10.1172/JCI190958
Trends Biochem Sci. 2025 Oct 02. pii: S0968-0004(25)00222-1. [Epub ahead of print]

Interconnectivity of mitochondrial protein biogenesis and quality control.

Abi S Ghifari, Carmela Vazquez-Calvo, Andreas Carlström, Martin Ott.

  Mitochondrial protein homeostasis (proteostasis) keeps the mitochondrial proteome functional. Thus, proteostasis is essential for mitochondrial activity and overall cellular functions, and a reduction in its function corresponds with diseases and aging in humans. Recent studies in various model organisms highlight components and mechanisms of mitochondrial proteostasis from biogenesis, through assembly, to turnover. Key findings include the identification of new components and mechanistic insights into protein import and mitochondrial translation processes, the interconnectivity of protein biogenesis and quality control, and proteolytic degradation machineries. In this review we discuss these advances that improve our current understanding of the inner workings and significance of the mitochondrial proteostasis network in maintaining functional mitochondria.

Keywords:  mitochondria; proteases; protein import; proteolysis; proteostasis; translation

DOI:  https://doi.org/10.1016/j.tibs.2025.09.004
Sci Adv. 2025 Oct 03. 11(40): eady9234

Mitochondrial ROS trigger interorganellular signaling and prime ER processes to establish enhanced plant immunity.

Yang Yang, Yan Zhao, Wei Zhao, Yingqi Zhang, Hongmei Wang, Murray Grant, Patrick Schäfer, Yuling Meng, Weixing Shan.

Reactive oxygen species (ROS) are key signaling molecules in plant development and immunity, but current understanding is primarily focused on apoplastic and chloroplastic ROS. Mitochondria are also a key source of intracellular ROS, yet their contribution to plant immunity is poorly characterized. Here, we studied mitochondrial ROS (mROS) function in plant-pathogen interactions, deploying genetically encoded sensors, assorted fluorescent markers, and genetic approaches to track mROS, specifically H2O2, dynamics and identify interorganelle contact sites. We unexpectedly found a mitochondria-endoplasmic reticulum (ER) ROS signal cascade functioning independently of apoplastic and chloroplastic ROS in plant immunity. mROS initiate immune responses induced by the oomycete pathogen Phytophthora parasitica and promote mitochondria-ER association. These enhanced mitochondria-ER membrane associations are required for transfer of mROS signals and initiation of extensive unfolded protein responses. We conclude that mROS transfer via mitochondria-ER membranes to the ER lumen is an underappreciated yet essential component in plant defense.

DOI: https://doi.org/10.1126/sciadv.ady9234
Nat Methods. 2025 Oct 03.

All-at-once RNA folding with 3D motif prediction framed by evolutionary information.

Aayush Karan, Elena Rivas.

Structural RNAs exhibit a vast array of recurrent short three-dimensional (3D) elements found in loop regions involving non-Watson-Crick interactions that help arrange canonical double helices into tertiary structures. Here we present CaCoFold-R3D, a probabilistic grammar that predicts these RNA 3D motifs (also termed modules) jointly with RNA secondary structure over a sequence or alignment. CaCoFold-R3D uses evolutionary information present in an RNA alignment to reliably identify canonical helices (including pseudoknots) by covariation. Here we further introduce the R3D grammars, which also exploit helix covariation that constrains the positioning of the mostly noncovarying RNA 3D motifs. Our method runs predictions over an almost-exhaustive list of over 50 known RNA motifs ('everything'). Motifs can appear in any nonhelical loop region (including three-way, four-way and higher junctions) ('everywhere'). All structural motifs as well as the canonical helices are arranged into one single structure predicted by one single joint probabilistic grammar ('all-at-once'). Our results demonstrate that CaCoFold-R3D is a valid alternative for predicting the all-residue interactions present in a RNA 3D structure. CaCoFold-R3D is fast and easily customizable for novel motif discovery and shows promising value both as a strong input for deep learning approaches to all-atom structure prediction as well as toward guiding RNA design as drug targets for therapeutic small molecules.

DOI: https://doi.org/10.1038/s41592-025-02833-w
Nat Commun. 2025 Sep 29. 16(1): 8578

Visualization of liquid-liquid phase transitions using a tiny G-quadruplex binding protein.

Bikash R Sahoo, Xiexiong Deng, Ee Lin Wong, Nathan Clark, Harry J Yang, Alexey Kovalenko, Vivekanandan Subramanian, Bryan B Guzman, Sarah E Harris, Budheswar Dehury, Emi Miyashita, J Damon Hoff, Vojč Kocman, Hirohide Saito, Daniel Dominguez, Janez Plavec, James C A Bardwell.

