bims-proteo Biomed News
on Proteostasis
Issue of 2025–06–15
forty-five papers selected by
Eric Chevet, INSERM
  1. Loss of FIC-1-mediated AMPylation activates the UPRER and upregulates cytosolic HSP70 chaperones to suppress polyglutamine toxicity.
  2. Proximity biotinylation at the host- Shigella interface reveals UFMylation as an antibacterial pathway.
  3. Phosphoribosyl ubiquitination of SNARE proteins regulates autophagy during Legionella infection.
  4. SEL1L Regulates ER Homeostasis in Sertoli Cells but Is Dispensable for Their Function.
  5. In-Cell Approach to Evaluate E3 Ligases for Use in Targeted Protein Degradation.
  6. Identification of Actionable Targeted Protein Degradation Effector Sites through Site-Specific Ligand Incorporation-Induced Proximity (SLIP).
  7. Covalent Recruitment of NEDD4 for Targeted Protein Degradation: Rational Design of Small Molecular Degraders.
  8. The Mechanism of Histone Ubiquitylation by the ASB9-CUL5 Ubiquitin Ligase.
  9. ADP ribosylation factor-like GTPase 6-interacting protein 5 (ARL6IP5): a prenylated Rab acceptor protein 1 (PRA1) family protein that shapes the ER membrane and regulates ER-phagy.
  10. Ubiquitin-Proteasome System Dysregulation in Alzheimer's Disease Impacts Protein Abundance.
  11. Reciprocal Regulation Between the SCF FBXO24 Ubiquitin E3 Ligase and FoxP1 Protein.
  12. Transcriptome-wide RNA accessibility mapping reveals structured RNA elements and pervasive conformational rearrangements under stress.
  13. ATP13A1 engages SEC61 to facilitate substrate-specific translocation.
  14. Impact of ATF6 deletion on the embryonic brain development.
  15. Organization of the Proteostasis Network of Membraneless Organelles.
  16. An adaptive organelle triad houses lipid droplets for dynamic regulation.
  17. GPC3-mediated lysosome-targeting chimeras (GLTACs) for targeted degradation of membrane proteins.
  18. Aging and diet alter the protein ubiquitylation landscape in the mouse brain.
  19. Mitochondrial presequences harbor variable strengths to maintain organellar function.
  20. Switchable client specificity in a dual functional chaperone coordinates light-harvesting complex biogenesis.
  21. The Flagellin-Specific Chaperone FliS of Borrelia burgdorferi Controls the Cytoplasmic Pool of Flagellins at the Level of Translation Initiation, Secretion, and Proteolysis.
  22. Loss of UFMylation supports prostate cancer metastasis and rewires cell metabolism towards hexosamine biosynthesis.
  23. A Diffusion-Based Framework for Designing Molecules in Flexible Protein Pockets.
  24. Structural insights into tRNA recognition of the human FTSJ1-THADA complex.
  25. Pan-viral ORFs discovery using massively parallel ribosome profiling.
  26. Integrative Chemical Genetics Platform Identifies Condensate Modulators Linked to Neurological Disorders.
  27. Rational design and discovery of potent PROTAC degraders of ASK1: a targeted therapy in MASH.
  28. AbEpiTope-1.0: Improved antibody target prediction by use of AlphaFold and inverse folding.
  29. CD44 Deficiency Induces Combinatory NRF2 Inhibition and Endoplasmic Reticulum Stress-Associated Dyserythropoiesis.
  30. CRISPR screens and quantitative proteomics reveal remodeling of the aryl hydrocarbon receptor-driven proteome through PARP7 activity.
  31. Translation suppresses exogenous target RNA-mediated microRNA decay.
  32. The lysosomal membrane protein LAMP2B mediates microlipophagy to target obesity-related disorders.
  33. SP140-RESIST pathway regulates interferon mRNA stability and antiviral immunity.
  34. Tumorous cholesterol biosynthesis curtails anti-tumor immunity by preventing MTOR-TFEB-mediated lysosomal degradation of CD274/PD-L1.
  35. Small molecule disruption of RARα/NCoR1 interaction inhibits chaperone-mediated autophagy in cancer.
  36. Derepressing nuclear pyruvate dehydrogenase induces therapeutic cancer cell reprogramming.
  37. The RNA-stability-independent role of the RNA m6A reader YTHDF2 in promoting protein translation to confer tumor chemotherapy resistance.
  38. TANGO2 binds crystallin alpha B and its loss causes desminopathy.
  39. Unraveling mitochondrial influence on mammalian pluripotency via enforced mitophagy.
  40. Integrin-Specific Signaling Drives ER Stress-Dependent Atherogenic Endothelial Activation.
  41. Early proteasome downregulation and dysfunction drive proteostasis failure in Alzheimer's disease.
  42. Redundancy of the OST catalytic subunit facilitates therapeutic targeting of N-glycosylation.
  43. Massively parallel genetic perturbation suggests the energetic structure of an amyloid-β transition state.
  44. An improved model for prediction of de novo designed proteins with diverse geometries.
  45. The NAMPT enzyme employs a switch that directly senses AMP/ATP and regulates cellular responses to energy stress.
  1. PLoS Genet. 2025 Jun 13. 21(6): e1011723
    Loss of FIC-1-mediated AMPylation activates the UPRER and upregulates cytosolic HSP70 chaperones to suppress polyglutamine toxicity.
    Kate M Van Pelt, Matthias C Truttmann.
      Targeted regulation of cellular proteostasis machinery represents a promising strategy for the attenuation of pathological protein aggregation. Recent work suggests that the unfolded protein response in the endoplasmic reticulum (UPRER) directly regulates the aggregation and toxicity of expanded polyglutamine (polyQ) proteins. However, the mechanisms underlying this phenomenon remain poorly understood. In this study, we report that perturbing ER homeostasis in Caenorhabditis elegans through the depletion of either BiP ortholog, hsp-3 or hsp-4, causes developmental arrest in worms expressing aggregation-prone polyQ proteins. This phenotype is rescued by the genetic deletion of the conserved UPRER regulator, FIC-1. We demonstrate that the beneficial effects of fic-1 knock-out (KO) extend into adulthood, where the loss of FIC-1-mediated protein AMPylation in polyQ-expressing animals is sufficient to prevent declines in fitness and lifespan. We further show that loss of hsp-3 and hsp-4 leads to distinct, but complementary transcriptomic responses to ER stress involving all three UPRER stress sensors (IRE-1, PEK-1, and ATF-6). We identify the cytosolic HSP70 family chaperone F44E5.4, whose expression is increased in fic-1-deficient animals upon ER dysregulation, as a key effector suppressing polyQ toxicity. Over-expression of F44E5.4, but not other HSP70 family chaperones, is sufficient to rescue developmental arrest in polyQ-expressing embryos upon hsp-3 knock-down. We further show that knock-down of ire-1 or atf-6 blocks the upregulation of F44E5.4 in fic-1-deficient worms. Taken together, our findings support a model in which the loss of FIC-1-mediated AMPylation engages UPRER signaling to upregulate cytosolic chaperone activity in response to polyQ toxicity.
    DOI:  https://doi.org/10.1371/journal.pgen.1011723
  2. bioRxiv. 2025 May 29. pii: 2025.05.29.656827. [Epub ahead of print]
    Proximity biotinylation at the host- Shigella interface reveals UFMylation as an antibacterial pathway.
    Ana T López-Jiménez, Fabien Théry, Kathryn Wright, Hannah Painter, Shelby T Hoffmeister, Lucas Jarche, Jeremy Benjamin, Gerbrand J van der Heden van Noort, Dominik Brokatzky, Margarida C Gomes, Sydney L Miles, Damián Lobato-Márquez, John Rohde, Jonathan N Pruneda, Francis Impens, Serge Mostowy.
      Host cells contest invasion by intracellular bacterial pathogens with multiple strategies that recognise and / or damage the bacterial surface. To identify novel host defence factors targeted to intracellular bacteria, we developed a versatile proximity biotinylation approach coupled to quantitative mass spectrometry that maps the host-bacterial interface during infection. Using this method, we discovered that intracellular Shigella and Salmonella become targeted by UFM1-protein ligase 1 (UFL1), an E3 ligase that catalyses the covalent attachment of Ubiquitin-fold modifier 1 (UFM1) to target substrates in a process called UFMylation. We show that Shigella antagonises UFMylation in a dual manner: first, using its lipopolysaccharide (LPS) to shield from UFL1 recruitment; second, preventing UFM1 decoration by the bacterial effector IpaH9.8. Absence of UFMylation leads to an increase of bacterial burden in both human cells and zebrafish larvae, suggesting that UFMylation is a highly conserved antibacterial pathway. Contrary to canonical ubiquitylation, the protective role of UFMylation is independent of autophagy. Altogether, our proximity mapping of the host-bacterial interface identifies UFMylation as an ancient antibacterial pathway and holds great promise to reveal other cell-autonomous immunity mechanisms.
    DOI:  https://doi.org/10.1101/2025.05.29.656827
  3. EMBO J. 2025 Jun 12.
    Phosphoribosyl ubiquitination of SNARE proteins regulates autophagy during Legionella infection.
    Rukmini Mukherjee, Anshu Bhattacharya, Ines Tomaskovic, João Mello-Vieira, Melinda Elaine Brunstein, Marion Başoğlu, Tineke Veenendaal, Henry Bailey, Thomas Colby, Mohit Misra, Stefan Eimer, Judith Klumperman, Christian Münch, Ivan Matic, Ivan Dikic.
      Legionella pneumophila is an intracellular pathogen that causes Legionnaires' disease. The bacteria release effector proteins, some of which remodel host autophagic-lysosomal pathways. One such effector is RavZ, which delipidates ATG8 proteins, making compromising autophagy in Legionella-infected cells. Here we show that SidE effectors also affect these pathways, by mediating phosphoribosyl-ubiquitination (PR-Ub) of the autophagic SNARE proteins STX17 and SNAP29. STX17 modification induces recruitment of STX17-positive membranes from the endoplasmic reticulum to Legionella-containing phagosomes, forming replicative vacuoles. Using proximity labeling, biochemistry and Legionella infection studies, we define a mechanism by which autophagy is hijacked by bacteria to recruit ER membranes to the bacterial vacuole, via a structure bearing autophagy markers but not fusing with lysosomes. Mass-spectrometric identification of PR-Ub sites and mutational studies show that phosphoribosyl-ubiquitination of STX17 alters its interaction with ATG14L, which causes ER membranes to be recruited to the bacterial vacuole in a PI3K-dependent manner. On the other hand, phosphoribosyl-ubiquitination of SNAP29 inhibits the formation of the autophagosomal SNARE complex (STX17-SNAP29-VAMP8) via steric hindrance, thus preventing the fusion of bacterial vacuoles with lysosomes.
