bims-proteo Biomed News
on Proteostasis
Issue of 2025–04–27
38 papers selected by
Eric Chevet, INSERM
  1. NEMF-mediated CAT tailing facilitates translocation-associated quality control.
  2. A hollow TFG condensate spatially compartmentalizes the early secretory pathway.
  3. ECLIPSE mediates selective degradation of inner nuclear membrane protein in plants.
  4. PERK-dependent reciprocal crosstalk between ER and non-centrosomal microtubules coordinates ER architecture and cell shape.
  5. Proteostasis landscapes of cystic fibrosis variants reveal drug response vulnerability.
  6. ByeTAC: Bypassing E-Ligase-Targeting Chimeras for Direct Proteasome Degradation.
  7. Pooled tagging and hydrophobic targeting of endogenous proteins for unbiased mapping of unfolded protein responses.
  8. TRIM52 maintains cellular fitness and is under tight proteolytic control by multiple giant E3 ligases.
  9. Targeted protein degradation in oncology: novel therapeutic opportunity for solid tumours?
  10. Deciphering disordered regions controlling mRNA decay in high-throughput.
  11. ATP2A2 regulates STING1/MITA-driven signal transduction including selective autophagy.
  12. Aberrant phase separation drives membranous organelle remodeling and tumorigenesis.
  13. Treatment of acute myeloid leukemia models by targeting a cell surface RNA-binding protein.
  14. Optimization of the PROTAC linker region of the proteasome substrate receptor hRpn13 rationalized structural modeling with molecular dynamics.
  15. Glucose sensing and the unfolded protein response.
  16. Cryo-EM structure of the AAA+ SPATA5 complex and its role in human cytoplasmic pre-60S maturation.
  17. Localizatome: a database for stress-dependent subcellular localization changes in proteins.
  18. Genotoxic stress triggers Scd6-dependent regulation of translation to modulate the DNA damage response.
  19. UBL7 is indispensable for spermiogenesis through protecting critical factors from excessive degradation by proteasomes.
  20. Endoplasmic reticulum stress: A key player in immune cell regulation and autoimmune disorders.
  21. The structural and functional dynamics of BiP and Grp94: opportunities for therapeutic discovery.
  22. The ubiquitin ligase NKLAM promotes apoptosis and suppression of cell growth.
  23. EvoWeaver: large-scale prediction of gene functional associations from coevolutionary signals.
  24. TOM20-driven E3 ligase recruitment regulates mitochondrial dynamics through PLD6.
  25. Endoplasmic reticulum facilitates the coordinated division of Salmonella-containing vacuoles.
  26. Defective autophagy in CD4 T cells drives liver fibrosis via type 3 inflammation.
  27. Evolution of tumor stress response during cytoreductive surgery for ovarian cancer.
  28. Global analysis of protein and small-molecule substrates of ubiquitin-like proteins (UBLs).
  29. Mass balance approximation of unfolding boosts potential-based protein stability predictions.
  30. Natural Spy Chaperone Mimic: Tailored Nanochaperone with Electrostatic-Hydrophobic Synergy To Enhance Protein Folding Regulation.
  31. SLAYER: a computational framework for identifying synthetic lethal interactions through integrated analysis of cancer dependencies.
  32. The evolution and diversification of the Hsp90 co-chaperone system.
  33. A high throughput compatible workflow for the biochemical identification and characterisation of molecular glues.
  34. Werner syndrome exonuclease promotes gut regeneration and causes age-associated gut hyperplasia in Drosophila.
  35. High throughput screening assay for the identification of ATF4 and TFEB activating compounds.
  36. RiboParser/RiboShiny: an integrated platform for comprehensive analysis and visualization of Ribo-seq data.
  37. Rab32 regulates Golgi structure and cell migration through Protein Kinase A-mediated phosphorylation of Optineurin.
  38. A Common CTRB misfolding variant associated with pancreatic cancer risk causes ER stress and inflammation in mice.
  1. J Cell Biol. 2025 Jun 02. pii: e202408199. [Epub ahead of print]224(6):
    NEMF-mediated CAT tailing facilitates translocation-associated quality control.
    Amanda Ennis, Lihui Wang, Yue Xu, Layla Saidi, Xiaorong Wang, Clinton Yu, Sijung Yun, Lan Huang, Yihong Ye.
      Ribosome stalling during co-translational translocation at the ER causes translocon clogging and impairs ER protein biogenesis. Mammalian cells resolve translocon clogging via a poorly characterized translocation-associated quality control (TAQC) process. Here, we combine a genome-wide CRISPR screen with live-cell imaging to dissect the molecular linchpin of TAQC. We show that TAQC substrates translated from mRNAs bearing a ribosome-stalling poly(A) sequence are degraded by lysosomes and the proteasome. By contrast, the degradation of defective nascent chains encoded by nonstop (NS) mRNAs involves an unconventional ER-associated protein degradation (ERAD) mechanism depending on ER-to-Golgi trafficking, KDEL-mediated substrate retrieval at the Golgi, and a tRNA-binding factor NEMF that appends an aggregation-prone carboxyl tail to stalled NS nascent chains. We propose that NEMF-mediated CAT tailing targets a subset of TAQC substrates via Golgi retrieval for ERAD, safeguarding ER homeostasis.
    DOI:  https://doi.org/10.1083/jcb.202408199
  2. Nat Commun. 2025 Apr 19. 16(1): 3715
    A hollow TFG condensate spatially compartmentalizes the early secretory pathway.
    Savannah M Bogus, William R Wegeng, Miguel Ruiz, Sindy R Chavez, Samantha N Cheung, Khalid S M Noori, Ingrid R Niesman, Andreas M Ernst.
      In the early secretory pathway, endoplasmic reticulum (ER) and Golgi membranes form a nearly spherical interface. In this ribosome-excluding zone, bidirectional transport of cargo coincides with a spatial segregation of anterograde and retrograde carriers by an unknown mechanism. We show that at physiological conditions, the Trk-fused gene (TFG) self-organizes to form a hollow, anisotropic condensate that matches the dimensions of the ER-Golgi interface and is dynamically regulated across the cell cycle. Regularly spaced hydrophobic residues in TFG control the condensation mechanism and result in a porous condensate surface. We find that TFG condensates act as a molecular sieve capable of allowing access of anterograde coats (COPII) to the condensate interior while restricting retrograde coats (COPI). We propose that a hollow TFG condensate structures the ER-Golgi interface to create a diffusion-limited space for anterograde transport. We further propose that TFG condensates optimize membrane flux by insulating secretory carriers in their lumen from retrograde carriers outside TFG cages.
    DOI:  https://doi.org/10.1038/s41467-025-59118-1
  3. J Exp Bot. 2025 Apr 23. pii: eraf167. [Epub ahead of print]
    ECLIPSE mediates selective degradation of inner nuclear membrane protein in plants.
    Enrico Calvanese, Min Jia, Olivia Xie, Yangnan Gu.
      The inner nuclear membrane (INM) hosts a unique set of membrane proteins essential for nuclear functions. Proteolytic removal of mislocalized or defective membrane proteins is of critical importance for maintaining the homeostasis and integrity of the INM. Previous studies revealed that INM protein degradation depends on a specialized ubiquitin-proteasome system termed INM-Associated Degradation (INMAD) in plants, requiring the CDC48 complex and the 26S proteasome for membrane protein retrotranslocation and destruction, respectively. However, adaptor proteins that link membrane substrates to the CDC48/proteasome degradation machinery remain missing in the pathway. Here, we report the discovery of ECLIPSE, a previously uncharacterized protein that may serve as such a molecular bridge in the degradation of the conserved INM protein SUN1. We demonstrate that ECLIPSE physically associates with CDC48 and exhibits strong transcriptional co-regulation with multiple established plant INMAD components. Mechanistically, ECLIPSE may act as an adaptor through its dual-domain architecture: its C-terminal PUB domain mediates direct interaction with CDC48, while its N-terminal Ubiquitin-Associated domain recognizes ubiquitinated INM substrates. Genetic and biochemical analyses further established that ECLIPSE is required for SUN1 protein degradation in Arabidopsis, supporting its role in the turnover of at least some inner nuclear membrane proteins in plants.