Liquid-liquid phase transitions govern a wide range of protein-protein and protein-RNA interactions. Although the importance of multivalency and protein disorder in driving these transitions is clear, there is limited knowledge concerning the structural basis of phase transitions or the conformational changes that accompany this process. In this work, we found that a small human protein, SERF2, is important for the formation of stress granules. We determined the solution NMR structure ensemble of SERF2. We show that SERF2 specifically interacts with non-canonical tetrahelical RNA structures called G-quadruplexes, structures linked to stress granule formation. The biophysical amenability of both SERF2 and RNA G4 quadruplexes have allowed us to characterize the multivalent protein-RNA interactions involved in liquid-liquid phase transitions, the role that protein disorder plays in these transitions, identify the specific contacts involved, and describe how these interactions impact the structural dynamics of the components enabling a detailed understanding of the structural transitions involved in early stages of ribonucleoprotein condensate formation.

DOI: https://doi.org/10.1038/s41467-025-63597-7
Annu Rev Cell Dev Biol. 2025 Oct;41(1): 479-504

Mechanisms of Messenger RNA Packaging and Export.

Rupert Faraway, Daniel Zenklusen, Clemens Plaschka.

  The packaging and export of messenger RNA (mRNA) are essential cellular pathways that bridge the nuclear and cytoplasmic phases of eukaryotic gene expression. During their nuclear maturation, mRNAs are packaged by proteins into mRNA ribonucleoproteins (mRNPs). Other proteins then assist in the export of mRNPs into the cytoplasm for translation. Together, these proteins play critical roles in compacting the mRNA, defining mRNA identity, preventing unwanted interactions, and orchestrating mRNA transport through the nuclear pore complex (NPC). Here, we review decades of genetics and biochemistry alongside recent structural and functional insights and outline a general framework for the late stages of nuclear mRNA biogenesis and export. We also highlight open questions, including the mechanisms of mRNP packaging, mRNP export through the NPC, and the regulation, quality control, and exploitation of the pathway.

Keywords:  RNA biology; gene expression; mRNA nuclear export; mRNA packaging; molecular biology

DOI:  https://doi.org/10.1146/annurev-cellbio-101123-045256
J Biol Chem. 2025 Sep 29. pii: S0021-9258(25)02625-0. [Epub ahead of print] 110773

Missense mutations in intrinsically disordered protein regions link pathogenicity and phase separation.

Oliver L Kipp, Karen A Lewis, Loren E Hough, Steven T Whitten.

The impact of missense genetic variations on protein function is often enigmatic, especially for mutations that map to intrinsically disordered regions (IDRs). Given the functional importance of phase separation of IDRs, it has been proposed that mutations that modulate phase separation might preferentially lead to disease. To examine this idea, we used the robust predictability of phase-separating (PS) IDRs and annotation of disease-associated proteins and mutations to map the correlation between disease and phase separation. Consistent with previous work linking phase separation to cancer and autism spectrum disorder, we find a higher prevalence of predicted phase separation behavior in disease-associated proteins than typical for human proteins. We map the prevalence of phase separation across a wide range of diseases, finding that many, but not all, show an enrichment of phase separation in the proteins associated with them. Strikingly, the pathogenic mutation rate in predicted PS IDRs was elevated three-fold relative to IDRs not predicted to phase separate. Substitutions involving arginine and the aromatic types were among the most pathogenic for PS IDRs, while substitutions involving serine, threonine, and alanine the most benign. We applied these trends to mutations of uncertain clinical significance and predict that half found in PS IDRs are likely pathogenic. We find that phosphorylation sites were enriched in PS IDRs when compared to other protein regions, though mutations at such sites were mostly benign. Pathogenicity was highest for mutations in predicted PS IDRs when also found in a short linear motif, known mediators of protein-protein interactions.

DOI: https://doi.org/10.1016/j.jbc.2025.110773
Commun Chem. 2025 Sep 29. 8(1): 284

Nanoscale profiling of evolving intermolecular interactions in ageing FUS condensates.

Alyssa Miller, Zenon Toprakcioglu, Seema Qamar, Peter St George-Hyslop, Francesco Simone Ruggeri, Tuomas P J Knowles, Michele Vendruscolo.