    Keywords:   Legionella pneumophila ; Autophagy; Syntaxin17; Ubiquitin; Xenophagy
    DOI:  https://doi.org/10.1038/s44318-025-00483-4
  4. Mol Biol Cell. 2025 Jun 11. mbcE25030101
    SEL1L Regulates ER Homeostasis in Sertoli Cells but Is Dispensable for Their Function.
    Nusrat Jahan Tushi, You Lu, Zhibing Zhang, Shengyi Sun.
      Endoplasmic reticulum (ER)-associated protein degradation (ERAD) plays a vital role in maintaining ER homeostasis by degrading misfolded ER proteins. The SEL1L-HRD1 complex, the most evolutionarily conserved branch of ERAD, has been implicated in various physiological processes in both mice and humans, including cellular stress responses, immune function, and development. However, its role in Sertoli cells, which are critical for supporting spermatogenesis, remains unexplored. Here, we show that Sertoli cell SEL1L is not essential for their function or spermatogenesis. SEL1L and HRD1 proteins are expressed in Sertoli cells, and the deletion of SEL1L in Sertoli cells reduces HRD1 protein levels and impairs ERAD function. This leads to elevated ER stress responses and increased expression of ER chaperones, suggesting a potential compensatory adaptation to maintain ER homeostasis. Despite these changes, Sertoli cell-specific Sel1L deletion does not disrupt testicular histology, sperm count, or male fertility. These findings reveal the adaptation of Sertoli cells to SEL1L and ERAD dysfunction and highlight their ability to sustain spermatogenesis under ER stress.
    DOI:  https://doi.org/10.1091/mbc.E25-03-0101
  5. J Am Chem Soc. 2025 Jun 10.
    In-Cell Approach to Evaluate E3 Ligases for Use in Targeted Protein Degradation.
    Yunan Zheng, Anamika Singh, Zeqi Niu, Violeta Marin, Jonathon Young, Paul Richardson, Marcus L Hemshorn, Richard B Cooley, P Andrew Karplus, Kedar Puvar, Scott E Warder, Anil Vasudevan, Justin M Reitsma, Ryan A Mehl.
      A major challenge in evaluating the suitability of ∼700 known and putative E3 ligases for target protein degradation (TPD) is the lack of ligase-specific binders. Here, we use genetic code expansion (GCE) to express in living cells an E3 ligase with a site-specifically encoded, tetrazine-containing noncanonical amino acid (Tet-ncAA). Then, using click chemistry, we conjugate the incorporated Tet with a strained trans-cyclooctene (sTCO) tethered to a neosubstrate protein binder. The resulting covalent E3 ligase-binder construct can then be evaluated for the TPD of the neosubstrate. We first demonstrate that cereblon (CRBN) has a rather high plasticity for TPD by studying CRBN containing Tet-ncAA at a variety of surface positions. When these CRBN forms are covalently tethered to an sTCO-linker-JQ1 reagent, they all successfully recruit BRD2/4 for degradation, with the efficiency depending on the placement of the Tet-ncAA and the linker length. The results highlight the ability of this approach to map E3 surfaces and identify optimal TPD interfaces and pockets. Applying this strategy to speckle-type POZ protein (SPOP), an E3 ligase with no known specific ligand, we demonstrate that multiple sites on its surface can support TPD, revealing the potential for PROTAC-type development. This E3-ligand-free degrader (ELF degrader) platform preserves the native state of E3 ligases, enables the interrogation of any E3 surface region in live cells, and is applicable to a broad range of E3 ligases. ELF degraders represent a versatile approach to define functional degron sites, guide degrader design, and unlock new E3 ligases, those without known ligands, for therapeutic applications.
    DOI:  https://doi.org/10.1021/jacs.5c02741
  6. J Am Chem Soc. 2025 Jun 10.
    Identification of Actionable Targeted Protein Degradation Effector Sites through Site-Specific Ligand Incorporation-Induced Proximity (SLIP).
    Zhangping Xiao, Efthymios S Gavriil, Fangyuan Cao, Xinyue Zhang, Stan Xiaogang Li, Sergei Kotelnikov, Patrycja Michalska, Friederike Marte, Chloe Huang, Yudi Lu, Yunxuan Zhang, Erika Bernardini, Dima Kozakov, Edward W Tate.
      Targeted protein degradation (TPD) is a rapidly emerging and potentially transformative therapeutic modality. However, the large majority of >600 known ubiquitin ligases have yet to be exploited as TPD effectors by proteolysis-targeting chimeras (PROTACs) or molecular glue degraders (MGDs). We report here a chemical-genetic platform, Site-specific Ligand Incorporation-induced Proximity (SLIP), to identify actionable ("PROTACable") sites on any potential effector protein in intact cells. SLIP uses genetic code expansion to encode copper-free "click" ligation at a specific effector site in intact cells, enabling the in situ formation of a covalent PROTAC-effector conjugate against a target protein of interest. Modification at actionable effector sites drives degradation of the targeted protein, establishing the potential of these sites for TPD. Using SLIP, we systematically screened dozens of sites across E3 ligases and E2 enzymes from diverse classes, identifying multiple novel potentially PROTACable effector sites which are competent for TPD. SLIP adds a powerful approach to the proximity-induced pharmacology (PIP) toolbox, enabling future effector ligand discovery to fully enable TPD and other emerging PIP modalities.
    DOI:  https://doi.org/10.1021/jacs.5c01420
  7. J Am Chem Soc. 2025 Jun 12.
    Covalent Recruitment of NEDD4 for Targeted Protein Degradation: Rational Design of Small Molecular Degraders.
    Xiaoqiang He, Shihan Zeng, Yalei Wen, Tao Yang, Chaoming Huang, Yifang Li, Zhang Zhang, Ke Ding, Tongzheng Liu, Yi Tan, Zhengqiu Li.
      Targeted protein degradation (TPD) has emerged as a promising therapeutic strategy for treating various diseases. However, current small molecule degraders predominantly rely on a limited set of E3 ubiquitin ligases, such as CRBN and VHL, which restricts their applications. Here, we report that incorporation of the 2H-azirine chemical handle into the EGFRL858R/T790M/C797S inhibitor induced remarkable degradation of the targeted protein. Proteomic profiling and functional validation confirmed that the NEDD4 E3 ligase was covalently recruited by 2H-azirine through engagement of C1286 residue, facilitating target degradation. Furthermore, the 2H-azirine moiety demonstrated versatility by acting as a small molecular degrader when conjugated to various ligands, effectively mediating the degradation of CDK4, PDE5, BTK and Brd4. More importantly, using the identical protein ligand scaffold, we demonstrated that the 2H-azirine based probe can degrade proteins resistant to degradation by CRBN or VHL recruitment. This approach provides a rational strategy for developing novel small molecular degraders that target alternative E3 ubiquitin ligases. Notably, these degraders significantly outperformed their parent kinase inhibitor in suppressing cancer cell growth.
    DOI:  https://doi.org/10.1021/jacs.4c18083
  8. bioRxiv. 2025 May 26. pii: 2025.05.21.655409. [Epub ahead of print]
    The Mechanism of Histone Ubiquitylation by the ASB9-CUL5 Ubiquitin Ligase.
    Calvin P Lin, Nathan H Lee, Francis X Alipranti, Harry Li, Elizabeth A Komives.
      The E3 ligase substrate receptor ankyrin and SOCS box protein 9 (ASB9) was shown to bind over 10 different proteins including metabolic enzymes such as creatine kinase, filament proteins such as vimentin, and histones. In previous work, we characterized the ASB9-Cullin 5 E3 ligase (ASB9-CRL) ubiquitylation of creatine kinase and showed that ubiquitylation required the ring-between-ring ligase, ARIH2. Here we characterize the ASB9-CRL ubiquitylation of histones and show that histones H3 and H4 are polyubiquitylated by the ASB9-CRL whereas histones H2A and H2B are not. Many, but not all lysines in the histones are ubiquitylated suggesting some substrate specificity. Binding experiments show that the ligase-histone interaction is highly electrostatic and the neddylated ASB9-CRL binds with highest affinity. Only free histones are ubiquitylated. When the histones are in nucleosomes or in complex with the chaperone Asf1, they are not ubiquitylated. Only K48 and K63 polyubiquitin chains were observed, suggesting that the ubiquitylation probably drives protein degradation. The presence of ASB9 in specific cell types correlates with situations in which free histones H3 and H4 need to be degraded. In this work, we demonstrate that the ASB9-CRL is the ligase that facilitates degradation of histones H3 and H4. In addition, this work represents the first example of Cullin 5 mediated ubiquitylation that does not require a ring-between-ring "helper" ligase.
    DOI:  https://doi.org/10.1101/2025.05.21.655409
  9. Autophagy Rep. 2025 ;4(1): 2513466
    ADP ribosylation factor-like GTPase 6-interacting protein 5 (ARL6IP5): a prenylated Rab acceptor protein 1 (PRA1) family protein that shapes the ER membrane and regulates ER-phagy.
    Yasunori Yamamoto, Toshiaki Sakisaka.
      The prenylated Rab acceptor protein 1 (PRA1) domain is a conserved domain encompassing four transmembrane domains (TMDs). ARL6IP5 (ADP ribosylation factor-like GTPase 6-interacting protein 5) is a member of the PRA1 family and interacts with the reticulon-homology domain (RHD)-containing proteins including ARL6IP1 (ADP ribosylation factor-like GTPase 6-interacting protein 1) and FAM134B. The RHD is a conserved domain encompassing two short hairpin TMDs and acts as a membrane-shaping unit for endoplasmic reticulum (ER) morphology and ER-phagy. However, the involvement of ARL6IP5 in ER morphology and ER-phagy remains unclear. We recently characterized ARL6IP5 as an ER membrane-shaping protein and found that ARL6IP5 constricts the ER membrane in a manner similar to ARL6IP1, possibly via short hairpin configuration of the TMDs in the PRA1 domain. ARL6IP5 also plays a redundant role with ARL6IP1 in shaping the ER membrane. Importantly, depletion of ARL6IP5 impaired FAM134B-meadited ER-phagy, which is reminiscent of ARL6IP1 depletion. These results suggested that ARL6IP5 acts as an ER membrane-shaping protein that regulates ER-phagy, underscoring the importance of the PRA1 domain. Although ARL6IP5 and ARL6IP1 are confusable protein names and seem to have similar roles in ER-phagy, we clarify in this punctum that they are distinct classes of ER membrane-shaping proteins.
    Keywords:  ARL6IP1; ARL6IP5; ER-phagy; Endoplasmic reticulum; prenylated Rab acceptor protein 1 domain; reticulon-homology domain
    DOI:  https://doi.org/10.1080/27694127.2025.2513466
  10. bioRxiv. 2025 May 29. pii: 2025.05.29.656728. [Epub ahead of print]
    Ubiquitin-Proteasome System Dysregulation in Alzheimer's Disease Impacts Protein Abundance.
