bims-proteo Biomed News
on Proteostasis
Issue of 2025–07–20
47 papers selected by
Eric Chevet, INSERM
  1. Structural basis and pathological implications of the dimeric OS9-SEL1L-HRD1 ERAD Core Complex.
  2. The contribution of native protein complexes to targeted protein degradation.
  3. Dynamic regulation of the COPII interactome and collagen trafficking by site-specific glycosylation of Sec24D.
  4. Membralin Selects Foreign Glycoproteins from the Endoplasmic Reticulum to Lysosomes for Degradation.
  5. Optogenetics-enabled discovery of integrated stress response modulators.
  6. Degrons: defining the rules of protein degradation.
  7. Elaboration of molecular glues that target TRIM21 into TRIMTACs that degrade protein aggregates.
  8. The endoplasmic reticulum proteostasis network and bone disease.
  9. Functional rescue of a fatal ERAD mutation via alternative splicing.
  10. Nuclear and cytosolic J-domain proteins provide synergistic control of Hsf1 at distinct phases of the heat shock response.
  11. FMRP drives mRNP targets into translationally silenced complexes.
  12. Conformational plasticity of a BiP-GRP94 chaperone complex.
  13. Synergistic Control of Mitochondrial Dynamics and Function by ERAD and Autophagy in Brown Adipocytes.
  14. Design of intrinsically disordered region binding proteins.
  15. Neuronal SEL1L-HRD1 ERAD regulates one-carbon metabolism and is essential for motor function and survival.
  16. Dissecting Branch-Specific Unfolded Protein Response Activation in Drug-Tolerant BRAF-Mutant Melanoma using Data-Independent Acquisition Mass Spectrometry.
  17. Endogenous retrovirus-like proteins recruit UBQLN2 to stress granules and shape their functional biology.
  18. The ribosome ubiquitination code: fine-tuning translation under stress.
  19. Stress testing reveals selective vulnerabilities in protein homeostasis.
  20. Scouring the human Hsp70 network uncovers diverse chaperone safeguards buffering TDP-43 toxicity.
  21. Drug Repurposing Screen Identifies an HRI Activating Compound that Promotes Adaptive Mitochondrial Remodeling in MFN2-deficient Cells.
  22. Influenza A virus induces PI4P production at the endoplasmic reticulum in an ATG16L1-dependent manner to promote the egress of viral ribonucleoproteins.
  23. Mechanistic insights into PROTAC-mediated degradation through an integrated framework of molecular dynamics, free energy landscapes, and quantum mechanics: A case study on kinase degraders.
  24. Molecular basis for the interactions of eIF2β with eIF5, eIF2B, and 5MP1 and their regulation by CK2.
  25. Assessing the Suitability of Deubiquitylases As Substrates For Targeted Protein Degradation.
  26. LCK-targeting molecular glues overcome resistance to inhibitor-based therapy in T-cell acute lymphoblastic leukemia.
  27. Chronic integrated stress response causes dysregulated cholesterol synthesis in white matter disease.
  28. Integrating structural homology with deep learning to achieve highly accurate protein-protein interface prediction for the human interactome.
  29. Intracellular pathogen effector reprograms host gene expression by inhibiting mRNA decay.
  30. Glucose restriction shapes pre-metastatic innate immune landscapes in the lung through exosomal TRAIL.
  31. Interpreting ribosome dynamics during mRNA translation.
  32. Valosin-Containing Protein (VCP) in Cilia Morphology: a novel target in ADPKD.
  33. Scaffold-hopping for molecular glues targeting the 14-3-3/ERα complex.
  34. Accurate PROTAC-targeted degradation prediction with DegradeMaster.
  35. De novo designed protein guiding targeted protein degradation.
  36. Chaperone saturation mediates translation and protein folding efficiency.
  37. Emerging roles for integrated stress response signaling in homeostasis.
  38. DDX3X acts as a selective dual switch regulator of mRNA translation in acute ER stress.
  39. Hac1p-based inverse secretory pathway engineering (Hi-SPE) of Pichia pastoris for improved glucose oxidase production.
  40. Susceptibility to inflammatory bowel diseases promotes invasive carcinomas in a murine model of ATF6-driven colon cancer.
  41. CryoEM-enabled visual proteomics reveals de novo structures of oligomeric protein complexes.
  42. Targeting the canonical ER stress IRE1α/XBP1 pathway counteracts pancreatic cancer-induced skeletal muscle wasting.
  43. Cancer-associated SPOP mutations enlarge nuclear size and facilitate nuclear envelope rupture upon farnesyltransferase inhibitor treatment.