Protein condensates can exist in different states with distinct material properties, corresponding to specific cellular functions. These material properties, however, remain difficult to characterise, in part due to the technical challenges associated with studying condensed states. Here, to address this problem, we combine a microfluidic sample deposition technique that preserves the solution properties of condensates on surfaces with a nanometre-resolution spatial mapping method to characterise the time-dependent material properties of condensates of the fused in sarcoma (FUS) protein. This approach revealed two distinct phase transitions within FUS condensates. We first observed a spatially heterogeneous disorder-to-order transition initiating at the condensate interfaces and associated with intermolecular β-sheet formation. This process was then followed by the gelation of the condensate core, arising from an increase in the density of intermolecular interactions between intrinsically disordered regions. Overall, this study identifies molecular conformations associated with emergent phases of FUS condensates, and establishes a technology platform to understand the role of nanometre-scale phase changes in protein condensates.

DOI: https://doi.org/10.1038/s42004-025-01659-z
Nat Commun. 2025 Oct 02. 16(1): 8804

Dual membrane receptor degradation via folate receptor targeting chimera.

Zhen Wang, Zhixin Li, Jenny Högström, Hiroyuki Inuzuka, Rui Jing, Peiqiang Yan, Tao Hou, Yihang Qi, Daoyuan Huang, Jingchao Wang, Ting Wu, Xiaoying Shi, Bolin Liu, Taru Muranen, Dingpeng Zhang, Wenyi Wei.

Cancer drug resistance poses a significant challenge in oncology, often driven by intricate cross-talk among membrane-bound receptors that compromise mono-targeted therapies. We develop a dual membrane receptor degradation strategy leveraging Folate Receptor α (FRα) to address this issue. Folate Receptor α Targeting Chimeras-dual (FolTAC-dual) are engineered degraders designed to selectively and simultaneously degrade distinct receptor pairs: (1) EGFR/HER2 and (2) PD-L1/VISTA. Through modular optimization of modality configurations and geometries, we identify the "string" format as the most effective construct. Mechanistic studies demonstrate an ~85% increase in EGFR-binding affinity compared to the conventional knob-into-hole design, likely contributing to the improved efficiency of dual-target degradation. Proof-of-concept studies reveal that EGFR and HER2 FolTAC-dual effectively counteracts resistance in Trastuzumab/Lapatinib-resistant HER2-positive breast cancer models, while PD-L1 and VISTA FolTAC-dual rejuvenates immune responses in PD-L1 antibody-resistant syngeneic mouse models. These findings establish FolTAC-dual as a promising dual-degradation platform for clinical translation.

DOI: https://doi.org/10.1038/s41467-025-63882-5
Cell. 2025 Oct 01. pii: S0092-8674(25)01037-2. [Epub ahead of print]

Sequencing-free whole-genome spatial transcriptomics at single-molecule resolution.

Yubao Cheng, Shengyuan Dang, Yuan Zhang, Yanbo Chen, Ruihuan Yu, Miao Liu, Shengyan Jin, Ailin Han, Samuel Katz, Siyuan Wang.

  Recent breakthroughs in spatial transcriptomics technologies have enhanced our understanding of diverse cellular identities, spatial organizations, and functions. Yet existing spatial transcriptomics tools are still limited in either transcriptomic coverage or spatial resolution, hindering unbiased, hypothesis-free transcriptomic analyses at high spatial resolution. Here, we develop reverse-padlock amplicon-encoding fluorescence in situ hybridization (RAEFISH), an image-based spatial transcriptomics method with whole-genome coverage and single-molecule resolution in intact tissues. We demonstrate the spatial profiling of transcripts from 23,000 human or 22,000 mouse genes in single cells and tissue sections. Our analyses reveal transcript-specific subcellular localization, cell-type-specific and cell-type-invariant zonation-dependent transcriptomes, and gene programs underlying preferential cell-cell interactions. Finally, we further develop our technology for the direct spatial readout of guide RNAs (gRNAs) in an image-based, high-content CRISPR screen. Overall, these developments offer a broadly applicable technology that enables high-coverage, high-resolution spatial profiling of both long and short, native and engineered RNAs in many biomedical contexts.

Keywords:  high content CRISPR screen; highly multiplexed RNA imaging; spatial transcriptomics

DOI:  https://doi.org/10.1016/j.cell.2025.09.006
iScience. 2025 Oct 17. 28(10): 113514

Integrated multi-dimensional comparison of proteomic profiles across 54 cancer cell lines.

Wenhao Shi, Tianlong He, Nan Wang, Annan Qian, Yuqiao Liu, Shaojun Tang, Yiying Zhu.