    Keywords:  CDC48; ECLIPSE; INM-Associated Degradation (INMAD); Inner nuclear membrane (INM); PLANT UBX DOMAIN-CONTAINING (PUX) protein; SUN1; UBIQUITIN REGULATORY X (UBX); UBIQUITIN-PROTEASOME SYSTEM (UPS); adaptor proteins; membrane-associated protein degradation
    DOI:  https://doi.org/10.1093/jxb/eraf167
  4. Cell Rep. 2025 Apr 15. pii: S2211-1247(25)00361-4. [Epub ahead of print] 115590
    PERK-dependent reciprocal crosstalk between ER and non-centrosomal microtubules coordinates ER architecture and cell shape.
    Miguel Sánchez-Álvarez, Fidel Nicolás Lolo, Heba Sailem, Giulio Fulgoni, Patricia Pascual-Vargas, Lucía Agüera, Mauro Catalá-Montoro, Mar Arias-García, Juan Antonio López, Jesús Vázquez, Miguel Ángel Del Pozo, Chris Bakal.
      The architecture of the endoplasmic reticulum (ER) is a key determinant of its function. Its dynamics are linked to those of the cytoskeleton, but our understanding of how this coordination occurs and what its functional relevance is, limited. Here, we report that the unfolded protein response (UPRER) transducer EIF2AK3/PERK (eukaryotic translation initiation factor 2-alpha kinase 3/protein kinase R-like endoplasmic reticulum kinase) is essential for acute-stress-induced peripheral redistribution and remodeling of the ER through eukaryotic initiation factor 2 alpha (eIF2α) phosphorylation and translation initiation shutdown. PERK-mediated eIF2α phosphorylation can be bypassed by blocking polysome assembly, depleting microtubule (MT)-anchoring ER proteins such as p180/RRBP1 (ribosome-binding protein 1), or disrupting the MT cytoskeleton. Specific disruption of non-centrosomal MTs, but not centrosome depletion, rescues ER redistribution in PERK-deficient cells. Conversely, PERK deficiency stabilizes non-centrosomal MTs against proteasomal degradation, promoting polarized protrusiveness in epithelial cells and neuroblasts. Thus, PERK coordinates ER architecture and homeostasis with cell morphogenesis by coupling ER remodeling and non-centrosomal MT stability and dynamics.
    Keywords:  CP: Cell biology; EIF2AK3/PERK; cell polarity; endoplasmic reticulum; integrated stress response; non-centrosomal microtubules
    DOI:  https://doi.org/10.1016/j.celrep.2025.115590
  5. Proc Natl Acad Sci U S A. 2025 Apr 29. 122(17): e2418407122
    Proteostasis landscapes of cystic fibrosis variants reveal drug response vulnerability.
    Eli Fritz McDonald, Minsoo Kim, John A Olson, Jens Meiler, Lars Plate.
      Cystic fibrosis (CF) is a lethal genetic disorder caused by variants in CF transmembrane conductance regulator (CFTR). Many variants are treatable with correctors, which enhance the folding and trafficking of CFTR. However, approximately 3% of persons with CF harbor poorly responsive variants. Here, we used affinity purification mass spectrometry proteomics to profile the protein homeostasis (proteostasis) changes of CFTR variants during correction to assess modulated interactions with protein folding and maturation pathways. Responsive variant interactions converged on similar proteostasis pathways during correction. In contrast, poorly responsive variants subtly diverged, revealing a partial restoration of protein quality control surveillance and partial correction. Computational structural modeling showed that corrector VX-445 failed to confer enough NBD1 stability to poor responders. NBD1 secondary stabilizing mutations rescued poorly responsive variants, revealing structural vulnerabilities in NBD1 required for treating poor responders. Our study provides a framework for discerning the underlying protein quality control and structural defects of CFTR variants not reached with existing drugs to expand therapeutics to all susceptible CFTR variants.
    Keywords:  correctors; cystic fibrosis; drug response; proteostasis; variants
    DOI:  https://doi.org/10.1073/pnas.2418407122
  6. J Med Chem. 2025 Apr 19.
    ByeTAC: Bypassing E-Ligase-Targeting Chimeras for Direct Proteasome Degradation.
    Cody A Loy, Eslam M H Ali, Laurence J Seabrook, Timothy J Harris, Kate A Kragness, Lauren Albrecht, Darci J Trader.
      The development of targeted protein degradation by recruiting a protein of interest to a ubiquitin ligase to facilitate its degradation has become a powerful therapeutic tool. The potential of this approach is limited to proteins that can be readily ubiquitinated and relies on having a ligand with the various E3 ligases. Here, we describe a new methodology for targeted protein degradation that directly recruits a protein of interest to the proteasome for degradation. We generated bifunctional molecules that incorporate a small molecule ligand into a subunit on the 26S proteasome that recruits the protein directly for degradation. ByeTAC degradation requires binding to Rpn-13, a nonessential ubiquitin receptor of the 26S proteasome, and the protein of interest and does not have to rely on the E ligase cascade for ubiquitination. The ByeTAC methodology demonstrates the application of directly recruiting a protein to the proteasome via interactions with Rpn-13 for degradation.
    DOI:  https://doi.org/10.1021/acs.jmedchem.5c00485
  7. Mol Cell. 2025 Apr 18. pii: S1097-2765(25)00305-3. [Epub ahead of print]
    Pooled tagging and hydrophobic targeting of endogenous proteins for unbiased mapping of unfolded protein responses.
    Stephanie E Sansbury, Yevgeniy V Serebrenik, Tomer Lapidot, David G Smith, George M Burslem, Ophir Shalem.
      To achieve system-level insights into proteome organization, regulation, and function, we developed an approach to generate complex cell pools with endogenously tagged proteins amenable to high-throughput visualization and perturbation. Pooled imaging coupled to in situ barcode sequencing identified the subcellular localization of each HaloTag-tagged protein, and subsequent ligand-induced misfolding of the library followed by single-cell RNA sequencing revealed responses to spatially restricted protein misfolding. These datasets characterized protein quality control responses in previously uninterrogated cellular compartments, and cross-compartment analyses revealed mutually exclusive rather than collaborative responses, whereby the heat shock response (HSR) is induced in some compartments and repressed in others where autophagy genes are induced. We further assign protein quality control functions to previously uncharacterized genes based on shared transcriptional responses to protein misfolding across cellular compartments. Altogether, we present an efficient method for large-scale studies of proteome dynamics, function, and homeostasis.
    Keywords:  hydrophobic targeting; in situ sequencing; pooled tagging; protein localization; protein misfolding; proteostasis
    DOI:  https://doi.org/10.1016/j.molcel.2025.04.002
  8. Nat Commun. 2025 Apr 24. 16(1): 3894
    TRIM52 maintains cellular fitness and is under tight proteolytic control by multiple giant E3 ligases.
    Alexandra Shulkina, Kathrin Hacker, Julian F Ehrmann, Valentina Budroni, Ariane Mandlbauer, Johannes Bock, Daniel B Grabarczyk, Genevieve Edobor, Luisa Cochella, Tim Clausen, Gijs A Versteeg.
      Tripartite motif 52 (TRIM52) exhibits strong positive selection in humans, yet is lost in many other mammals. In contrast to what one would expect for such a non-conserved factor, TRIM52 loss compromises cell fitness. We set out to determine the cellular function of TRIM52. Genetic and proteomic analyses revealed TRIM52 physically and functionally interacts with the DNA repair machinery. Our data suggest that TRIM52 limits topoisomerase 2 adducts, thereby preventing cell-cycle arrest. Consistent with a fitness-promoting function, TRIM52 is upregulated in various cancers, prompting us to investigate its regulatory pathways. We found TRIM52 to be targeted for ultra-rapid proteasomal degradation by the giant E3 ubiquitin ligases BIRC6, HUWE1, and UBR4/KCMF1. BIRC6 mono-ubiquitinates TRIM52, with subsequent extension by UBR4/KCMF1. These findings suggest a role for TRIM52 in maintaining genome integrity, and regulation of its own abundance through multi-ligase degradation.
    DOI:  https://doi.org/10.1038/s41467-025-59129-y
  9. Mol Oncol. 2025 Apr 23.
    Targeted protein degradation in oncology: novel therapeutic opportunity for solid tumours?
    Noé Herbel, Sophie Postel-Vinay.