    Mahlon Collins, Corinna Friedrich, Megan Elcheikhali, Peyton Stewart, Jason Derks, Theresa Connors-Stewart, Kirstin Altig, Alexandra Melloni, Aleksandra Petelski, Derek Oakley, Bradley Hyman, Nikolai Slavov.
      Alzheimer's disease (AD) is a relentlessly progressive, fatal neurodegenerative disorder associated with widespread aberrant proteomic changes. The full extent of protein dysfunctions in AD and their impact on cellular physiology remains unknown. Here, we used plexDIA, an approach that parallelizes the acquisition of samples and peptides, to characterize proteomic changes in AD. Using human dorsolateral prefrontal cortex tissue, we identified 281 differentially abundant proteins in AD. By systematically analyzing cellular compartment-specific shifts in protein abundance, we identified an AD-specific decrease in levels of the 20S proteasome, the catalytic core of the cell's primary protein degradation pathway. This alteration was accompanied by widespread decreases in proteasome subunit stoichiometries. Many proteasome substrate proteins were negatively correlated with 20S levels and increased in AD, suggesting that reduced 20S levels leads to abnormal protein accumulation. By analyzing proteins increased in AD, we identify key properties of such proteins. Namely, they have highly specific subcellular localizations and fast degradation rates, they contain signal sequences that allow them to be targeted for proteasomal degradation, and they are targeted by quality control pathways that recognize mislocalized proteins. Furthermore, we identify coherent sets of ubiquitin system enzymes, proteins that target substrates for proteasomal degradation, whose levels robustly discriminate AD from non-AD samples. One subset exhibited consistent increases in AD, while another exhibited consistent decreases, revealing complex alterations to the ubiquitin system in AD. Taken together, our results suggest that decreased ubiquitin-proteasome system capacity and impaired clearance of short-lived and mislocalized proteins contribute substantially to proteopathic burden in AD.
    DOI:  https://doi.org/10.1101/2025.05.29.656728
  11. bioRxiv. 2025 May 28. pii: 2025.05.23.653845. [Epub ahead of print]
    Reciprocal Regulation Between the SCF FBXO24 Ubiquitin E3 Ligase and FoxP1 Protein.
    Abigail Maloy, Sydney Walter, Arthur Mascilli, David C Klein, Santana M Lardo, James Londino, Toru Nyunoya, John McDyer, Mia Sun, Xuemei Zeng, Nathan Yates, Pamela Cantrell, Sarah J Hainer, Rama K Mallampalli, Divay Chandra.
      Forkhead Box Protein P1 (FoxP1) is a crucial transcriptional repressor essential for the development of the brain and heart. In adults, FoxP1 protein levels are dysregulated in a variety of disorders, including chronic obstructive pulmonary disease (COPD), atherosclerosis, and heart failure, where they causally contribute to disease pathogenesis. Although independent investigators have reported that FoxP1 protein is ubiquitinated, and E3 ligases have been identified for other FoxP family proteins, the identity of the E3 ligase that controls FoxP1 protein stability has remained unknown. Here, we identify FBXO24, a subunit of the Skp-Cullin-F-box (SCF) ubiquitin E3 ligase complex, as the regulator of FoxP1 ubiquitination and stability. Specifically, FBXO24 regulates K48 and K63 ubiquitination, complexes with, and co-localizes to the nucleus with FoxP1 protein in lung epithelial cells. Depleting FBXO24 reverses the unfolded protein response and cell death triggered by loss of FoxP1 protein in lung epithelium, suggesting a protective role. Additionally, FBXO24 knockout mice exhibit elevated FoxP1 levels in the lung and heart and reduced unfolded protein response activity after short-term cigarette smoke exposure. Intriguingly, we also uncovered bidirectional regulation, whereby FoxP1 protein binds to the FBXO24 promoter to suppress FBXO24 transcription. To our knowledge, this is the first evidence that a substrate for an E3 ligase can also regulate the E3 ligase and, therefore, control levels of other substrates, revealing new regulatory networks. Targeting FBXO24 may offer a therapeutic strategy for COPD, atherosclerosis, and heart failure by stabilizing FoxP1 levels in the heart and lungs and mitigating harmful downstream effects.
    DOI:  https://doi.org/10.1101/2025.05.23.653845
  12. bioRxiv. 2025 Jun 05. pii: 2025.06.05.658101. [Epub ahead of print]
    Transcriptome-wide RNA accessibility mapping reveals structured RNA elements and pervasive conformational rearrangements under stress.
    Kelsey Farenhem, Troy W Whitfield, Alina Chouloute, Ankur Jain.
      RNA structure plays a central role in post-transcriptional gene regulation, modulating RNA stability, translation, and interactions with RNA-binding proteins (RBPs). However, capturing RNA conformations at scale remains challenging. Here, we introduce DMS-TRAM-seq (Dimethyl Sulfate-Transcriptome-wide RNA Accessibility Mapping by sequencing), which probes RNA structure across nearly the entire transcriptome. Using DMS-TRAM-seq, we generated secondary structure predictions for over 9,000 human transcripts, including hundreds of non-coding RNAs, and identified more than 700 previously unannotated, high-confidence structured elements. Importantly, the enhanced coverage provided by DMS-TRAM-seq enabled comparative analyses, revealing RNAs that undergo structural rearrangements in response to cellular perturbations. Integration with RBP motifs and ribosome profiling uncovered altered RNA-RBP interactions during oxidative stress and showed that translation inhibition broadly drives RNAs toward their thermodynamically favored conformations. DMS-TRAM-seq enables interrogation of the RNA structurome and its plasticity at an unprecedented scale, opening new directions for elucidating the structural basis of RNA regulation.
    DOI:  https://doi.org/10.1101/2025.06.05.658101
  13. Sci Adv. 2025 Jun 13. 11(24): eadt1346
    ATP13A1 engages SEC61 to facilitate substrate-specific translocation.
    Xiaoyan Yang, Yi Li, Chengxi Yang, Tingting Li, Zhiyu Fang, Zhigang Feng, Jun Liao, Yan Zou.
      The accurate targeting of proteins to their designated cellular compartments is essential for maintaining proper cellular architecture and function. However, interpreting and sorting the highly variable targeting sequences in secreted and membrane proteins present a substantial challenge for achieving precise localization within the secretory pathway. In this study, we demonstrate that atypical signal sequences, characterized by high hydrophobicity and/or the absence of characteristic charges, are recognized by the signal recognition particle and targeted to the endoplasmic reticulum in a reverse orientation. These misoriented signal sequences are subsequently dislocated by the P5A-ATPase ATP13A1 and delivered to SEC61 for further translocation. Using cryo-electron microscopy, we determined the structures of human ATP13A1 in multiple conformations (3.40- to 3.87-angstrom resolution), revealing key residues within its substrate-binding pocket that engage signal sequences through polar interactions. Collectively, our findings elucidate a comprehensive, substrate-specific translocation pathway that ensures both high efficiency and fidelity in protein subcellular localization.
    DOI:  https://doi.org/10.1126/sciadv.adt1346
  14. iScience. 2025 Jun 20. 28(6): 112569
    Impact of ATF6 deletion on the embryonic brain development.
    Loc Dinh Nguyen, Ly Huong Nguyen, Dat Xuan Dao, Tsuyoshi Hattori, Mika Takarada-Iemata, Hiroshi Ishii, Takashi Tamatani, Hiroshi Kawasaki, Yohei Shinmyo, Kenta Onoue, Shigenobu Yonemura, Jun Zhang, Masato Miyake, Seiichi Oyadomari, Kazutoshi Mori, Osamu Hori.
      Although the unfolded protein response (UPR) is activated during brain development, its roles remain unclear. Here, we report that deletion of activating transcription factor 6 (ATF6), consisting of ATF6α and ATF6β, in the developing brain caused microcephaly and neonatal death in mice. Analysis of Atf6a/Atf6b double conditional knockout (dcKO) brains revealed diverse neuronal phenotypes, such as reduced neurogenesis, increased cell death, impaired cortical layer formation, and axon projection defects. Furthermore, hypervasculature, glial defects, and neuroinflammation were observed in dcKO brains. Notably, hypervasculature was detected at E14.5, when endoplasmic reticulum (ER) stress was morphologically unclear, but the UPR was activated to a greater extent in dcKO brains. Expression profiles revealed reduced levels of molecular chaperones in the ER and enhanced levels of PERK- and IRE1-downstream molecules, including VEGFA, in dcKO brains. Administration of a chemical chaperone 4-phenylbutyric acid partially rescued dcKO mice, suggesting roles of ATF6 for improving proteostasis and for coordinating the UPR.
    Keywords:  Cell biology; Developmental biology; Neuroscience
    DOI:  https://doi.org/10.1016/j.isci.2025.112569
  15. Adv Sci (Weinh). 2025 Jun 11. e00233
    Organization of the Proteostasis Network of Membraneless Organelles.
    Christine M Lim, Yuqi Bian, Alicia González Díaz, Frank Pun, Alex Zhavoronkov, Richard I Morimoto, Michele Vendruscolo.
      Membraneless organelles (MLOs) are dynamic macromolecular condensates that act as crucibles to modulate cellular processes. Since MLOs form in the absence of lipid membranes, it is important to understand how their effective regulation is achieved by the protein homeostasis (proteostasis) system. To address this question, a comprehensive mapping of the proteostasis network (PN) of MLOs, comprising over 220 000 protein-protein interactions is reported. This analysis reveals how regulatory proteins (PN proteins) occupy central roles in the overall protein-protein interaction network of MLOs. It is then investigated which branches of the PN are most important in the regulation of MLOs, finding that the anabolic component, which makes up ≈30% of the PN, is more closely involved than the catabolic component, which makes up the remaining ≈70% of the PN. It is also found that translation-related PN proteins and molecular chaperones play central roles in MLO regulation. Finally, how specificity may be achieved despite shared PN components is explored. These findings suggest that HSP70 chaperones function as generic MLO regulators, while client-specific HSP70 co-chaperones confer specificity to the chaperone action. These results identify the composition of the PN of MLOs, rationalize its organization, and reveal central roles of molecular chaperones in protein regulation within MLOs.
    Keywords:  liquid‐liquid phase separation; membraneless organelles; protein condensation; protein homeostasis
    DOI:  https://doi.org/10.1002/advs.202500233
  16. Cell Rep. 2025 Jun 11. pii: S2211-1247(25)00584-4. [Epub ahead of print]44(6): 115813
    An adaptive organelle triad houses lipid droplets for dynamic regulation.
    Hong Qiu, Can Miao, Cunqi Ye.