  Human cancer cell lines are essential model systems in biomedical research. We conducted multi-level proteomics analyses on 54 widely used cancer cell lines derived from various tissues using two prominent proteomics technologies: mass spectrometry (MS) and reverse-phase protein array (RPPA). Our analysis identified 10,088 proteins, 33,161 phosphorylation sites across 7,469 phosphoproteins, and 56,320 site-specific glycans on 14,228 glycosylation sites from 5,966 glycoproteins, along with 305 drug-relevant protein and phosphoprotein targets. Analysis of this rich dataset yielded numerous biological insights, including protein features that distinguish tissue origins and cell line-specific kinase activation patterns, reflecting signaling diversity across cancer types. These findings may inform therapeutic strategies and support rational model system selection. Additionally, MS and RPPA showed consistent fold-change estimation and provided complementary views of proteome and signaling variation. This comprehensive resource facilitates biomarker discovery, signaling analysis, and translational oncology research across diverse human tumor types.

Keywords:  Cancer; Integrative aspects of cell biology; Proteomics

DOI:  https://doi.org/10.1016/j.isci.2025.113514
Comput Struct Biotechnol J. 2025 ;27 4040-4047

DrugDomain 2.0: Comprehensive database of protein domains-ligands/drugs interactions across the whole Protein Data Bank.

Kirill E Medvedev, R Dustin Schaeffer, Nick V Grishin.

  Proteins carry out essential cellular functions - signaling, metabolism, transport - through the specific interaction of small molecules and drugs within their three-dimensional structural domains. Protein domains are conserved folding units that, when combined, drive evolutionary progress. The Evolutionary Classification Of protein Domains (ECOD) places domains into a hierarchy explicitly built around distant evolutionary relationships, enabling the detection of remote homologs across the proteomes. Yet no single resource has systematically mapped domain-ligand interactions at the structural level. To fill this gap, we introduce DrugDomain v2.0, an updated comprehensive resource, that extends earlier releases by linking evolutionary domain classifications (ECOD) to ligand binding events across the entire Protein Data Bank. We also leverage AI-driven predictions from AlphaFold to extend domain-ligand annotations to human drug targets lacking experimental structures. DrugDomain v2.0 catalogs interactions with over 37,000 PDB ligands and 7560 DrugBank molecules, integrates more than 6000 small-molecule-associated post-translational modifications, and provides context for 14,000 + PTM-modified human protein models featuring docked ligands. The database encompasses 43,023 unique UniProt accessions and 174,545 PDB structures. The DrugDomain data is available online: https://drugdomain.cs.ucf.edu/ and https://github.com/kirmedvedev/DrugDomain.

Keywords:  Database; Drug discovery; Drugs; Protein domains; Protein-drug interaction; Small molecules

DOI:  https://doi.org/10.1016/j.csbj.2025.09.018
Nat Commun. 2025 Sep 29. 16(1): 8552

In situ secondary structure imaging of protein phase separation and aggregation by hyperspectral stimulated Raman scattering microscopy.

Ruifeng Sun, Yanrong Zhuang, Yi Lin, Fanghao Hu.

Biomolecular condensates enable the coordination of cellular activities with high spatiotemporal selectivity. Many techniques have been developed to characterize protein condensate. However, direct visualization of protein structure in phase-separated condensate remains underexplored. Here we develop in situ quantitative imaging of secondary structure in protein condensates by stimulated Raman scattering (SRS) microscopy. Characteristic spectra of four secondary structures are obtained from protein amide I vibration analysis. Hyperspectral SRS imaging reveals significant enrichments and disordered to ordered structural changes during phase separation of ALS-related proteins. Time-lapse imaging of protein aging process directly visualizes heterogeneous β-sheet formation on the condensate surface. And secondary structures of mutant proteins are imaged to correlate amino acid sequence to phase separation property. Live-cell label-free imaging of protein structure is further demonstrated to exhibit pronounced heterogeneity in subcellular aggregates. Therefore, our technique provides crucial molecular-level information to investigate protein phase separation and its transition in pathological aggregation.

DOI: https://doi.org/10.1038/s41467-025-63894-1
Commun Biol. 2025 Oct 03. 8(1): 1421

How SecB maintains clients in a translocation competent state.

Guillaume Roussel, Jochem H Smit, Dries Smets, Spyridoula Karamanou, Anastassios Economou.

Bacterial secretory proteins must remain soluble and non-folded until they reach the SecYEG translocase. Preprotein intrinsic features and chaperones can delay protein folding. The SecB chaperone is known to delay folding of some proteins, however the link between the folding state of a client and its interaction with SecB, until the client-transfer to the translocase remains elusive. This study unravels how a model client, maltose binding protein (MBP), is kept in a non-folded state by SecB. Using single-molecule FRET and hydrogen-deuterium exchange mass spectrometry, we describe in detail the folding pathway of the client and demonstrate that SecB acts first as an unfoldase, reverting partial folding and then as holdase, preventing folding. The presence of an SP delays the folding and stabilizes the client to SecB interaction. Single-point mutations that abolish formation of some foldons drastically increase the lifetime of the SecB-bound state. Towards delivery to the translocase, SecA interacts with the MBP:SecB complex forming a quaternary super-assembly thus, further stabilizing the disordered state of the client. Collectively, our study demonstrates the interplay between secretory chaperones and a model client, with SecB combining unfoldase and holdase activities to retain the client in a translocation-competent state while SecA secures this complex until later translocation.