      Targeted and immune therapies have improved patient outcomes in selected diseases. Still, resistance inevitably occurs, and a significant portion of the proteome remains undruggable due to target localisation, structural or functional constraints. Targeted protein degraders (TPDs) represent a promising strategy to expand druggable targets by redirecting the ubiquitin-proteasome system to selectively degrade proteins of interest (POI). TPDs include proteolysis-targeting chimeras (PROTACs), which are heterobifunctional molecules that create a ternary complex with the POI and the E3 ligase, and molecular glues (MGs), which are monovalent small molecules that create an interface between an E3 ligase and the POI. Here, we provide a viewpoint on novel therapeutic opportunities offered by TPDs, notably through the targeting of previously undruggable proteins or overcoming some resistance mechanisms. We further present challenges that will need to be addressed in order to optimise clinical development, including dose optimisation, patient selection and drug delivery.
    Keywords:  E3 ligase; PROTAC; molecular glue; targeted protein degradation; undruggable proteome
    DOI:  https://doi.org/10.1002/1878-0261.70034
  10. Nature. 2025 Apr 23.
    Deciphering disordered regions controlling mRNA decay in high-throughput.
    Joseph H Lobel, Nicholas T Ingolia.
      Intrinsically disordered regions within proteins drive specific molecular functions despite lacking a defined structure1,2. Although disordered regions are integral to controlling mRNA stability and translation, the mechanisms underlying these regulatory effects remain unclear3. Here we reveal the molecular determinants of this activity using high-throughput functional profiling. Systematic mutagenesis across hundreds of regulatory disordered elements, combined with machine learning, reveals a complex pattern of molecular features important for their activity. The presence and arrangement of aromatic residues strongly predicts the ability of seemingly diverse protein sequences to influence mRNA stability and translation. We further show how many of these regulatory elements exert their effects by engaging core mRNA decay machinery. Our results define molecular features and biochemical pathways that explain how disordered regions control mRNA expression and shed light on broader principles within functional, unstructured proteins.
    DOI:  https://doi.org/10.1038/s41586-025-08919-x
  11. Autophagy. 2025 Apr 23.
    ATP2A2 regulates STING1/MITA-driven signal transduction including selective autophagy.
    Xue Yang, Linyue Lv, Yuelan Zhang, Zhuyou Zhang, Shaowei Zeng, Xinyi Zhang, Qinyang Wang, Martin Dorf, Shitao Li, Bishi Fu.
      STING1/MITA not only induces innate immune responses but also triggers macroautophagy/autophagy to selectively degrade signaling molecules. However, the molecular mechanisms regulating STING1-mediated selective autophagy remain unclear. Here, we first report that ATP2A2 directly interacts with STING1, regulating STING1-mediated innate immune response by modulating its polymerization and trafficking, thereby inhibiting DNA virus infection. Notably, while screening for reticulophagy receptors involved in STING1-mediated selective autophagy, we identified SEC62 as an important receptor protein in STING1-mediated reticulophagy. Mechanistically, SEC62 strengthens its interaction with STING1 upon activation and concurrently facilitates STING1-mediated reticulophagy upon starvation, which are dependent on ATP2A2. Furthermore, knocking down SEC62 in WT cells inhibits STING1-mediated MAP1LC3B/LC3B lipidation and autophagosome formation, an effect that is lost in ATP2A2 knockout cells, suggesting that SEC62's role in STING1-mediated selective autophagy is ATP2A2 dependent. Thus, our findings identify the reticulophagy receptor SEC62 as a novel receptor protein regulating STING1-mediated selective autophagy, providing new insight into the mechanism regarding a reticulophagy receptor in the process of STING1-induced selective autophagy.
    Keywords:  ATP2A2; Antiviral; SEC62; STING1/MITA; innate immunity; selective autophagy
    DOI:  https://doi.org/10.1080/15548627.2025.2496786
  12. Mol Cell. 2025 Apr 18. pii: S1097-2765(25)00304-1. [Epub ahead of print]
    Aberrant phase separation drives membranous organelle remodeling and tumorigenesis.
    Xinyu Wang, Amin Jiang, Quan Meng, Tao Jiang, Huaide Lu, Xiaohan Geng, Zikuo Song, Xinyao Hu, Zhu Yu, Wencong Xu, Chao Ning, Yajing Lin, Dong Li.
      Membrane remodeling is essential for numerous cellular functions. Although liquid-liquid phase separation (LLPS) of intrinsically disordered region (IDR)-rich proteins could drive dramatic membrane remodeling of artificial giant unilamellar vesicles, it remains elusive whether LLPS-mediated membrane-remodeling functions in live cells and what role it plays in specific bioprocesses. Here, we show that three IDR-rich integral transmembrane fusion proteins (MFPs), generated by chromosomal translocations, can lead to de novo remodeling of their located membranous organelles. Taking FUS-CREB3L2, prevalent in low-grade fibromyxoid sarcoma (LGFMS), as a proof of concept, we recorded super-resolution long-time imaging of endoplasmic reticulum (ER) remodeling dynamics as accumulating FUS-CREB3L2, meanwhile causing spontaneous ER stress to hijack the X-box-binding protein 1 (XBP1) pathway. We further reveal the underlying mechanisms of how FUS-CREB3L2 transduces its tumorigenic signals and aberrant LLPS effects from the ER membrane into the nucleus autonomously, which activates hundreds of LGFMS-specific genes de novo compared with CREB3L2, thus sufficiently reprogramming the cells into an LGFMS-like status.
    Keywords:  ER stress; FUS-CREB3L2; fusion proteins; membrane remodeling; phase separation; spontaneous regulated intramembrane proteolysis
    DOI:  https://doi.org/10.1016/j.molcel.2025.04.001
  13. Nat Biotechnol. 2025 Apr 23.
    Treatment of acute myeloid leukemia models by targeting a cell surface RNA-binding protein.
    Benson M George, Maria Eleftheriou, Eliza Yankova, Jonathan Perr, Peiyuan Chai, Gianluca Nestola, Karim Almahayni, Siân Evans, Aristi Damaskou, Helena Hemberger, Charlotta G Lebedenko, Justyna Rak, Qi Yu, Ece Bapcum, James Russell, Jaana Bagri, Regan F Volk, Malte Spiekermann, Richard M Stone, George Giotopoulos, Brian J P Huntly, Joanna Baxter, Fernando Camargo, Jie Liu, Balyn W Zaro, George S Vassiliou, Leonhard Möckl, Jorge de la Rosa, Ryan A Flynn, Konstantinos Tzelepis.
      Immunotherapies for acute myeloid leukemia (AML) and other cancers are limited by a lack of tumor-specific targets. Here we discover that RNA-binding proteins and glycosylated RNAs (glycoRNAs) form precisely organized nanodomains on cancer cell surfaces. We characterize nucleophosmin (NPM1) as an abundant cell surface protein (csNPM1) on a variety of tumor types. With a focus on AML, we observe csNPM1 on blasts and leukemic stem cells but not on normal hematopoietic stem cells. We develop a monoclonal antibody to target csNPM1, which exhibits robust anti-tumor activity in multiple syngeneic and xenograft models of AML, including patient-derived xenografts, without observable toxicity. We find that csNPM1 is expressed in a mutation-agnostic manner on primary AML cells and may therefore offer a general strategy for detecting and treating AML. Surface profiling and in vivo work also demonstrate csNPM1 as a target on solid tumors. Our data suggest that csNPM1 and its neighboring glycoRNA-cell surface RNA-binding protein (csRBP) clusters may serve as an alternative antigen class for therapeutic targeting or cell identification.
    DOI:  https://doi.org/10.1038/s41587-025-02648-2
  14. J Biol Chem. 2025 Apr 18. pii: S0021-9258(25)00369-2. [Epub ahead of print] 108520
    Optimization of the PROTAC linker region of the proteasome substrate receptor hRpn13 rationalized structural modeling with molecular dynamics.
    Xiuxiu Lu, Venkata R Sabbasani, Bakar Hassan, Rolf E Swenson, Kylie J Walters.