      Cell organelles compartmentalize metabolic reactions and require inter-organelle communications to coordinate metabolic activities in fluctuating nutrient environments. While membrane contacts enable this communication by facilitating metabolite exchange, the functional organization of organelles through these contacts remains underexplored. Here, we show that excess lactate induces severe metabolic stress under nutrient deprivation in the budding yeast Saccharomyces cerevisiae, necessitating a rapid life cycle of lipid droplets (LDs) for cellular adaptation. This process uncovers a previously uncharacterized subcellular architecture-an organelle triad-comprising the vacuole, LDs, and the nuclear endoplasmic reticulum (ER). The vacuole undergoes expansion and deformation, enveloping the entire nucleus that is encircled by an orbit of LDs. Formation of this organelle triad depends on the timely and abundant expression of membrane-tethering proteins that mediate vacuole-LD contact sites and nuclear ER-vacuole junctions. This dynamic and reversible subcellular organization ensures efficient LD metabolism to support cell survival under nutrient stress.
    Keywords:  CP: Cell biology; LDO proteins; lipid droplet; membrane contact; nutrient stress; nvj1; subcellular architecture; the nucleus–vacuole junction; vac8; vacuole deformation
    DOI:  https://doi.org/10.1016/j.celrep.2025.115813
  17. Acta Pharm Sin B. 2025 Apr;15(4): 2156-2169
    GPC3-mediated lysosome-targeting chimeras (GLTACs) for targeted degradation of membrane proteins.
    Yuxin Fang, Yaojin Zhu, Wei Wang, Zhewei Xia, Shipeng He, Guoqiang Dong, Chunquan Sheng.
      Membrane protein degradation is a cutting-edge field in targeted protein degradation (TPD). Herein, we developed glypican-3 (GPC3)-mediated lysosome-targeting chimeras (GLTACs) as a novel strategy for the targeted degradation of tumor-specific membrane proteins. GLTACs utilize tumor-specific expression and endocytosis properties of GPC3 to degrade membrane proteins. By conjugating a GPC3-targeting peptide with the ligand of protein of interest (POI), GLTACs induce the formation of a ternary complex that is internalized into lysosomes, leading to the degradation of the POI. The effectiveness and specificity of GLTACs were validated by designing PD-L1, c-Met, and FGFR1 degraders. In particular, GLTAC WP0 potently degraded PD-L1 and induced T-cell-mediated tumor killing against HepG2 cells, highlighting the potential therapeutic applications. The development of GLTAC technology expands the scope of TPD strategies and opens new avenues for discovering novel therapeutic modalities against challenging protein targets.
    Keywords:  Glypican-3; Lysosomal degradation; Membrane protein; PD-L1; Targeted protein degradation
    DOI:  https://doi.org/10.1016/j.apsb.2025.02.037
  18. Nat Commun. 2025 Jun 06. 16(1): 5266
    Aging and diet alter the protein ubiquitylation landscape in the mouse brain.
    Antonio Marino, Domenico Di Fraia, Diana Panfilova, Amit Kumar Sahu, Alberto Minetti, Omid Omrani, Emilio Cirri, Alessandro Ori.
      Post-translational modifications (PTMs) regulate protein homeostasis, but how aging impacts PTMs remains unclear. Here, we used mass spectrometry to reveal changes in hundreds of protein ubiquitylation, acetylation, and phosphorylation sites in the mouse aging brain. We show that aging has a major impact on protein ubiquitylation. 29% of the quantified ubiquitylation sites were affected independently of protein abundance, indicating altered PTM stoichiometry. Using iPSC-derived neurons, we estimated that 35% of ubiquitylation changes observed in the aged brain can be attributed to reduced proteasome activity. Finally, we tested whether protein ubiquitylation in the brain can be influenced by dietary intervention. We found that one cycle of dietary restriction and re-feeding modifies the brain ubiquitylome, rescuing some but exacerbating other ubiquitylation changes observed in old brains. Our findings reveal an age-dependent ubiquitylation signature modifiable by dietary intervention, providing insights into mechanisms of protein homeostasis impairment and highlighting potential biomarkers of brain aging.
    DOI:  https://doi.org/10.1038/s41467-025-60542-6
  19. bioRxiv. 2025 May 29. pii: 2025.05.26.655807. [Epub ahead of print]
    Mitochondrial presequences harbor variable strengths to maintain organellar function.
    Youmian Yan, Baigalmaa Erdenepurev, Ian Collinson, Natalie M Niemi.
      Hundreds of mitochondrial-destined proteins rely on N-terminal presequences for organellar targeting and import. While generally described as positively charged amphipathic helices, presequences lack a consensus motif and thus likely promote the import of proteins into mitochondria with variable efficiencies. Indeed, the concept of presequence "strength" critically underlies biological models such as stress sensing, yet a quantitative analysis of what dictates "strong" versus "weak" presequences is lacking. Furthermore, the extent to which presequence strength affects mitochondrial function and cellular fitness remains unclear. Here, we capitalize on the high-throughput and kinetic nature of the MitoLuc mitochondrial protein import assay to quantify multiple aspects of presequence strength. We find that select presequences, including those that regulate the mitochondrial unfolded protein response (UPR mt ), are sufficient to impart differential import efficiencies during mitochondrial uncoupling. Surprisingly, we find that presequences beyond those classically associated with stress signaling promote highly variable import efficiency in stressed and basal (i.e., non-stressed) conditions in vitro, suggesting that presequence strength may influence a broader array of processes than currently appreciated. We exploit this variability to demonstrate that only presequences that promote robust import in vitro can fully rescue defects in respiratory growth in Complex IV-deficient yeast, suggesting that presequence strength dictates metabolic potential. Collectively, our findings demonstrate that presequence strength can describe numerous metrics, such as total imported protein, maximal import velocity, or sensitivity to uncoupling, suggesting that the annotation of presequences as "weak" versus "strong" requires more nuanced characterization than is typically performed. Importantly, we find that such variability in presequence strength meaningfully affects cellular fitness in processes beyond stress signaling, suggesting that organisms may broadly exploit presequence strength to fine-tune mitochondrial import and thus organellar homeostasis.
    DOI:  https://doi.org/10.1101/2025.05.26.655807
  20. Sci Adv. 2025 Jun 13. 11(24): eadu5791
    Switchable client specificity in a dual functional chaperone coordinates light-harvesting complex biogenesis.
    Alex R Siegel, Gerard Kroon, Changqi Zhao, Peng Wang, Peter E Wright, Shu-Ou Shan.
      The proper assembly of light-harvesting complexes (LHCs) is critical for photosynthesis and requires the biogenesis of light-harvesting chlorophyll a,b-binding proteins (LHCPs) to be coordinated with chlorophyll (Chl) biosynthesis. The mechanism underlying this coordination is not well understood. Here, we show that a conserved molecular chaperone, chloroplast signal recognition particle 43-kDa protein (cpSRP43), provides a molecular thermostat that helps maintain this coordination. cpSRP43 undergoes a conformational rearrangement between a well-folded closed state and a partially disordered open state. Closed cpSRP43 is dedicated to the biogenesis of LHCPs, whereas open cpSRP43 protects multiple Chl biosynthesis enzymes from heat-induced destabilization. Rising temperature shifts cpSRP43 to the open state, enabling it to protect heat-destabilized Chl biosynthesis enzymes. Our results reveal the molecular basis of a posttranslational mechanism for the thermoadaptation of LHC biogenesis. They also demonstrate how an adenosine triphosphate-independent chaperone uses conformational dynamics to switch its activity and client selectivity, thereby adapting to different proteostatic demands under shifting environmental conditions.
    DOI:  https://doi.org/10.1126/sciadv.adu5791
  21. Mol Microbiol. 2025 Jun 09.
    The Flagellin-Specific Chaperone FliS of Borrelia burgdorferi Controls the Cytoplasmic Pool of Flagellins at the Level of Translation Initiation, Secretion, and Proteolysis.
    Ching Wooen Sze, Kai Zhang, Michael J Lynch, Wangbiao Guo, Jun Liu, Brian R Crane, Chunhao Li.
      The flagellin-specific chaperone FliS has been studied in externally flagellated bacteria; however, its role in spirochetes, a group of bacteria that possess unique internalized flagella (termed endo- or periplasmic flagella), remains unexplored. Here, we investigate the function of FliS in the Lyme disease spirochete Borrelia burgdorferi. Using loss-of-function studies, combined with biochemical assays and cryo-electron tomography, we demonstrate that FliS deletion selectively reduces FlaB expression, the major flagellin protein, resulting in non-motile mutants with defective flagellar filaments. Mechanistically, we show that FlaB interacts with both FliS and FliW, the latter being an allosteric repressor of the RNA-binding protein CsrA, which inhibits FlaB translation. These four components form a regulatory circuit that fine-tunes FlaB levels and flagellar assembly via a partner-switching mechanism. Deletion of fliS disrupts FlaB secretion, leading to its cytoplasmic accumulation, sequestration of FliW, and subsequent release of CsrA to suppress FlaB synthesis. Accumulation of cytoplasmic FlaB also triggers its degradation to prevent toxicity. Our findings reveal a post-transcriptional regulatory mechanism governing flagellar assembly in B. burgdorferi, an evolutionary outlier that lacks the canonical transcriptional cascade controlling flagellar biosynthesis in most bacteria.
    Keywords:   Borrelia burgdorferi ; FlaB; FliS; FliW; Lyme disease; flagellin
    DOI:  https://doi.org/10.1111/mmi.15380
  22. bioRxiv. 2025 Jun 04. pii: 2025.06.02.657324. [Epub ahead of print]
    Loss of UFMylation supports prostate cancer metastasis and rewires cell metabolism towards hexosamine biosynthesis.
    Laura Bozal-Basterra, María-Camila Salazar, Ana Margarida Ferreira Campos, Margherita Demicco, Daniel R Schmidt, Klaudia Sobczak, June Ereño-Orbea, Patricia Altea-Manzano, Ainara Miranda Villanueva, Belén Martínez La Osa, Saioa Garcia-Longarte, María Ponce-Rodriguez, Isabel Mendizabal, Onintza Carlevaris, Ianire Astobiza, Natalia Martin-Martin, Amaia Zabala-Letona, Ana Talamillo, Juan Fernández-García, Mikel Azkargorta, Ibon Iloro, Rosa Barrio, Félix Elortza, Sarah-Maria Fendt, Matthew G Vander Heiden, Jesús-Jiménez Barbero, James D Sutherland, Arkaitz Carracedo.
      The acquisition of metastatic features in tumor cells encompasses genetic and non-genetic adaptation, including reprogramming of cellular metabolism. Here we show that loss of UFMylation reroutes glucose metabolism, promotes invasive capacity and supports prostate cancer metastasis. Through transcriptome-based bioinformatics analysis, we identified a reduction in the ubiquitin-like modifier UFM1 and its ligase UFL1 in metastatic prostate cancer. We demonstrate that loss of UFMylation results in enhanced cancer cell dissemination and a switch from cellular proliferation to invasion. Using biotin-based proteomics, we identified phosphofructokinase (PFKAP) as an unprecedented UFMylation substrate. Consistent with UFMylation playing a role in the regulation of phosphofructokinase activity, loss of UFMylation reduced glucose metabolism in favour of hexosamine biosynthesis, which resulted in elevated glycosylation of proteins relevant for cell invasion. These results reveal a role for UFMylation in the regulation of phosphofructokinase and glucose metabolism to support prostate cancer metastasis.