  44. Polyglycine-mediated aggregation of FAM98B disrupts tRNA processing in GGC repeat disorders.
  45. A proximity-labeling-based approach to directly detect mRNA delivery to specific subcellular locations.
  46. The proteostatic landscape of healthy human oocytes.
  47. Somatic mutations in IRE1α regulate keratinocyte migration and survival by differentially activating Rho GTPases.
  1. bioRxiv. 2025 Jun 15. pii: 2025.06.13.659592. [Epub ahead of print]
    Structural basis and pathological implications of the dimeric OS9-SEL1L-HRD1 ERAD Core Complex.
    Liangguang Leo Lin, Emir Maldosevic, Linyao Elina Zhou, Ahmad Jomaa, Ling Qi.
      The SEL1L-HRD1 complex represents the most conserved branch of endoplasmic reticulum (ER)-associated degradation (ERAD), a critical pathway that clears misfolded proteins to maintain ER proteostasis. However, the molecular organization and pathogenic mechanisms of mammalian ERAD have remained elusive. Here, we report the first cryo-EM structure of the core mammalian ERAD complex, comprising the ER lectin OS9, SEL1L, and the E3 ubiquitin ligase HRD1. The structure, validated by mutagenesis and crosslinking assays, reveals a dimeric assembly of the core complex in which SEL1L and OS9 form a claw-like configuration in the ER lumen that mediates substrate engagement, while HRD1 dimerizes within the membrane to facilitate substrate translocation. Pathogenic SEL1L mutations at the SEL1L-OS9 (Gly585Asp) and SEL1L-HRD1 (Ser658Pro) interfaces disrupt complex formation and impair ERAD activity. A newly identified disease-associated HRD1 variant (Ala91Asp), located in transmembrane helix 3, impairs HRD1 dimerization and substrate processing, underscoring the functional necessity of this interface and HRD1 dimerization. Finally, the structure also reveals two methionine-rich crevices flanking the HRD1 dimer, suggestive of substrate-conducting channels analogous to those in the ER membrane protein complex (EMC). These findings establish a structural framework for mammalian ERAD and elucidate how mutations destabilizing this machinery contribute to human disease.
    SUMMARY: The dimeric structure of the human SEL1L-HRD1 ERAD core complex reveals key architectural and functional principles underlying the recognition and processing of misfolded proteins linked to human disease.
    DOI:  https://doi.org/10.1101/2025.06.13.659592
  2. bioRxiv. 2025 Jun 17. pii: 2025.06.17.660125. [Epub ahead of print]
    The contribution of native protein complexes to targeted protein degradation.
    Lorraine Glennie, Nicole Curnutt, Tyrell Cartwright, Karen Dunbar, Brune Le Chatelier, Nicola T Wood, Thomas J Macartney, Christina M Woo, Gopal P Sapkota.
      Targeted protein degradation (TPD) destroys proteins of interest (POIs) by hijacking the cellular proteolytic machinery. Most proteins in cells exist and function as part of multi-protein or macromolecular complexes, thereby allowing a single protein to control multiple biological processes. Therefore, when a small molecule degrader induces proximity between an E3 ligase and the POI, the macromolecular context of the POI potentially influences the degradation outcomes of the POI and of the complex components. Here, we explore degradation of the eight CK1α-SACK1(A-H) (formerly known as FAM83A-H) complexes initiated by molecular glue degraders primarily designed to target Ser/Thr kinase CK1α. We demonstrate that lenalidomide-derived degraders DEG-77 and SJ3149, which selectively target the CK1α isoform, co-degrade multiple SACK1(A-H) proteins. We show that the degradation of SACK1(A-H) proteins by DEG-77 and SJ3149 requires CK1α, the CUL4A CRBN E3 ligase complex and the proteasome. In cells derived from palmoplantar keratoderma patients harbouring the CK1α-binding deficient SACK1G R265P mutation, DEG-77 targets CK1α and mitotic SACK1D but not SACK1G R265P , highlighting the requirement for CK1α-SACK1(A-H) interaction to achieve co-degradation. Our study underscores the importance of POI context in TPD and reinforces the potential for selectively targeting specific protein complexes for degradation.
    DOI:  https://doi.org/10.1101/2025.06.17.660125
  3. bioRxiv. 2025 Jun 13. pii: 2025.06.13.659590. [Epub ahead of print]
    Dynamic regulation of the COPII interactome and collagen trafficking by site-specific glycosylation of Sec24D.