      Proteasome substrate receptor hRpn13 is a promising target for cancer therapy. hRpn13 PROTACs induce apoptosis by targeting the hRpn13 proteolytic product hRpn13Pru, which contains an intact ubiquitin- and proteasome-binding Pru domain. We generated a PROTAC series based on hRpn13Pru-targeting XL5 by varying the linker that connects it to a warhead against the VHL-based ubiquitin E3 ligase machinery. Among eight tested derivatives, XL5-VHL-7 with a -(CH2)5- alkyl linker promoted hRpn13Pru degradation and induced cellular apoptosis with 2-fold improved potency compared to the original PROTAC. By using this PROTAC series with slight chemical modifications in the linker region, we were able to evaluate the efficacy of structural modeling with molecular dynamics for refining PROTACs. Overall, we found that the experimental data correlated with efficacy predictions based on molecular dynamics and structural modeling. Moreover, we could observe hRpn13:PROTAC:VHL complexes by 2D NMR that support the structural modeling and stronger affinity of XL5-VHL-7 compared to the original hRpn13 PROTAC. Our NMR data further indicate that hRpn13 Pru affinity for XL5-VHL-7 is higher within the VHL complex present than with XL5-VHL-7 alone. Altogether, we develop an hRpn13 PROTAC with 2-fold increased potency by optimizing the linker and demonstrate the current benefit and limitations for including modeling with molecular dynamics to aid PROTAC optimization.
    Keywords:  NMR; PROTAC; hRpn13; molecular dynamics; molecular modeling; proteasome; protein degradation
    DOI:  https://doi.org/10.1016/j.jbc.2025.108520
  15. FEBS J. 2025 Apr 24.
    Glucose sensing and the unfolded protein response.
    Mabel Cruz-Rodríguez, Eric Chevet, Cristina Muñoz-Pinedo.
      The unfolded protein response (UPR) is activated primarily upon alteration of protein folding in the endoplasmic reticulum (ER). This occurs under physiological situations that cause an abrupt increase in protein synthesis, or under redox and metabolic stresses. Among the latter, hyperglycemia and glucose scarcity have been identified as major modulators of UPR signaling. Indeed, the first mammalian UPR effector, the glucose-regulated protein 78, also known as BiP, was identified in response to glucose deprivation. Tunicamycin, arguably the most commonly used drug to induce ER stress responses in vitro and in vivo, is an inhibitor of N-glycosylation. We compile here evidence that the UPR is activated upon physiological and pathological conditions that alter glucose levels and that this is mostly mediated by alterations of protein N-glycosylation, ATP levels, or redox balance. The three branches of the UPR transduced by PERK/ATF4, IRE1/XBP1s, and ATF6, as well as non-canonical ER sensors such as SCAP/SREBP, sense ER protein glycosylation status driven by glucose and other glucose-derived metabolites. The outcomes of UPR activation range from restoring protein N-glycosylation and protein folding flux to stimulating autophagy, organelle recycling, and mitochondrial respiration, and in some cases, cell death. Anabolic responses to glucose levels are also stimulated by glucose through components of the UPR. Therefore, the UPR should be further studied as a potential biomarker and mediator of glucose-associated diseases.
    Keywords:  ATF6; IRE1; PERK; glucose; glycosylation; nutrient sensing; starvation; unfolded protein response
    DOI:  https://doi.org/10.1111/febs.70113
  16. Nat Commun. 2025 Apr 23. 16(1): 3806
    Cryo-EM structure of the AAA+ SPATA5 complex and its role in human cytoplasmic pre-60S maturation.
    Yuhao Dai, Damu Wu, Ningning Li, Chengying Ma, Yunyang Zhang, Ning Gao.
      Eukaryotic ribosome biogenesis is an energy-consuming process involving many ATPase-driven steps. In yeast, AAA+ protein Drg1 releases an assembly factor Rlp24, a placeholder for Rpl24, from pre-60S particles just exported to cytosol. The equivalent process in human cells involves SPATA5 (Drg1 homolog) and additional factors. However, the mechanistic details remain unclear. Here we reveal that SPATA5 forms a 4:2:2:2 complex with SPATA5L1, C1orf109, and CINP. This complex features an N-terminal ring made of C1orf109, CINP and NTDs of SPATA5/SPATA5L1, and two hexameric AAA+ ATPase rings. Intriguingly, a conserved cysteine C672 in the P-loop of SPATA5 is sulfinylated, generating an inactive conformation incompatible with ATP binding. We also obtained a cryo-EM structure of pre-60S-bound SPATA5 complex. Different from yeast, the recognition of the pre-60S particle is mediated by human-specific factor CINP, through two distinct sets of interactions: one with GTPBP4 and the other with ES27A. Taken together, these data provide structural basis for understanding the cytoplasmic maturation of the pre-60S, and reveal human-specific features that might be harnessed for therapeutic purposes.
    DOI:  https://doi.org/10.1038/s41467-025-58894-0
  17. Database (Oxford). 2025 Apr 21. pii: baaf028. [Epub ahead of print]2025
    Localizatome: a database for stress-dependent subcellular localization changes in proteins.
    Takahide Matsushima, Yuki Naito, Tomoki Chiba, Ryota Kurimoto, Keiko Itano, Koji Ochiai, Koichi Takahashi, Naoki Goshima, Hiroshi Asahara.
      Understanding protein subcellular localization and its dynamic changes is crucial for elucidating cellular function and disease mechanisms, particularly under stress conditions, where protein localization changes can modulate cellular responses. Currently available databases provide insights into protein localization under steady-state conditions; however, stress-related dynamic localization changes remain poorly understood. Here, we present the Localizatome, a comprehensive database that captures stress-induced protein localization dynamics in living cells. Using an original high-throughput microscopy system and machine learning algorithms, we analysed the localization patterns of 10 287 fluorescent protein-fused human proteins in HeLa cells before and after exposure to oxidative stress. Our analysis revealed that 1910 proteins exhibited oxidative stress-dependent localization changes, particularly forming distinct foci. Among them, there were stress granule assembly factors and autophagy-related proteins, as well as components of various signalling pathways. Subsequent characterization identified some specific amino acid motifs and intrinsically disordered regions associated with stress-induced protein redistribution. The Localizatome provides open access to these data through a web-based interface, supporting a wide range of studies on cellular stress response and disease mechanisms. Database URL https://localizatome.embrys.jp/.
    DOI:  https://doi.org/10.1093/database/baaf028
  18. EMBO Rep. 2025 Apr 24.
    Genotoxic stress triggers Scd6-dependent regulation of translation to modulate the DNA damage response.
    Gayatri Mohanan, Raju Roy, Hélène Malka-Mahieu, Swati Lamba, Lucilla Fabbri, Sidhant Kalia, Anusmita Biswas, Sylvain Martineau, Céline M Labbé, Stéphan Vagner, Purusharth I Rajyaguru.
      The role of mRNA translation and decay in the genotoxic stress response remains poorly explored. Here, we identify the role of yeast RGG motif-containing RNA binding protein Scd6 and its human ortholog LSM14A in genotoxic stress response. Scd6 localizes to cytoplasmic puncta upon cell treatment with various genotoxic agents. Scd6 genetically interacts with SRS2, a DNA helicase with an anti-recombination role in DNA damage repair under HU stress. Scd6 directly interacts with the SRS2 mRNA to repress its translation in cytoplasmic granules upon HU stress in an eIF4G1-independent manner. Scd6-SRS2 interaction is modulated by arginine methylation and the LSm-domain of Scd6, which acts as a cis-regulator of Scd6 arginine methylation. LSM14A regulates the translation of mRNAs encoding key NHEJ (Non-homologous end-joining) proteins such as RTEL1 (SRS2 functional homolog) and LIG4. NHEJ activity in yeast and mammalian cells is regulated by Scd6 and LSM14A, respectively. Overall, this report unveils the role of RNA binding proteins in regulating the translation of specific mRNAs coding for DNA damage response proteins upon genotoxic stress.
    Keywords:  DNA Damage; Genotoxic Stress; RNA Binding Protein; RNA Granules; mRNA Translation
    DOI:  https://doi.org/10.1038/s44319-025-00443-3
  19. Nat Commun. 2025 Apr 23. 16(1): 3803
    UBL7 is indispensable for spermiogenesis through protecting critical factors from excessive degradation by proteasomes.
    Tianyi Yuan, Jiajun Yang, Dan Xu, Huiqi Li, Wanping Min, Fengchao Wang.