    DOI:  https://doi.org/10.1101/2025.06.02.657324
  23. bioRxiv. 2025 May 30. pii: 2025.05.27.656443. [Epub ahead of print]
    A Diffusion-Based Framework for Designing Molecules in Flexible Protein Pockets.
    Jian Wang, Dong Yan Zhang, Shreshty Budakoti, Nikolay V Dokholyan.
      The design of molecules for flexible protein pockets represents a significant challenge in structure-based drug discovery, as proteins often undergo conformational changes upon ligand binding. While deep learning-based approaches have shown promise in molecular generation, they typically treat protein pockets as rigid structures, limiting their ability to capture the dynamic nature of protein-ligand interactions. Here, we introduce YuelDesign, a novel diffusion-based framework specifically developed to address this challenge. YuelDesign employs a new protein encoding scheme with a fully connected graph representation to encode protein pocket flexibility, a systematic denoising process that refines both atomic properties and coordinates, and a specialized bond reconstruction module tailored for de novo generated molecules. Our results demonstrate that YuelDesign generates molecules with favorable drug-likeness and low synthetic complexity. The generated molecules also exhibit diverse chemical functional groups, including some not even present in the training set. Redocking analysis reveals that the generated molecules exhibit docking energies comparable to native ligands. Additionally, a detailed analysis of the denoising process shows how the model systematically refines molecular structures through atom type transitions, bond dynamics, and conformational adjustments. Overall, YuelDesign presents a versatile framework for generating novel molecules tailored to flexible protein pockets, with promising implications for drug discovery applications.
    DOI:  https://doi.org/10.1101/2025.05.27.656443
  24. Commun Biol. 2025 Jun 07. 8(1): 893
    Structural insights into tRNA recognition of the human FTSJ1-THADA complex.
    Kensuke Ishiguro, Atsushi Fujimura, Mikako Shirouzu.
      tRNA undergoes various post-transcriptional modifications in the anticodon loop. FTSJ1, a protein conserved among most eukaryotes, mediates 2'-O-methylations at position 32 (Nm32) or position 34 (Nm34), complexed with THADA or WDR6, respectively. These methylations are crucial for accurate translation and cellular growth. FTSJ1 mutations are associated with non-syndromic X-linked intellectual disability. Although the structure of the FTSJ1-WDR6 complex in yeast has been solved, the structural details of the FTSJ1-THADA complex formation and substrate recognition remain unclear. Herein, using cryo-electron microscopy, we solve the high-resolution structure of FTSJ1-THADA with or without a tRNA substrate. FTSJ1 binds to THADA via its C-terminal region, with a unique interaction mode distinct from the FTSJ1-WDR6 complex. The tRNA substrate is anchored inside THADA, and key THADA residues for THADA-tRNA interaction are identified via structural and biochemical analyses. These findings demonstrate how FTSJ1 and THADA form a complex to mediate Nm32 modification in various tRNAs.
    DOI:  https://doi.org/10.1038/s42003-025-08278-3
  25. Science. 2025 Jun 12. 388(6752): 1218-1224
    Pan-viral ORFs discovery using massively parallel ribosome profiling.
    Shira Weingarten-Gabbay, Matthew R Bauer, Alexandra C Stanton, Yingpu Yu, Catherine A Freije, Nicole L Welch, Chloe K Boehm, Susan Klaeger, Eva K Verzani, Daniel López, Lisa E Hensley, Karl R Clauser, Steven A Carr, Jennifer G Abelin, Charles M Rice, Pardis C Sabeti.
      Defining viral proteomes is crucial to understanding viral life cycles and immune recognition but the landscape of translated regions remains unknown for most viruses. We have developed massively parallel ribosome profiling (MPRP) to determine open reading frames (ORFs) across tens of thousands of designed oligonucleotides. MPRP identified 4208 unannotated ORFs in 679 human-associated viral genomes. We found viral peptides originating from detected noncanonical ORFs presented on class-I human leukocyte antigen in infected cells and hundreds of upstream ORFs that likely modulate translation initiation of viral proteins. The discovery of viral ORFs across a wide range of viral families-including highly pathogenic viruses-expands the repertoire of vaccine targets and reveals potential cis-regulatory sequences.
    DOI:  https://doi.org/10.1126/science.ado6670
  26. bioRxiv. 2025 Jun 08. pii: 2025.06.07.658469. [Epub ahead of print]
    Integrative Chemical Genetics Platform Identifies Condensate Modulators Linked to Neurological Disorders.
    Dylan Poch, Chandrayee Mukherjee, Sunanda Mallik, Vanessa Todorow, E F Elsiena Kuiper, Nalini Dhingra, Yulia V Surovtseva, Christian Schlieker.
      Aberrant biomolecular condensates are implicated in multiple incurable neurological disorders, including Amyotrophic Lateral Sclerosis (ALS), Frontotemporal Dementia (FTD), and DYT1 dystonia. However, the role of condensates in driving disease etiology remains poorly understood. Here, we identify myeloid leukemia factor 2 (MLF2) as a disease-agnostic biomarker for phase transitions, including stress granules and nuclear condensates associated with dystonia. Exploiting fluorophore-derivatized MLF2 constructs, we developed a high-content platform and computational pipeline to screen modulators of NE condensates across chemical and genetic space. We identified RNF26 and ZNF335 as protective factors that prevent the buildup of nuclear condensates sequestering K48-linked polyubiquitinated proteins. Chemical screening identified four FDA-approved drugs that potently modulate condensates by resolving polyubiquitinated cargo and MLF2 accumulation. Our exploratory integrated chemical-genetics approach suggests that modulation of zinc, and potentially autophagy and oxidative stress, is critical for condensate modulation and nuclear proteostasis, offering potential therapeutic strategies for neurological disorders. Application of our platform to a genome-wide CRISPR KO screen identified strong enrichment of candidate genes linked to primary microcephaly and related neurodevelopmental disorders. Two hypomorphic microcephaly-associated alleles of ZNF335 failed to rescue nuclear condensate accumulation in ZNF335 KO cells, suggesting that aberrant condensates and impaired nuclear proteostasis may contribute to the pathogenesis of microcephaly.
    HIGHLIGHTS: MLF2 emerges as a disease-agnostic condensate biomarker co-localizing with TDP-43 and G3BP1FDA-approved drugs target condensates linked to perturbed proteostasis.RNF26 and ZNF335 are identified as modulators of nuclear phase transitions.Microcephaly patient disease alleles fail to counteract aberrant condensates.
    DOI:  https://doi.org/10.1101/2025.06.07.658469
  27. RSC Med Chem. 2025 Jun 06.
    Rational design and discovery of potent PROTAC degraders of ASK1: a targeted therapy in MASH.
    Himadri Sekhar Sarkar, Abhishek Sen, Israful Hoque, Uddipta Ghosh Dastidar, Soupayan Pal, Dipika Sarkar, Partha Chakrabarti, Arindam Talukdar.
      Metabolic dysfunction-associated steatohepatitis (MASH) is a progressive liver disease marked by hepatic steatosis, inflammation, and fibrosis, with limited therapeutic options. This study introduces a novel PROTAC-based strategy for the selective degradation of apoptosis signal-regulating kinase 1 (ASK1), a key mediator of MASH pathology. We first developed dASK1 (35), a cereblon (CRBN)-based PROTAC, which successfully formed a stable ternary complex with ASK1, facilitating its rapid and sustained degradation via the ubiquitin-proteasome pathway. In vitro evaluations demonstrated potent ASK1 degradation in the 10-100 nM range (70% degradation at 100 nM) in HepG2 and HEK293A cell lines, validating the efficacy of dASK1 (35). To enhance the degradation mechanism and explore broader E3 ligase utility, we designed and synthesized dASK1-VHL (60), leveraging the von Hippel-Lindau (VHL) E3 ligase, known for its regulatory functions in hepatic physiology. We optimized the linker length through molecular docking and MMGBSA calculations, achieving efficient ASK1-VHL engagement and stable ternary complex formation. Detailed ADME and pharmacokinetic studies confirmed that dASK1-VHL (60) exhibited enhanced solubility, moderate clearance, and improved bioavailability, making it suitable for in vivo application. In an MCD diet-induced murine model of MASH, dASK1-VHL (60) effectively reduced ASK1 protein levels, suppressed p38 MAPK activation, and decreased hepatic lipid content, indicating significant therapeutic benefits. This work underscores the importance of rational PROTAC design, precise linker engineering, and innovative E3 ligase selection in optimizing target protein degradation. Our findings pave the way for developing VHL-based PROTACs, offering a novel therapeutic approach for metabolic and inflammatory liver diseases.
    DOI:  https://doi.org/10.1039/d5md00252d
  28. Sci Adv. 2025 Jun 13. 11(24): eadu1823
    AbEpiTope-1.0: Improved antibody target prediction by use of AlphaFold and inverse folding.
    Joakim Nøddeskov Clifford, Eve Richardson, Bjoern Peters, Morten Nielsen.
      B cell epitope prediction tools are crucial for designing vaccines and disease diagnostics. However, predicting which antigens a specific antibody binds to and their exact binding sites (epitopes) remains challenging. Here, we present AbEpiTope-1.0, a tool for antibody-specific B cell epitope prediction, using AlphaFold for structural modeling and inverse folding for machine learning models. On a dataset of 1730 antibody-antigen complexes, AbEpiTope-1.0 outperforms AlphaFold in predicting modeled antibody-antigen interface accuracy. By creating swapped antibody-antigen complex structures for each antibody-antigen complex using incorrect antibodies, we show that predicted accuracies are sensitive to antibody input. Furthermore, a model variant optimized for antibody target prediction-differentiating true from swapped complexes-achieved an accuracy of 61.21% in correctly identifying antibody-antigen pairs. The tool evaluates hundreds of structures in minutes, providing researchers with a resource for screening antibodies targeting specific antigens. AbEpiTope-1.0 is freely available as a web server and software.
    DOI:  https://doi.org/10.1126/sciadv.adu1823
  29. FASEB J. 2025 Jun 15. 39(11): e70695
    CD44 Deficiency Induces Combinatory NRF2 Inhibition and Endoplasmic Reticulum Stress-Associated Dyserythropoiesis.
    Ran Yang, Donglin Yu, Jiuqiang Ren, Jiaying Yin, Jing Li, Xuehui Liu, Xiang Lv.