DOI: https://doi.org/10.1038/s42003-025-08821-2
Am J Physiol Gastrointest Liver Physiol. 2025 Sep 29.

Pioglitazone Reduces Hepatic Alpha-1 Antitrypsin Accumulation Through Autophagy and AMPK Activation in Alpha-1 Antitrypsin Deficient Mice.

Yuanqing Lu, Regina Oshins, Nesmine R Maptue, Qingyang Shen, Chalermchai Khemtong, Kenneth Cusi, Mark Brantly, Nazli Khodayari.

  Background: Alpha-1 antitrypsin deficiency (AATD) is a genetic disorder characterized by accumulation of misfolded Z α-1 antitrypsin (ZAAT) in hepatocytes, leading to liver injury and metabolic dysfunction. There is no therapy to reduce ZAAT accumulation and restore proteostasis. Pioglitazone activates AMP-activated protein kinase (AMPK), enhance autophagy, and modulate ER stress responses, suggesting a potential effect on ZAAT clearance. Our objective is to examine whether pioglitazone can protect against AATD-mediated liver disease. Methods: Huh7.5 cells expressing ZAAT (HuhZ) and Pi*Z transgenic mice were used to investigate pioglitazone treatment on hepatic ZAAT accumulation, autophagy activation, and AMPK signaling. Histological, molecular, and metabolic analyses were conducted to assess changes in ZAAT content, autophagy markers, AMPK phosphorylation, and proteostasis. Results: Pioglitazone significantly reduced intracellular ZAAT and decreased lipid droplet accumulation in HuhZ cells. Pioglitazone markedly lowered hepatic ZAAT content in Pi*Z mice, suggesting enhanced degradation. This reduction was mediated through the AMPK pathway, indicated by increased phosphorylation of AMPK and ULK1. Pioglitazone induced autophagy, shown by decreased p62 and increased ATG5 and LC3B-II. This is indicative of enhanced autophagy. Although total hepatic AAT levels were reduced, PASD-positive ZAAT aggregates exhibited only a downward trend, suggesting these may be more resistant to clearance. Conclusion: These findings demonstrate pioglitazone reduces hepatic ZAAT accumulation by activating AMPK and inducing autophagy in AATD-associated liver disease, supporting its potential for therapeutic repurposing. As pioglitazone is FDA-approved with benefits for metabolic liver health, further studies are warranted to evaluate efficacy in restoring proteostasis and reducing hepatic ZAAT.

Keywords:  alpha-1 antitrypsin deficiency; autophagy; pioglitazone

DOI:  https://doi.org/10.1152/ajpgi.00272.2025
J Cell Physiol. 2025 Sep;240(9): e70094

Holding Out for a Model: Rhomboid Superfamily in Vertebrate Development and Disease.

Saroj Gourkanti, Yazmin Munoz, Jacqueline Cheung, Rosa M Chavez, Devanshi Agarwal, Taylor J Schoen, Kristina Solorio-Kirpichyan, Sonya E Neal.

  The rhomboid superfamily, comprising both proteases and pseudoproteases, has emerged as a central regulator of membrane biology, mediating diverse functions including protein quality control, signal transduction, trafficking, and more. While molecular mechanisms of rhomboid activity have been well-characterized in invertebrate and cell-based systems, their physiological role in vertebrate development remains limited and continues to evolve. Here, we review recent advances in cell culture systems and vertebrate models that uncover the developmental and disease-relevant functions of rhomboid family members, including RHBDLs, iRhoms, PARL, and Derlins. We outline their roles in embryogenesis, tissue regeneration, neurodevelopment, and immune signaling, alongside their pathological involvement in cancer, neurodegeneration, and metabolic disorders. We also emphasize the limitations posed by early embryonic lethality in knockout models and advocate for tissue-specific vertebrate models to dissect rhomboid-dependent pathways in vivo. Understanding how rhomboid proteins coordinate developmental processes will not only reveal fundamental principles of membrane-associated processes, but also open new avenues for therapeutic targeting in disease.

Keywords:  development; membrane; rhomboid proteins; vertebrate model

DOI:  https://doi.org/10.1002/jcp.70094