      Spermiogenesis is a tightly regulated process to produce mature sperm cells. The ubiquitin-proteasome system (UPS) plays a crucial role in controlling protein half-life and is essential for spermiogenesis. Recently, proteins containing ubiquitin-like domains and ubiquitin-associated domains (UBL-UBA proteins) have emerged as novel regulators within the UPS. In this study, we demonstrate that UBL7, a testis-enriched UBL-UBA protein, is indispensable for sperm formation. Deficiency of UBL7 leads to severe malformations of both the sperm tail and head. Mechanistically, UBL7 interacts with the valosin-containing protein (VCP) complex and proteasomes, and shuttles substrates between them. Notably, UBL7 slows down the degradation rates of substrates involved in endoplasmic reticulum-associated degradation (ERAD) within cells. Through a two-step immunoprecipitation method, we identify several essential factors in spermatids that are protected by UBL7, including factors involved in the development of manchette (such as IFT140), head-tail coupling apparatus (such as SPATA20) and cytoplasmic droplets (such as HK1 and SLC2a3). In summary, our findings highlight UBL7 as a guardian that protects crucial factors from excessive degradation and thereby ensures successful spermiogenesis.
    DOI:  https://doi.org/10.1038/s41467-025-59209-z
  20. Semin Immunol. 2025 Apr 22. pii: S1044-5323(25)00026-0. [Epub ahead of print]78 101954
    Endoplasmic reticulum stress: A key player in immune cell regulation and autoimmune disorders.
    Marion Moreews, Mikael C I Karlsson.
      The endoplasmic reticulum (ER) is a large organelle, found in all eukaryotes, that is essential for normal cellular function. This function encompasses protein folding and quality control, post-translational modifications, lipid regulation, and the storage of intracellular calcium, among others. These diverse processes are essential for maintaining proteome stability. Therefore, a robust surveillance system is established under stress to ensure cell homeostasis. Sources of stress can originate from the cellular environment, including nutrient deprivation, hypoxia, and low pH, as well as from endogenous signals within the cell, such as metabolic challenges and increased demands for protein production. When cellular homeostasis is altered by one of these triggers, ER primary functions are altered which leads to the accumulation of misfolded proteins. These impaired proteins trigger the activation of the Unfolded Protein Response (UPR) pathway. This response aims at reducing ER stress by implementing the induction of complex programs to restore cell homeostasis. However, extended ER stress can modify the UPR response, shifting its signals from promoting survival to triggering pathways that reprogram or eliminate affected cells.
    Keywords:  Autoimmunity; B cells; ER stress
    DOI:  https://doi.org/10.1016/j.smim.2025.101954
  21. Trends Pharmacol Sci. 2025 Apr 18. pii: S0165-6147(25)00044-6. [Epub ahead of print]
    The structural and functional dynamics of BiP and Grp94: opportunities for therapeutic discovery.
    Ikponwmosa Obaseki, Chioma C Ndolo, Ayodeji A Adedeji, Hannah O Popoola, Andrea N Kravats.
      Binding immunoglobulin protein (BiP) and glucose-regulated protein 94 (Grp94) are endoplasmic reticulum (ER)-localized molecular chaperones that ensure proper protein folding and maintain protein homeostasis. However, overexpression of these chaperones during ER stress can contribute to disease progression in numerous pathologies. Although these chaperones represent promising therapeutic targets, their inhibition has been challenged by gaps in understanding of targetable chaperone features and their complex biology. To overcome these challenges, a new assay has been developed to selectively target BiP, and compounds that exploit subtle conformational changes of Grp94 have been designed. This review summarizes recent advances in elucidating structural and functional dynamics of BiP and Grp94. We explore leveraging this information to develop novel therapeutic interventions. Finally, given the recent advances in computing, we discuss how machine learning methods can be used to accelerate drug discovery efforts.
    Keywords:  BiP; Grp94; drug design; endoplasmic reticulum stress; molecular chaperones
    DOI:  https://doi.org/10.1016/j.tips.2025.03.004
  22. J Biol Chem. 2025 Apr 22. pii: S0021-9258(25)00376-X. [Epub ahead of print] 108527
    The ubiquitin ligase NKLAM promotes apoptosis and suppression of cell growth.
    Paul A Willard, Jacki Kornbluth.
      Natural Killer Lytic Associated Molecule (NKLAM), also known as RNF19b, is a member of the RING-in between-RING-RING (RBR) E3 ubiquitin ligase family and plays a pivotal role in immune regulation. We identified a critical cysteine residue at position 301 essential for NKLAM's ubiquitin ligase function. Site-directed mutagenesis of this residue to serine or alanine abrogated the ligase activity of NKLAM. Utilizing inducible expression systems in two different cell lines, HEK293 embryonic kidney cells and K562 myeloid leukemia cells, we demonstrated that wild-type (WT) NKLAM, but not the catalytically inactive NKLAM alanine mutant (C301A), inhibited cellular proliferation, as evidenced by reduced cell numbers and decreased metabolic activity. Moreover, NKLAM expression led to a significant decrease in the abundance and stability of the proto-oncogene c-Myc, a key regulator of proliferation. NKLAM facilitated the proteasomal degradation of c-Myc, with a reduction in c-Myc half-life from 27 minutes to 12 minutes and restoration of c-Myc levels upon proteasome inhibition. Notably, prolonged NKLAM expression induced apoptosis, measured by annexin-V staining and caspase activation. Strikingly, the serine mutant, C301S, while lacking ubiquitin ligase activity, induced apoptosis comparable to WT NKLAM, highlighting an alternative pathway for NKLAM-mediated inhibition of cellular homeostasis. Our findings indicate that NKLAM is a cytolytic protein with multifaceted roles in cellular proliferation and apoptosis.
    Keywords:  E3 ubiquitin ligase; NKLAM; RBR ubiquitin ligase; RNF19b; apoptosis; cell death; cell metabolism; c‐Myc; natural killer cells
    DOI:  https://doi.org/10.1016/j.jbc.2025.108527
  23. Nat Commun. 2025 Apr 24. 16(1): 3878
    EvoWeaver: large-scale prediction of gene functional associations from coevolutionary signals.
    Aidan H Lakshman, Erik S Wright.
      The known universe of uncharacterized proteins is expanding far faster than our ability to annotate their functions through laboratory study. Computational annotation approaches rely on similarity to previously studied proteins, thereby ignoring unstudied proteins. Coevolutionary approaches hold promise for injecting new information into our knowledge of the protein universe by linking proteins through 'guilt-by-association'. However, existing coevolutionary algorithms have insufficient accuracy and scalability to connect the entire universe of proteins. We present EvoWeaver, a method that weaves together 12 signals of coevolution to quantify the degree of shared evolution between genes. EvoWeaver accurately identifies proteins involved in protein complexes or separate steps of a biochemical pathway. We show the merits of EvoWeaver by partly reconstructing known biochemical pathways without any prior knowledge other than that available from genomic sequences. Applying EvoWeaver to 1545 gene groups from 8564 genomes reveals missing connections in popular databases and potentially undiscovered links between proteins.
    DOI:  https://doi.org/10.1038/s41467-025-59175-6
  24. Nat Chem Biol. 2025 Apr 22.
    TOM20-driven E3 ligase recruitment regulates mitochondrial dynamics through PLD6.
    Anat Raiff, Shidong Zhao, Aizat Bekturova, Colin Zenge, Shir Mazor, Xinyan Chen, Wenwen Ru, Yaara Makaros, Tslil Ast, Alban Ordureau, Chao Xu, Itay Koren.
      Mitochondrial homeostasis is maintained through complex regulatory mechanisms, including the balance of mitochondrial dynamics involving fusion and fission processes. A central player in this regulation is the ubiquitin-proteasome system (UPS), which controls the degradation of pivotal mitochondrial proteins. In this study, we identified cullin-RING E3 ligase 2 (CRL2) and its substrate receptor, FEM1B, as critical regulators of mitochondrial dynamics. Through proteomic analysis, we demonstrate here that FEM1B controls the turnover of PLD6, a key regulator of mitochondrial dynamics. Using structural and biochemical approaches, we show that FEM1B physically interacts with PLD6 and that this interaction is facilitated by the direct association of FEM1B with the mitochondrial import receptor TOM20. Ablation of FEM1B or disruption of the FEM1B-TOM20 interaction impairs PLD6 degradation and induces mitochondrial defects, phenocopying PLD6 overexpression. These findings underscore the importance of FEM1B in maintaining mitochondrial morphology and provide further mechanistic insights into how the UPS regulates mitochondrial homeostasis.
    DOI:  https://doi.org/10.1038/s41589-025-01894-4
  25. mBio. 2025 Apr 24. e0011425
    Endoplasmic reticulum facilitates the coordinated division of Salmonella-containing vacuoles.
    Umesh Chopra, Priyanka Bhansali, Subba Rao Gangi Setty, Dipshikha Chakravortty.