      The expression of the transmembrane protein CD44 gradually declines during terminal erythropoiesis, marking the differentiating stages of erythroblasts. Despite its significance, the specific role and functional mechanisms of CD44 in erythropoiesis remain poorly understood. In this study, we found that CD44 knockout (CD44KO) mice displayed bone marrow dyserythropoiesis and an exacerbation of phenylhydrazine-induced hemolytic anemia. Single-cell sequencing analysis revealed activated endoplasmic reticulum (ER) stress in CD44KO erythroblasts. Specifically, the PERK/eIF2α/ATF4/CHOP pathway was upregulated, accompanied by enhanced autophagy and subsequent cell apoptosis. Mechanistically, CD44 deficiency suppressed both the PI3K/AKT/NRF2 and p62/NRF2 pathways, consequently downregulating GPX1 and GPX4 expression, resulting in significantly elevated levels of reactive oxygen species (ROS) and lipid oxidation in erythroid cells. Furthermore, the oxidizing agent H2O2 induced ER stress dose-dependently, inhibiting in vitro erythroid differentiation of mouse fetal liver cells. Notably, CD44KO erythroblasts demonstrated heightened sensitivity to H2O2 treatment. Our findings elucidate the role of CD44 in maintaining redox homeostasis and proteostasis during terminal erythropoiesis, providing valuable insights into how ROS-induced ER stress contributes to pathogenic erythropoiesis.
    Keywords:  CD44; NRF2; autophagy; endoplasmic reticulum stress; erythropoiesis
    DOI:  https://doi.org/10.1096/fj.202500228R
  30. Proc Natl Acad Sci U S A. 2025 Jun 17. 122(24): e2424985122
    CRISPR screens and quantitative proteomics reveal remodeling of the aryl hydrocarbon receptor-driven proteome through PARP7 activity.
    Andrii Gorelik, Joao A Paulo, Christina B Schroeter, Melanie Lad, Abigail Shurr, Chara Mastrokalou, Samrah Siddiqi, Osamu Suyari, John Brognard, David Walter, Jason Matthews, Timothy M Palmer, Steven P Gygi, Ivan Ahel.
      PARP7 is an enzyme that uses donor substrate NAD+ to attach a single ADP-ribose moiety onto proteins related to immunity, transcription, and cell growth and motility. Despite the importance of PARP7 in these processes, PARP7 signaling networks remain underresearched. Here, we used genome-wide CRISPR screens and multiplex quantitative proteomics in distinct lung cancer cell lines treated with a PARP7 inhibitor to better understand PARP7 molecular functions. We find that manipulating the aryl hydrocarbon receptor (AHR) transcriptional activity mediates PARP7 inhibitor sensitivity and triggers robust changes to the AHR-controlled proteome (AHR-ome). One of the striking features of such AHR-ome remodeling was the downregulation of filamins A and B concurrent with the induction of the corresponding E3 ubiquitin ligase ASB2. We also show that suppressor of cytokine signaling 3 (SOCS3) crosstalks to AHR. Inhibition of PARP7 in SOCS3 knockout cells leads to reduced viability compared to wild-type cells treated with a PARP7 inhibitor. Our results reveal signaling interplay between PARP7, AHR, and SOCS3 and establish an invaluable resource to study the role of PARP7 in the regulation of AHR signaling and innate immunity through its ADP-ribosyl transferase activity.
    Keywords:  ADP-ribosylation; CRISPR; PARP; aryl hydrocarbon receptor; proteomics
    DOI:  https://doi.org/10.1073/pnas.2424985122
  31. Nat Commun. 2025 Jun 06. 16(1): 5257
    Translation suppresses exogenous target RNA-mediated microRNA decay.
    Tianqi Li, Lu Li, Nicholas M Hiers, Peike Sheng, Yuzhi Wang, Conner M Traugot, Jessi F Effinger-Morris, Pitchaporn Akaphan, Yanyan Liu, Jiang Bian, Kotaro Fujii, Mingyi Xie.
      MicroRNAs (miRNAs) interact with the target mRNAs to induce translational repression and mRNA degradation. Interestingly, miRNAs themselves can turnover rapidly when binding to a target RNA with extensive complementarity, a phenomenon called target-directed miRNA degradation (TDMD). To date, all validated TDMD "triggers" can induce miRNA degradation reside in non-coding regions of the RNA. We found that TDMD triggers placed in the 3' untranslated region (UTR) of a reporter degraded miRNAs more effectively than those in the coding sequence (CDS). Inhibiting translation of the reporter enhanced miRNA degradation by the CDS trigger, indicating that ribosome-free CDS triggers are more accessible to miRNAs. By small RNA sequencing, we explored mammalian miRNAs sensitive to global translation status. Yet, no endogenous CDS trigger could be confidently assigned to these miRNAs. Our work revealed the intricate relationship between translation and TDMD, and explains the paucity of effective TDMD triggers in the CDS.
    DOI:  https://doi.org/10.1038/s41467-025-60374-4
  32. Cell Rep. 2025 Jun 10. pii: S2211-1247(25)00600-X. [Epub ahead of print]44(6): 115829
    The lysosomal membrane protein LAMP2B mediates microlipophagy to target obesity-related disorders.
    Ryohei Sakai, Shu Aizawa, Hyeon-Cheol Lee-Okada, Katsunori Hase, Hiromi Fujita, Hisae Kikuchi, Yukiko U Inoue, Takayoshi Inoue, Chihana Kabuta, Takehiko Yokomizo, Tadafumi Hashimoto, Keiji Wada, Tatsuo Mano, Ikuko Koyama-Honda, Tomohiro Kabuta.
      Lifestyle diseases, such as obesity, diabetes, and metabolic syndrome, are leading health problems, most of which are related to abnormal lipid metabolism. Lysosomes can degrade lipid droplets (LDs) via microautophagy, but the regulatory factors and physiological significance of this process are not fully understood. Here, we report the molecular mechanism and pathophysiological roles of microlipophagy, regulated by the lysosomal membrane protein LAMP2B. Our study reveals that LAMP2B interacts with phosphatidic acid, facilitating lysosomal-LD interactions and enhancing lipid hydrolysis via microlipophagy depending on endosomal sorting complexes required for transport. Correlative light and electron microscopy demonstrates direct LD uptake into lysosomes at contact sites. Moreover, LAMP2B overexpression in mice prevents high-fat diet-induced obesity, insulin resistance, and adipose tissue inflammation; liver lipidomics analysis suggests enhanced triacylglycerol hydrolysis. Overall, the findings of this study elucidate the mechanism of microlipophagy, which could be promising for the treatment of obesity and related disorders.
    Keywords:  CP: Cell biology; CP: Metabolism; LAMP2B; lipid droplet; lysosome; microautophagy; microlipophagy
    DOI:  https://doi.org/10.1016/j.celrep.2025.115829
  33. Nature. 2025 Jun 11.
    SP140-RESIST pathway regulates interferon mRNA stability and antiviral immunity.
    Kristen C Witt, Adam Dziulko, Joohyun An, Filip Pekovic, Arthur Xiuyuan Cheng, Grace Y Liu, Ophelia Vosshall Lee, David J Turner, Azra Lari, Moritz M Gaidt, Roberto Chavez, Stefan A Fattinger, Preethy Abraham, Harmandeep Dhaliwal, Angus Y Lee, Dmitri I Kotov, Laurent Coscoy, Britt A Glaunsinger, Eugene Valkov, Edward B Chuong, Russell E Vance.
      Type I interferons are essential for antiviral immunity1 but must be tightly regulated2. The conserved transcriptional repressor SP140 inhibits interferon-β (Ifnb1) expression through an unknown mechanism3,4. Here we report that SP140 does not directly repress Ifnb1 transcription. Instead, SP140 negatively regulates Ifnb1 mRNA stability by directly repressing the expression of a previously uncharacterized regulator that we call RESIST (regulated stimulator of interferon via stabilization of transcript; previously annotated as annexin 2 receptor). RESIST promotes Ifnb1 mRNA stability by counteracting Ifnb1 mRNA destabilization mediated by the tristetraprolin (TTP) family of RNA-binding proteins and the CCR4-NOT deadenylase complex. SP140 localizes within punctate structures called nuclear bodies that have important roles in silencing DNA-virus gene expression in the nucleus3. Consistent with this observation, we find that SP140 inhibits replication of the gammaherpesvirus MHV68. The antiviral activity of SP140 is independent of its ability to regulate Ifnb1. Our results establish dual antiviral and interferon regulatory functions for SP140. We propose that SP140 and RESIST participate in antiviral effector-triggered immunity5,6.
    DOI:  https://doi.org/10.1038/s41586-025-09152-2
  34. Autophagy. 2025 Jun 12.
    Tumorous cholesterol biosynthesis curtails anti-tumor immunity by preventing MTOR-TFEB-mediated lysosomal degradation of CD274/PD-L1.
    Huina Wang, Xiuli Yi, Di Qu, Xiangxu Wang, Hao Wang, Hengxiang Zhang, Yuqi Yang, Tianwen Gao, Weinan Guo, Chunying Li.
      Enhanced cholesterol biosynthesis is a hallmark metabolic characteristic of cancer, exerting an oncogenic role by supplying intermediate metabolites that regulate intracellular signaling pathways. The pharmacological blockade of cholesterol biosynthesis has been well documented as a promising therapeutic approach in cancer. Particularly, cholesterol biosynthesis is linked to macroautophagy/autophagy and lysosome metabolism, with the engagement of the critical autophagy regulators like MTOR to be fully activated by lysosomal cholesterol trafficking and accumulation. Previous studies have primarily focused on the role of cholesterol biosynthesis in tumor cell-intrinsic biological processes, whereas its involvement in tumor immune evasion and the underlying mechanisms related to autophagy or lysosome metabolism remain elusive. Herein, through bioinformatics analysis we discovered a negative correlation between cholesterol biosynthesis and the score of tumor-infiltrating lymphocytes in cancers. Inhibition of tumor cell cholesterol biosynthesis leads to increased infiltration and activation of CD8+ T cells in the tumor microenvironment, which is largely responsible for the impairment of tumor growth. Mechanistically, cholesterol biosynthesis inhibition impairs the activation of MTOR at lysosomes, thereby promoting the nuclear translocation of TFEB and downstream lysosome biosynthesis, facilitating the degradation of CD274/PD-L1 within lysosomes in tumor cells. Ultimately, the HMGCR-MTOR-LAMP1 axis that connects cholesterol, lysosome and tumor immunology, predicts poor response to immunotherapy and worse prognosis of patients with melanoma. These findings unveil an immunomodulatory role of tumorous cholesterol biosynthesis via the regulation of CD274 lysosomal degradation. Targeting cholesterol biosynthesis holds promise as a potential therapeutic strategy in cancer, particularly when combined with immune checkpoint blockade.