      Salmonella Typhimurium (STM) resides in a membrane-bound compartment called the Salmonella-containing vacuole (SCV) in several infected cell types where bacterial and SCV division occur synchronously to maintain a single bacterium per vacuole. However, the mechanism behind this synchronous fission is not well understood. Fission of intracellular organelles is known to be regulated by the dynamic tubular endoplasmic reticulum (ER). In this study, we evaluated the role of ER in controlling SCV division. Interestingly, Salmonella-infected cells show activation of the unfolded protein response (UPR) and expansion of ER tubules. Altering the expression of ER morphology regulators, such as reticulon-4a (Rtn4a) and CLIMP63, significantly impacted bacterial proliferation, suggesting a potential role of tubular ER in facilitating SCV division. Live-cell imaging revealed the marking of tubular ER at the center of 78% of SCV division sites. This study also explored the role of SteA (a known Salmonella effector in modulating membrane dynamics) in coordinating the SCV division. SteA resides on the SCV membranes and helps form membrane contact between SCV and ER. The colocalization of ER with SCV enclosing STMΔsteA was significantly reduced, compared with SCV of STM WT or STMΔsteA:steA. STMΔsteA shows profound defects in SCV division, resulting in multiple bacteria in a single vacuole with proliferation defects. In vivo, the STMΔsteA shows a defect in colonization in the spleen and liver and affects the initial survival rate of mice. Overall, this study suggests a coordinated role of bacterial effector SteA in promoting ER contact/association with SCVs and regulating SCV division.IMPORTANCEThis study highlights the essential role of the host endoplasmic reticulum in facilitating SCV division and maintaining a single bacterium per vacuole. The Salmonella effector SteA helps maintain the single bacterium per vacuole state. In the absence of SteA, Salmonella resides as multiple bacteria within a single large vacuole. The STMΔsteA shows reduced proliferation under in vitro conditions and exhibits colonization defects in vivo, highlighting the importance of this effector in Salmonella pathogenesis. These findings suggest that targeting SteA could provide a novel therapeutic approach to inhibit Salmonella pathogenicity.
    Keywords:  ER contact sites; ER tubules; Salmonella effectors; Salmonella-containing vacuole
    DOI:  https://doi.org/10.1128/mbio.00114-25
  26. Nat Commun. 2025 Apr 24. 16(1): 3860
    Defective autophagy in CD4 T cells drives liver fibrosis via type 3 inflammation.
    Rola Al Sayegh, Jinghong Wan, Charles Caër, Margot Azoulai, Maxime Gasperment, Sukriti Baweja, Marc-Anthony Chouillard, Janany Kandiah, Mathilde Cadoux, Morgane Mabire, Camille Pignolet, Tristan Thibault-Sogorb, Adel Hammoutene, Valérie Paradis, Loredana Saveanu, Rémy Nicolle, Hélène Gilgenkrantz, Emmanuel Weiss, Sophie Lotersztajn.
      Conventional CD4 T cells represent a major source of inflammatory mediators that drive progression of chronic liver disease to fibrosis and to end-stage cirrhosis. Identification of T cell pathways that limits the inflammatory response could thus have therapeutic relevance. Here we show, using both human samples and mouse models, that autophagy is deficient in CD4 T cells from patients with advanced fibrosis, and that loss of autophagy following genomic deletion of ATG5 in T cells is associated with the emergence of pathogenic IL-17A + IFN-γ + Th17 T cells that drive liver fibrosis in mice. Mechanistically, liver CD4 T cells lacking autophagy display a Th17 glycolytic phenotype associated with enhanced type 3 cytokine (i.e., IL-17A and GM-CSF) release, shifting hepatic myofibroblasts, hepatocytes and macrophages toward a proinflammatory phenotype. We also show that autophagy can be rescued in CD4 T cells from patients with extensive liver fibrosis, leading to decreased frequency of pathogenic Th17 cells and reduced GM-CSF levels; in addition, limited fibrosis is observed in mice in which Rubicon, a negative regulator of autophagy, is deleted specifically in their T cells. Our findings thus implicate autophagy in CD4 T cells as a key therapeutic target to control inflammation-driven fibrosis during chronic liver injury.
    DOI:  https://doi.org/10.1038/s41467-025-59218-y
  27. iScience. 2025 May 16. 28(5): 112317
    Evolution of tumor stress response during cytoreductive surgery for ovarian cancer.
    Aaron M Praiss, Lea A Moukarzel, Yingjie Zhu, Ana Leda F Longhini, Fatemeh Derakhshan, Timothy Hoang, Giulio Pesci, Hunter Green, Melih A Ozsoy, Etta Hanlon, Ryan Kahn, Melica Nourmoussavi Brodeur, Tiffany Sia, Nadeem R Abu-Rustum, Ginger Gardner, Kara Long Roche, Yukio Sonoda, Oliver Zivanovic, Dennis S Chi, Taha Merghoub, Rui Gardner, Britta Weigelt, Dmitriy Zamarin.
      Upfront treatment for patients with advanced high-grade serous ovarian cancer (HGSOC) includes a multi-hour cytoreductive surgery. Although the procedure is necessary for maximal tumor cytoreduction, understanding of the biology of systemic and intratumoral responses induced by surgical cytoreduction is limited. Through analysis of matched tumor and normal tissues and peripheral blood collected at multiple time points during cytoreductive surgery in patients with HGSOC, we demonstrate that surgery leads to rapid induction of systemic inflammatory response and activation of inflammatory signaling in the tumor and normal tissue, with interleukin-6 emerging as a dominant inflammatory pathway. A parallel study in a syngeneic murine HGSOC model recapitulated these findings and demonstrated accelerated tumor growth in response to surgery. This study highlights the previously unappreciated impact of specimen collection timing on the tumor signaling networks and provides insights into stress pathways activated by surgery, generating rationale for perioperative therapeutic interventions to reduce protumorigenic effects.
    Keywords:  Cancer; Cell biology
    DOI:  https://doi.org/10.1016/j.isci.2025.112317
  28. Mol Cell Proteomics. 2025 Apr 18. pii: S1535-9476(25)00073-8. [Epub ahead of print] 100975
    Global analysis of protein and small-molecule substrates of ubiquitin-like proteins (UBLs).
    Guang-Can Shao, Zhen-Lin Chen, Shan Lu, Qing-Cui Wu, Yao Sheng, Jing Wang, Yan Ma, Jian-Hua Sui, Hao Chi, Xiang-Bing Qi, Si-Min He, Li-Lin Du, Meng-Qiu Dong.
      Ubiquitin-like proteins (UBLs) constitute a family of evolutionarily conserved proteins that share similarities with ubiquitin in 3D structures and modification mechanisms. For most UBLs including Small-Ubiquitin-like Modifiers (SUMO), their modification sites on substrate proteins cannot be identified using the mass spectrometry-based method that has been successful for identifying ubiquitination sites, unless a UBL protein is mutated accordingly. To identify UBL modification sites without having to mutate UBL, we have developed a dedicated search engine pLink-UBL on the basis of pLink, a software tool for identification of cross-linked peptide pairs. pLink-UBL exhibited superior precision, sensitivity, and speed than "make-do" search engines such as MaxQuant, pFind, and pLink. For example, compared to MaxQuant, pLink-UBL increased the number of identified SUMOylation sites by 50 ∼ 300% from the same datasets. Additionally, we present a method for identifying small-molecule modifications of UBLs. This method involves antibody enrichment of a UBL C-terminal peptide following enrichment of a UBL protein, followed by LC-MS/MS analysis and a pFind 3 blind search to identify unexpected modifications. Using this method, we have discovered non-protein substrates of SUMO, of which spermidine is the major one for fission yeast SUMO Pmt3. Spermidine can be conjugated to the C-terminal carboxylate group of Pmt3 through its N1 or also likely, N8 amino group in the presence of SUMO E1, E2, and ATP. Pmt3-spermidine conjugation does not require E3 and can be reversed by SUMO isopeptidase Ulp1. SUMO-spermidine conjugation is present in mice and humans. Also, spermidine can be conjugated to ubiquitin in vitro by E1 and E2 in the presence of ATP. The above observations suggest that spermidine may be a common small molecule substrate of SUMO and possibly ubiquitin across eukaryotic species.
    Keywords:  UBL; antibody; mass spectrometry; pLink-UBL; spermidine; substrates
    DOI:  https://doi.org/10.1016/j.mcpro.2025.100975
  29. Protein Sci. 2025 May;34(5): e70134
    Mass balance approximation of unfolding boosts potential-based protein stability predictions.
    Ivan Rossi, Guido Barducci, Tiziana Sanavia, Paola Turina, Emidio Capriotti, Piero Fariselli.
      Predicting protein stability changes upon single-point mutations is crucial in computational biology, with applications in drug design, enzyme engineering, and understanding disease mechanisms. While deep-learning approaches have emerged, many remain inaccessible for routine use. In contrast, potential-like methods, including deep-learning-based ones, are faster, user-friendly, and effective in estimating stability changes. However, most of them approximate Gibbs free-energy differences without accounting for the free-energy changes of the unfolded state, violating mass balance and potentially reducing accuracy. Here, we show that incorporating mass balance as a first approximation of the unfolded state significantly improves potential-like methods. While many machine-learning models implicitly or explicitly use mass balance, our findings suggest that a more accurate unfolded-state representation could further enhance stability change predictions.
    Keywords:  Gibbs free energy; deep learning models; mass‐balance correction; potential‐like methods; protein stability prediction; single‐point mutations
    DOI:  https://doi.org/10.1002/pro.70134
  30. J Am Chem Soc. 2025 Apr 25.
    Natural Spy Chaperone Mimic: Tailored Nanochaperone with Electrostatic-Hydrophobic Synergy To Enhance Protein Folding Regulation.
    Shuyue Zhao, Shu Quan, Wei He, Linlin Xu, Haodong Hu, Zixuan Ma, Rujiang Ma, Fan Huang, Linqi Shi.
      Protein folding regulation is of great significance for maintaining protein structures and biological functions. This fundamental process is assisted by molecular chaperones, which act in inhibiting undesired protein aggregation and facilitating misfolded protein refolding. Inspired by the unique structure and ingenious mechanisms of natural Spy chaperones, we innovate a nanochaperone-guided protein folding strategy by rationally designed nanochaperones (nChaps) with customizable surface structures and properties. In this strategy, the nChaps with tunable charged surfaces can first rapidly capture different client proteins through long-range electrostatic attraction, similar to Spy. Subsequently, the captured proteins can be dynamically bound into the Spy-mimetic hydrophobic microdomains via short-range hydrophobic interactions. As a result, the client proteins are sequestered and stabilized in the chaperone-mimicking confined spaces on the surface of nChaps, thereby facilitating dynamic regulation of protein folding through an electrostatic-hydrophobic synergy mechanism. Moreover, benefiting from the adjustable charge and multiple hydrophobic microdomains, this biomimetic nChap potentiates protein stability at harsh temperatures and long-term storage, which is hardly achieved by natural Spy. Additionally, this strategy is applicable to 9 different proteins with varying isoelectric points and molecular weights, showing superior generality than Spy. Therefore, this work provides new perspectives in developing an advanced strategy for enhanced protein folding regulation.
    DOI:  https://doi.org/10.1021/jacs.5c01133
  31. NAR Genom Bioinform. 2025 Jun;7(2): lqaf052
    SLAYER: a computational framework for identifying synthetic lethal interactions through integrated analysis of cancer dependencies.
    Ziv Cohen, Ekaterina Petrenko, Alma Sophia Barisaac, Enas R Abu-Zhayia, Chen Yanovich-Ben-Uriel, Nabieh Ayoub, Dvir Aran.
      Synthetic lethality represents a promising therapeutic approach in precision oncology, yet systematic identification of clinically relevant synthetic lethal interactions remains challenging. Here, we present SLAYER (Synthetic Lethality AnalYsis for Enhanced taRgeted therapy), a computational framework that integrates cancer genomic data and genome-wide CRISPR knockout screens to identify potential synthetic lethal interactions. SLAYER employs parallel analytical approaches examining both direct mutation-dependency associations and pathway-mediated relationships across 1080 cancer cell lines. Our integrative method identified 682 putative interactions, which were refined to 148 high-confidence candidates through stringent filtering for effect size, druggability, and clinical prevalence. Systematic validation against protein interaction databases revealed an ∼14-fold enrichment of known associations among SLAYER predictions compared with random gene pairs. Through pathway-level analysis, we identified inhibition of the aryl hydrocarbon receptor (AhR) as potentially synthetically lethal with RB1 mutations in bladder cancer. Experimental studies demonstrated selective sensitivity to AhR inhibition in RB1-mutant versus wild-type bladder cancer cells, which probably operates through indirect pathway-mediated mechanisms rather than direct genetic interaction. In summary, by integrating mutation profiles, gene dependencies, and pathway relationships, our approach provides a resource for investigating genetic vulnerabilities across cancer types.
    DOI:  https://doi.org/10.1093/nargab/lqaf052
  32. Biol Chem. 2025 Apr 23.
    The evolution and diversification of the Hsp90 co-chaperone system.
    Sonja Engler, Johannes Buchner.
      The molecular chaperone Hsp90 is the central element of a chaperone machinery in the cytosol of eukaryotic cells that is characterized by a large number of structurally and functionally different co-chaperones that influence the core chaperone component in different ways and increase its influence on the proteome. From yeast to humans, the number of Hsp90 co-chaperones has increased from 14 to over 40, and new co-chaperones are still being discovered. While Hsp90 itself has only undergone limited changes in structure and mechanism from yeast to humans, its increased importance and contribution to different processes in humans is based on the evolution and expansion of the cohort of co-chaperones. In this review, we provide an overview of Hsp90 co-chaperones, focusing on their roles in regulating Hsp90 function and their evolution from yeast to humans.
    Keywords:  molecular chaperone; peptidyl prolyl isomerases; protein evolution; protein folding; protein homeostasis; protein interaction
    DOI:  https://doi.org/10.1515/hsz-2025-0112
  33. J Biol Chem. 2025 Apr 22. pii: S0021-9258(25)00375-8. [Epub ahead of print] 108526
    A high throughput compatible workflow for the biochemical identification and characterisation of molecular glues.
    Ryan Guilbert, Maxime Couturier, Yuanyuan Si, Daniel O' Donovan, David Longmire, Hazel Mak, Paul Clarkson, Argyrides Argyrou.
      Molecular glues are an emerging modality which induces or enhances an interaction between two proteins. Molecular glues can target proteins via proximity-induced degradation or sequestration and can, therefore, provide opportunities for therapeutic intervention to targets that cannot be modulated by traditional small molecule approaches. Due to their modest molecular weight, molecular glues may not encounter the bioavailability issues associated with PROTACs. Characterisation of molecular glues in hit finding and hit optimisation settings can be challenging, as both the affinity of the glue for the target protein and the resulting improvement in affinity between the proteins of interest need to be assessed in parallel. Here, we propose and validate a workflow to derive both key parameters from a classic concentration response experiment. Furthermore, we provide a method for the rational determination of optimum biochemical assay conditions to identify and characterise molecular glues.
    Keywords:  drug discovery; drug screening; fluorescence resonance energy transfer (FRET); high‐throughput screening (HTS); mathematical modelling; protein‐protein interaction
    DOI:  https://doi.org/10.1016/j.jbc.2025.108526
  34. PLoS Biol. 2025 Apr;23(4): e3003121
    Werner syndrome exonuclease promotes gut regeneration and causes age-associated gut hyperplasia in Drosophila.
    Kun Wu, Juanyu Zhou, Yiming Tang, Qiaoqiao Zhang, Lishou Xiong, Xiaorong Li, Zhangpeng Zhuo, Mei Luo, Yu Yuan, Xingzhu Liu, Zhendong Zhong, XiaoXin Guo, Zihua Yu, Xiao Sheng, Guanzheng Luo, Haiyang Chen.
      Human Werner syndrome (adult progeria, a well-established model of human aging) is caused by mutations in the Werner syndrome (WRN) gene. However, the expression patterns and functions of WRN in natural aging remain poorly understood. Despite the link between WRN deficiencies and progeria, our analyses of human colon tissues, mouse crypts, and Drosophila midguts revealed that WRN expression does not decrease but rather increases in intestinal stem cells (ISCs) with aging. Mechanistically, we found that the Drosophila WRN homologue (WRNexo) binds to Heat shock 70-kDa protein cognate 3 (Hsc70-3/Bip) to regulate the unfolded protein response of the endoplasmic reticulum (UPRER). Activation of the WRNexo-mediated UPRER in ISCs is required for ISC proliferation during injury repair. However, persistent DNA damage during aging leads to chronic upregulation of WRNexo in ISCs, where excessive WRNexo-induced ER stress drives age-associated gut hyperplasia in Drosophila. This study reveals how elevated WRNexo contributes to stem cell aging, providing new insights into organ aging and the pathogenesis of age-related diseases, such as colon cancer.