    Keywords:  Cholesterol; PD-L1; TFEB; immune evasion; lysosome; protein degradation
    DOI:  https://doi.org/10.1080/15548627.2025.2519066
  35. EMBO Mol Med. 2025 Jun 09.
    Small molecule disruption of RARα/NCoR1 interaction inhibits chaperone-mediated autophagy in cancer.
    Mericka McCabe, Rajanya Bhattacharyya, Rebecca Sereda, Olaya Santiago-Fernández, Rabia R Khawaja, Antonio Diaz, Kristen Lindenau, Deniz Gulfem Ozturk, Thomas P Garner, Simone Sidoli, Ana Maria Cuervo, Evripidis Gavathiotis.
      Chaperone-mediated autophagy (CMA), a type of selective degradation of cytosolic proteins in lysosomes, is commonly upregulated in cancer cells, contributing to their survival and growth. The lack of a specific target for CMA inhibition has limited CMA blockage to genetic manipulations or global lysosomal function inhibition. Here, using genetic modulation, transcriptional analysis, and functional studies, we demonstrate a regulatory role for the interaction of the retinoic acid receptor alpha (RARα) and its corepressor, the nuclear receptor corepressor 1 (NCoR1), on CMA in non-small cell lung cancer (NSCLC). By targeting the disruption of the NCoR1/RARα complex with a structure-based screening strategy, we identified compound CIM7, a potent and selective CMA inhibitor that has no effect on macroautophagy. CIM7 preferentially inhibits CMA in NSCLC cells over normal cells, reduces tumor growth in NSCLC cells, and demonstrates efficacy in an in vivo xenograft mouse model with no observed toxicity in blood or major tissues. These findings reveal a druggable mechanism for selective CMA inhibition and a first-in-class CMA inhibitor as a potential therapeutic strategy for NSCLC.
    Keywords:  Autophagy; CMA Inhibitor; NCoR1; Non-small Cell Lung Cancer; RARα
    DOI:  https://doi.org/10.1038/s44321-025-00254-y
  36. Cell Metab. 2025 Jun 09. pii: S1550-4131(25)00265-7. [Epub ahead of print]
    Derepressing nuclear pyruvate dehydrogenase induces therapeutic cancer cell reprogramming.
    Ting Zhao, Lingli He, Lai Ping Wong, Shenglin Mei, Jun Xia, Yanxin Xu, Jonathan G Van Vranken, Michael Mazzola, Lei Chen, Catherine Rhee, Tiancheng Fang, Tsuyoshi Fukushima, Leanne C Sayles, Matthew Diaz, J Alex B Gibbons, Raul Mostoslavsky, Steven P Gygi, Zhixun Dou, David B Sykes, Ruslan I Sadreyev, E Alejandro Sweet-Cordero, David T Scadden.
      Metabolites are essential substrates for epigenetic modifications. Although nuclear acetyl-coenzyme A (CoA) constitutes a small fraction of the whole-cell pool, it regulates cell fate by locally providing histone acetylation substrate. Here, we report a nucleus-specific acetyl-CoA regulatory mechanism that can be modulated to achieve therapeutic cancer cell reprogramming. Combining phenotypic chemical screen, genome-wide CRISPR screen, and proteomics, we identified that the nucleus-localized pyruvate dehydrogenase complex (nPDC) is constitutively inhibited by the nuclear protein ELMSAN1 through direct interaction. Pharmacologic inhibition of the ELMSAN1-nPDC interaction derepressed nPDC activity, enhancing nuclear acetyl-CoA generation and reprogramming cancer cells to a postmitotic state with diminished cell-of-origin signatures. Reprogramming was synergistically enhanced by histone deacetylase 1/2 inhibition, resulting in inhibited tumor growth, durably suppressed tumor-initiating ability, and improved survival in multiple cancer types in vivo, including therapy-resistant sarcoma patient-derived xenografts and carcinoma cell line xenografts. Our findings highlight the potential of targeting ELMSAN1-nPDC as an epigenetic cancer therapy.
    Keywords:  ELMSAN1; HDAC; ISX9; acetyl-CoA metabolism; cancer therapy; compartmentalized metabolism; epigenetic reprogramming; nuclear metabolism; pyruvate dehydrogenase complex; therapeutic reprogramming
    DOI:  https://doi.org/10.1016/j.cmet.2025.05.009
  37. Mol Cell. 2025 May 28. pii: S1097-2765(25)00454-X. [Epub ahead of print]
    The RNA-stability-independent role of the RNA m6A reader YTHDF2 in promoting protein translation to confer tumor chemotherapy resistance.
    Tao Liu, Dan Yang, Qinglv Wei, Yuan Wang, Lei Tian, Xiaoyi Liu, Yu Yang, Qingya Luo, Jie Xu, Yujiao Liu, Chenyue Yang, Xinzhao Zuo, Fatao Luo, Xin Luo, Hongyan Zhao, Li Li, Jing Xu, Jia Yu, Shoubao Ma, Jianhua Yu, Ping Yi.
      N6-methyladenosine (m6A) modification plays pivotal roles in myriad biological processes. The YTH domain family protein YTHDF2, recognized as an m6A "reader" protein, is primarily associated with the canonical function of facilitating RNA degradation. Nevertheless, the intricate non-decay regulatory mechanism exerted by YTHDF2 remains enigmatic. Here, using ovarian cancer as a model, we demonstrate that YTHDF2 forms a tangible interaction with the eukaryotic translation initiation factor eIF3F and the RNA helicase DDX1, thereby enhancing protein synthesis in tumor cells. Instead of promoting RNA degradation, YTHDF2 facilitates the translation of m6A-modified mRNAs encoding microtubule-associated proteins, which drives cancer progression and reduces the chemosensitivity of cancer cells to paclitaxel, a commonly used chemotherapy drug. Notably, through virtual screening, we identified a YTHDF2-specific small-molecule inhibitor. Therapeutic targeting of YTHDF2 with this inhibitor effectively suppresses protein translation in tumor cells and reverses paclitaxel resistance.
    Keywords:  YTHDF2; m(6)A; translational regulation; tumor resistance
    DOI:  https://doi.org/10.1016/j.molcel.2025.05.015
  38. Nat Commun. 2025 Jun 06. 16(1): 5261
    TANGO2 binds crystallin alpha B and its loss causes desminopathy.
    Maike Stentenbach, Laetitia A Hughes, Samuel V Fagan, Blake Payne, Danielle L Rudler, Stefan J Siira, Tim McCubbin, Anaëlle Chopin, Kara L Perks, Judith A Ermer, James Hendry, Teagan S Er, Shanti Balasubramaniam, Joel A Eliades, Livia C Hool, Nicolle H Packer, Edward S X Moh, Benjamin S Padman, Oliver Rackham, Aleksandra Filipovska.
      Mutations in the TANGO2 gene cause an autosomal recessive disorder characterised by developmental delay, stress-induced episodic rhabdomyolysis, and cardiac arrhythmias along with severe metabolic crises. Although TANGO2 mutations result in a well characterised disease pathology, the function of TANGO2 is still unknown. To investigate the function of TANGO2, we knocked out the TANGO2 gene in human cells and mice. We identify that loss of TANGO2 impairs intermediate filament structure, resulting in fragmented mitochondrial networks and formation of cup-like mitochondria. In male mice, loss of TANGO2 caused heart defects, reduced muscle function and glucose intolerance by remodelling of intermediate filaments, which altered the mitochondrial and cytoplasmic proteomes, N-glycosylation and nucleocytoplasmic O-GlcNAcylation. We identify that TANGO2 binds the small heat shock protein crystallin alpha B (CRYAB) to prevent the aggregation of the intermediate filament desmin and in the absence of TANGO2, mice develop desminopathy, which is consistent with features found in patients carrying mutations in either desmin or CRYAB.
    DOI:  https://doi.org/10.1038/s41467-025-60563-1
  39. Cell. 2025 Jun 05. pii: S0092-8674(25)00570-7. [Epub ahead of print]
    Unraveling mitochondrial influence on mammalian pluripotency via enforced mitophagy.
    Daniel A Schmitz, Seiya Oura, Leijie Li, Yi Ding, Rashmi Dahiya, Emily Ballard, Carlos Pinzon-Arteaga, Masahiro Sakurai, Daiji Okamura, Leqian Yu, Peter Ly, Jun Wu.
      Mitochondrial abundance and genome are crucial for cellular function, with disruptions often associated with disease. However, methods to modulate these parameters for direct functional dissection remain limited. Here, we eliminate mitochondria from pluripotent stem cells (PSCs) by enforced mitophagy and show that PSCs survived for several days in culture without mitochondria. We then leverage enforced mitophagy to generate interspecies PSC fusions that harbor either human or non-human hominid (NHH) mitochondrial DNA (mtDNA). Comparative analyses indicate that human and NHH mtDNA are largely interchangeable in supporting pluripotency in these PSC fusions. However, species divergence between nuclear and mtDNA leads to subtle species-specific transcriptional and metabolic variations. By developing a transgenic enforced mitophagy approach, we further show that reducing mitochondrial abundance leads to delayed development in pre-implantation mouse embryos. Our study opens avenues for investigating the roles of mitochondria in development, disease, and interspecies biology.
    Keywords:  cell fusion; great apes; interspecies composite; interspecies hybrid; metabolism; mitochondria; mitophagy; mtDNA; pluripotent stem cells
    DOI:  https://doi.org/10.1016/j.cell.2025.05.020
  40. bioRxiv. 2025 Jun 01. pii: 2025.05.31.654582. [Epub ahead of print]
    Integrin-Specific Signaling Drives ER Stress-Dependent Atherogenic Endothelial Activation.
    Cyrine Ben Dhaou, Zaki Al-Yafeai, G Ali Cruz-Marquez, Matthew L Scott, Brenna Pearson-Gallion, Elizabeth Cockerham, Hanna Li, Jonette M Peretik, Yuning Hong, Nirav Dhanesha, Arif Yurdagul, Oren Rom, Md Shenuarin Bhuiyan, A Wayne Orr.
      Atherogenic endothelial activation arises from both the local arterial microenvironment-characterized by altered extracellular matrix composition and disturbed blood flow-and soluble proinflammatory stimuli such as oxidized low-density lipoprotein (oxLDL). Fibronectin, a provisional extracellular matrix protein enriched at atheroprone sites, enhances endothelial activation and inflammation triggered by oxLDL and disturbed flow. Although endoplasmic reticulum (ER) stress contributes to vascular dysfunction, the role of matrix composition in regulating ER stress remains unknown. We show that oxLDL and disturbed flow induce ER stress selectively in endothelial cells adhered to fibronectin, whereas both stimuli fail to induce ER stress in cells on basement membrane proteins. This matrix-specific ER stress response requires integrin activation, as endothelial cells deficient for integrin activation (talin1 L325R mutation) fail to activate ER stress in response to disturbed flow and oxLDL and direct stimulation of integrin activation using CHAMP peptides is sufficient to trigger ER stress. Blunting endothelial expression of fibronectin-binding integrins (α5, αv) using siRNA prevents ER stress in response to atherogenic stimuli in vitro , whereas endothelial α5 and αv deletion reduces ER stress at atheroprone sites in vivo . The mechanisms driving integrin-dependent ER stress remain unclear, since matrix composition does not affect protein translation, unfolded protein accumulation, or superoxide production, and scavenging superoxide (TEMPOL) does not reduce integrin-dependent ER stress. Inhibiting ER stress with TUDCA reduces proinflammatory and metabolic gene expression (bulk RNAseq) but does not prevent NF-κB activation, a classic proinflammatory transcription factor. Rather, TUDCA prevents activation of c-jun N-terminal kinase (JNK) and c-jun activation, and blocking JNK (SP600126) or c-Jun activity (TAM67) prevents proinflammatory gene expression following both stimuli. Together, these findings offer new insight into how the arterial microenvironment contributes to atherogenesis, with fibronectin-binding integrin signaling promotes ER stress in response to mechanical and metabolic stressors, thereby amplying proinflammatory endothelial activation through JNK-c-Jun signaling.
    Highlights: Fibronectin promotes endothelial ER stress in response to oxLDL and disturbed flow via integrin α5β1 and αvβ3 signaling.Matrix-specific ER stress occurs independently of oxidative stress or misfolded protein accumulation, indicating a non-canonical UPR activation.ER stress amplifies proinflammatory gene expression through JNK-c-Jun signaling, without activating NF-κB.Targeting the integrin-ER stress-JNK axis may offer new therapeutic strategies for early atherosclerosis.
    DOI:  https://doi.org/10.1101/2025.05.31.654582
  41. Brain. 2025 Jun 09. pii: awaf222. [Epub ahead of print]
    Early proteasome downregulation and dysfunction drive proteostasis failure in Alzheimer's disease.
    Shan Jiang, Malavika Srikanth, Rossana Serpe, Shadi Yavari, Pallavi Gaur, Galen Andrew Collins, Rajesh Soni, Vilas Menon, Natura Myeku.
      Alzheimer's disease (AD) is characterized by the accumulation of pathogenic proteins, notably amyloid-beta and hyperphosphorylated tau, which disrupt neuronal function and contribute to cognitive decline. Although proteotoxic stress is well-established in AD, the role of the ubiquitin-proteasome system (UPS) in maintaining neuronal proteostasis, and how it becomes compromised during disease progression remains incompletely understood. Here we integrated multiple approaches to characterize proteasome function, composition, and regulation in post-mortem human AD brain tissue compared to age-matched controls. These included proteasome kinetic assays, affinity purification of intact 26S proteasomes, in-gel activity assays and proteomics. According to Braak staging, we further interrogated bulk RNA-seq and single-nucleus RNA-seq (sn-RNA-seq) datasets spanning the progression of AD pathology. Finally, we examined Nrf1/NFE2L1 binding and subcellular localization to understand the transcriptional regulation of proteasome genes in AD. We found that proteasome activity is significantly impaired in AD brains, affecting both 26S and 20S complexes. This reduction in proteolytic capacity persisted after proteasome purification, implicating intrinsic defects within the proteasome complex. Proteomic profiling of isolated proteasomes revealed diminished abundances of constitutive proteasome complexes and the co-purification of proteasomes with aggregation-prone substrates (e.g., tau, α-synuclein and SQSTM1/p62), suggesting proteasome entrapment in pathological aggregates. Transcriptomic analyses showed progressive downregulation of constitutive proteasome subunit genes in individuals along the Braak stage axis, with downregulation apparent even at the earliest Braak stages, in tissue without overt tau aggregation. Neurons were disproportionately affected, whereas non-neuronal cells did not show substantial differences in proteasome-related gene expression, possibly through immunoproteasome induction. Despite elevated NFE2L1 expression, a key transcription factor normally driving proteasome gene transcription, AD brains exhibited impaired Nrf1 nuclear localization, preventing the expected compensatory upregulation of proteasome components. Collectively, our findings suggest that proteasome dysfunction in AD arises early and deepens over the disease course. Intrinsic alterations in proteasome complexes, coupled with early transcriptional downregulation of proteasome subunits and disrupted Nrf1-mediated regulatory pathways, contribute to a vicious cycle of proteotoxic stress and neuronal vulnerability. Restoring proteasome function and enhancing Nrf1-driven transcriptional responses may represent promising therapeutic strategies to preserve proteostasis and mitigate neurodegeneration in AD.
    Keywords:  Alzheimer’s disease; Nrf1 (NFE2L1); RNA-sequencing; proteasome; proteostasis; tau aggregation
    DOI:  https://doi.org/10.1093/brain/awaf222
  42. Cell Chem Biol. 2025 May 30. pii: S2451-9456(25)00166-7. [Epub ahead of print]
    Redundancy of the OST catalytic subunit facilitates therapeutic targeting of N-glycosylation.
    Marta Baro, Hojin Lee, Vanessa Kelley, Rongliang Lou, Chatchai Phoomak, Katerina Politi, Caroline J Zeiss, Michael Van Zandt, Joseph N Contessa.
      Protein asparagine (N)-glycosylation, which promotes the folding and trafficking of cell surface receptors, has not traditionally been viewed as a viable target in oncology due to the essential and non-redundant enzymatic activities required for glycan synthesis and transfer. However, in mammals, an exception is the presence of the oligosaccharyltransferase (OST) catalytic subunit paralogs, STT3A and STT3B. In this study, we investigate the biological activity of OST inhibitors and develop a strategy for selectively inhibiting N-glycosylation that is optimized for its downstream effects on the EGFR glycoprotein. Small molecules with improved pharmacokinetic properties and selective preferences for STT3A or STT3B were synthesized, characterized in vitro, and advanced to in vivo testing. The lead compound from this series, NGI-189, induces tumor regression or growth delay in patient-derived and TKI-resistant EGFR-mutant lung cancer xenografts without causing toxicity. Collectively, these findings suggest that bioavailable OST inhibitors can be developed as therapeutic agents for oncology.
    Keywords:  EGFR; NSCLC; glycosylation; oligosaccharyltransferase; therapeutic resistance
    DOI:  https://doi.org/10.1016/j.chembiol.2025.05.005
  43. Sci Adv. 2025 Jun 13. 11(24): eadv1422
    Massively parallel genetic perturbation suggests the energetic structure of an amyloid-β transition state.
    Anna Arutyunyan, Mireia Seuma, Andre J Faure, Benedetta Bolognesi, Ben Lehner.
      Amyloid aggregates are pathological hallmarks of many human diseases, but how soluble proteins nucleate to form amyloids is poorly understood. Here, we use combinatorial mutagenesis, a kinetic selection assay, and machine learning to massively perturb the energetics of the nucleation reaction of amyloid-β (Aβ42), the protein that aggregates in Alzheimer's disease. In total, we measure the nucleation rates of >140,000 variants of Aβ42 to accurately quantify the changes in free energy of activation of the reaction for all possible amino acid substitutions in a protein and, in addition, to quantify >600 energetic interactions between mutations. Strong energetic couplings suggest that the Aβ42 nucleation reaction transition state is structured in a short C-terminal region, providing a structural model for the reaction that may initiate Alzheimer's disease. Using this approach it should be possible to reveal the energetic structures of additional amyloid transition states and, in combination with additional selection assays, protein transition states more generally.
    DOI:  https://doi.org/10.1126/sciadv.adv1422
  44. bioRxiv. 2025 Jun 06. pii: 2025.06.02.657515. [Epub ahead of print]
    An improved model for prediction of de novo designed proteins with diverse geometries.
    Benjamin Orr, Stephanie E Crilly, Deniz Akpinaroglu, Eleanor Zhu, Michael J Keiser, Tanja Kortemme.
      Nature uses structural variations on protein folds to fine-tune the geometries of proteins for diverse functions, yet deep learning-based de novo protein design methods generate highly regular, idealized protein fold geometries that fail to capture natural diversity. Here, using physics-based design methods, we generated and experimentally validated a dataset of 5,996 stable, de novo designed proteins with diverse non-ideal geometries. We show that deep learning-based structure prediction methods applied to this set have a systematic bias towards idealized geometries. To address this problem, we present a fine-tuned version of Alphafold2 that is capable of recapitulating geometric diversity and generalizes to a new dataset of thousands of geometrically diverse de novo proteins from 5 fold families unseen in fine-tuning. Our results suggest that current deep learning-based structure prediction methods do not capture some of the physics that underlie the specific conformational preferences of proteins designed de novo and observed in nature. Ultimately, approaches such as ours and further informative datasets should lead to improved models that reflect more of the physical principles of atomic packing and hydrogen bonding interactions and enable improved generalization to more challenging design problems.
    DOI:  https://doi.org/10.1101/2025.06.02.657515
  45. Mol Cell. 2025 Jun 05. pii: S1097-2765(25)00461-7. [Epub ahead of print]
    The NAMPT enzyme employs a switch that directly senses AMP/ATP and regulates cellular responses to energy stress.
    Yumeng Zu, Chou Wu, Feifei Li, Hong Yao, Yuyue Xia, Ruoxi Zhang, Lulu Li, Shuangquan Chen, Qi Shi, Shuang Xi, Huanhuan Pang, Minghui Liu, Leibo Wang, Sandi Terpack, Weihua Wang, She Chen, Hong Zhang, Yibing Wang, Maojun Yang, Shanjin Huang, Fuling Zhou, Yu Tang, Zeping Hu, Shilong Fan, Yefeng Tang, Young-Sam Lee, Gelin Wang.
      Nicotinamide adenine dinucleotide (NAD+) is a crucial compound in energy metabolism and cell signaling. Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme responsible for NAD+ biosynthesis from nicotinamide (NAM). Here, we report that NAMPT activity is inhibited by adenosine monophosphate (AMP) in response to energy stress. Our global metabolite-protein interaction mapping reveals that NAMPT differentially interacts with AMP from fasted mouse livers. Crystal structures of NAMPT-AMP show that AMP binds similarly to the NAMPT reaction product, nicotinamide mononucleotide (NMN). The inhibition of NAMPT by AMP can be relieved by NAMPT activators or adenosine triphosphate (ATP), likely in a competitive manner. Based on these findings, we further investigated upstream factors contributing to AMP accumulation and found that activation of purine synthesis unexpectedly promotes the rise of AMP during fasting. Notably, an increased AMP/ATP ratio correlates with NAD+ decline in ischemic stroke models, in which NAMPT activators can otherwise confer protection.
    Keywords:  AMP; ATP; NAD(+) biosynthesis; NAMPT; energy stress; fasting; ischemia; purine synthesis
    DOI:  https://doi.org/10.1016/j.molcel.2025.05.022
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