    DOI:  https://doi.org/10.1371/journal.pbio.3003121
  35. Autophagy Rep. 2025 ;pii: 2473765. [Epub ahead of print]4(1):
    High throughput screening assay for the identification of ATF4 and TFEB activating compounds.
    Daniel J Pfau, Ruslana Bryk.
      Macrophages act to defend against infection, but can fail to completely prevent bacterial replication and dissemination in an immunocompetent host. Recent studies have shown that activation of a host transcription factor, TFEB, a regulator of lysosomal biogenesis, could restrict intramacrophage replication of the human pathogen Mycobacterium tuberculosis and synergize with suboptimal levels of the antibiotic rifampin to reduce bacterial loads. Currently available small molecule TFEB activators lack selectivity and potency, but could be potentially useful in a variety of pathological conditions with suboptimal lysosomal activity. TFEB nuclear translocation and activation depend on its phosphorylation status which is controlled by multiple cellular pathways. We devised a whole cell, high throughput screening assay to identify small molecules that activate TFEB by establishing a stably transfected HEK293T reporter cell line for ATF4, a basic leucine zipper transcription factor induced by stress response and activated in parallel to TFEB. We optimized its use in vitro using compounds that target endoplasmic reticulum stress and intracellular calcium signaling. We report results from screening the commercially available LOPAC library and the Selleck Chemicals library modified to include only FDA-approved drugs and clinical research compounds. We identified twenty-one compounds across six clinical use categories that activate ATF4, and confirmed that two proteasome inhibitors promote TFEB activation. The results of this study provide an assay that could be used to screen for small molecules that activate ATF4 and TFEB and a potential list of compounds identified as activators of the ATF4 transcription factor in response to cellular stress.
    Keywords:  host-directed therapy; macrophage; mycobacteria; stress response; tuberculosis
    DOI:  https://doi.org/10.1080/27694127.2025.2473765
  36. J Genet Genomics. 2025 Apr 21. pii: S1673-8527(25)00119-5. [Epub ahead of print]
    RiboParser/RiboShiny: an integrated platform for comprehensive analysis and visualization of Ribo-seq data.
    Shuchao Ren, Yinan Li, Zhipeng Zhou.
      Translation is a crucial step in gene expression. Over the past decade, the development and application of Ribosome profiling (Ribo-seq) have significantly advanced our understanding of translational regulation in vivo. However, the analysis and visualization of Ribo-seq data remain challenging. Despite the availability of various analytical pipelines, improvements in comprehensiveness, accuracy, and user-friendliness are still necessary. In this study, we develop RiboParser/RiboShiny, a robust framework for analyzing and visualizing Ribo-seq data. Building on published methods, we optimize ribosome structure-based and start/stop-based models to improve the accuracy and stability of P-site detection, even in species with a high proportion of leaderless transcripts. Leveraging these improvements, RiboParser offers comprehensive analyses, including quality control, gene-level analysis, codon-level analysis, and the analysis of Ribo-seq variants. Meanwhile, RiboShiny provides a user-friendly and adaptable platform for data visualization, facilitating deeper insights into the translational landscape. Furthermore, the integration of standardized genome annotation renders our platform universally applicable to various organisms with sequenced genomes. This framework has the potential to significantly improve the precision and efficiency of Ribo-seq data interpretation, thereby deepening our understanding of translational regulation.
    Keywords:  Data visualization; Differentially translated genes; P-site detection; Ribo-seq; Ribosome profiling; Translation; Translation elongation speed; selective Ribo-seq
    DOI:  https://doi.org/10.1016/j.jgg.2025.04.010
  37. Proc Natl Acad Sci U S A. 2025 Apr 29. 122(17): e2502971122
    Rab32 regulates Golgi structure and cell migration through Protein Kinase A-mediated phosphorylation of Optineurin.
    Katherine M Johnson, Maxwell G Marley, Kristina Drizyte-Miller, Jing Chen, Hong Cao, Nourhan Mostafa, Micah B Schott, Mark A McNiven, Gina L Razidlo.
      Rab32 is a small GTPase and molecular switch implicated in vesicular trafficking. Rab32 is also an A-Kinase Anchoring Protein (AKAP), which anchors cAMP-dependent Protein Kinase (PKA) to specific subcellular locations and specifies PKA phosphorylation of nearby substrates. Surprisingly, we found that a form of Rab32 deficient in PKA binding (Rab32 L188P) relocalized away from the Golgi apparatus and induced a marked disruption in Golgi organization, assembly, and dynamics. Although Rab32 L188P did not cause a global defect in PKA activity, our data indicate that Rab32 facilitates the phosphorylation of a specific PKA substrate. We uncovered a direct interaction between Rab32 and the adaptor protein optineurin (OPTN), which regulates Golgi dynamics. Further, our data indicate that optineurin is phosphorylated by PKA at Ser342 in a Rab32-dependent manner. Critically, blocking phosphorylation at OPTN Ser342 leads to Golgi fragmentation, and a phospho-mimetic version of OPTN rescues Golgi defects induced by Rab32 L188P. Finally, Rab32 AKAP function and OPTN phosphorylation are required for Golgi repositioning during cell migration, contributing to tumor cell invasion. Together, these data reveal a role for Rab32 in regulating Golgi dynamics through PKA-mediated phosphorylation of OPTN.
    Keywords:  Golgi; Optineurin; Protein Kinase A; Rab32; migration
    DOI:  https://doi.org/10.1073/pnas.2502971122
  38. Gut. 2025 Apr 19. pii: gutjnl-2024-333406. [Epub ahead of print]
    A Common CTRB misfolding variant associated with pancreatic cancer risk causes ER stress and inflammation in mice.
    Cristina Bodas, Irene Felipe, Brice Chanez, Miguel Lafarga, Evangelina Lopez de Maturana, Jaime Martinez de Villarreal, Natalia Del Pozo, Marina Malumbres, Pierfrancesco Vargiu, Ana Cayuela, Isabel Peset, Katelyn Connelly, Jason Hoskins, Raúl Méndez, Laufey Amundadottir, Núria Malats, Sagrario Ortega, Francisco X Real.
       BACKGROUND: Genome-wide association studies have identified an exon 6 CTRB2 deletion variant proposed to increase pancreatic cancer risk.
    OBJECTIVE: To acquire evidence on its causal role, we developed and analysed a new mouse strain carrying an equivalent variant in Ctrb1, the mouse CTRB2 orthologue.
    DESIGN: We used CRISPR/Cas9 to introduce a 707 bp deletion encompassing Ctrb1 exon 6 (Ctrb1Δexon6 ). This mutation closely mimics the human variant. Mice carrying the mutant allele were profiled at 3 months to assess their phenotype.
    RESULTS: Ctrb1Δexon6 mutant mice express a truncated CTRB1 that accumulates in the endoplasmic reticulum (ER). The pancreas of homozygous mutant mice displays reduced chymotrypsin activity, total protein synthesis and amylase secretion. The histological aspect of the pancreas is inconspicuous but ultrastructural analysis shows evidence of dramatic ER stress and cytoplasmic and nuclear inclusions. Transcriptomic studies of the mutant pancreas reveal downregulation of the acinar programme and increased activity of ER stress-related and inflammatory pathways. Agr2 is one of the most upregulated genes in mutant pancreata. Heterozygous mice have an intermediate phenotype. Ctrb1Δexon6 mutant mice exhibit impaired recovery from acute caerulein-induced pancreatitis. Administration of tauroursodeoxycholic acid or sulindac partially alleviates the phenotype. A transcriptomic signature derived from the mutant pancreata is significantly enriched in normal human pancreas of CTRB2 exon 6 deletion variant carriers from the GTEx cohort.
    CONCLUSIONS: This mouse strain provides evidence that the exon 6 deletion causes ER stress and inflammation and is an excellent model to understand its contribution to pancreatic cancer and identify preventive strategies.
    Keywords:  CANCER GENETICS; EXPERIMENTAL PANCREATITIS; INFLAMMATION; PANCREATIC CANCER
    DOI:  https://doi.org/10.1136/gutjnl-2024-333406
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