    Tetsuya Hirata, Dharmendra Choudhary, Brittany J Bisnett, Erik J Soderblom, Ela W Knapik, Michael Boyce.
      Coat protein complex II (COPII) mediates anterograde trafficking from the endoplasmic reticulum (ER). While the core COPII machinery is well-characterized, how cells regulate COPII to accommodate large cargoes, including collagens, remains incompletely understood. Here, we show that the cargo-selecting COPII subunit Sec24D is modified by site-specific O-linked β- N -acetylglucosamine (O-GlcNAc) in its N-terminal intrinsically disordered region upon induction of collagen transport. These glycosylations are required for collagen trafficking in human cells and developing zebrafish. Crosslinking proteomics demonstrated that each O-GlcNAcylation influences the Sec24D interactome in a distinct way, revealing novel mediators of COPII function. In particular, Sec24D glycosylation is required for its interaction with myoferlin, which unexpectedly facilitates fusion of ER exit sites (ERES) and the ER-Golgi intermediate compartment (ERGIC) to enable collagen transport. Our results establish Sec24D O-GlcNAcylation as a dynamic regulator of COPII protein-protein interactions and collagen trafficking and identify myoferlin as a novel mediator of this process.
    DOI:  https://doi.org/10.1101/2025.06.13.659590
  4. Res Sq. 2025 Jun 23. pii: rs.3.rs-6498082. [Epub ahead of print]
    Membralin Selects Foreign Glycoproteins from the Endoplasmic Reticulum to Lysosomes for Degradation.
    Yong-Hui Zheng, Jing Zhang, Xiaoran Lu, Sunan Li, Tao Wang, Iqbal Ahmad.
      The endoplasmic reticulum (ER) plays a central role in protein synthesis and folding. Membralin is a multi-pass membrane protein involved in ER-associated degradation (ERAD). Here, we demonstrate that Membralin assembles a protein degradation machinery across the ER membrane, specifically targeting class I fusion proteins expressed by major human viruses. Membralin interacts with MAN1B1 and p97/VCP through its luminal and cytoplasmic loops, respectively. Importantly, Membralin also contains an LC3-interacting region (LIR) in its cytoplasmic tail. The expression of these viral glycoproteins induces ER stress, prompting MAN1B1 to trim mannose residues extensively. Subsequently, Membralin recruits p97/VCP and initiate ER-phagy via its LIR, leading to degradation. This pathway specifically recognizes dense N -glycans and is selective, as it does not degrade misfolded domestic proteins. Collectively, our study reveals a cell-autonomous immunity inside the ER orchestrated by Membralin, underscoring its important role in the clearance of foreign glycoproteins to maintain cellular homeostasis.
    DOI:  https://doi.org/10.21203/rs.3.rs-6498082/v1
  5. Cell. 2025 Jul 03. pii: S0092-8674(25)00690-7. [Epub ahead of print]
    Optogenetics-enabled discovery of integrated stress response modulators.
    Felix Wong, Alicia Li, Satotaka Omori, Ryan S Lach, Jose Nunez, Yunke Ren, Sean P Brown, Vipul Singhal, Brent R Lyda, Taivan Batjargal, Ethan Dickson, Jose Roberto Rodrigues Reyes, Juan Manual Uruena Vargas, Shalaka Wahane, Hahn Kim, James J Collins, Maxwell Z Wilson.
      The integrated stress response (ISR) is a conserved stress response that maintains homeostasis in eukaryotic cells. Modulating the ISR holds therapeutic potential for diseases including viral infection, cancer, and neurodegeneration, but few known compounds can do so without toxicity. Here, we present an optogenetic platform for the discovery of compounds that selectively modulate the ISR. Optogenetic clustering of PKR induces ISR-mediated cell death, enabling the high-throughput screening of 370,830 compounds. We identify compounds that potentiate cell death without cytotoxicity across diverse cell types and stressors. Mechanistic studies reveal that these compounds upregulate activating transcription factor 4 (ATF4), sensitizing cells to stress and apoptosis, and identify GCN2 as a molecular target. Additionally, these compounds exhibit antiviral activity, and one compound reduced viral titers in a mouse model of herpesvirus infection. Structure-activity and toxicology studies highlight opportunities to optimize therapeutic efficacy. This work demonstrates an optogenetic approach to drug discovery and introduces ISR potentiators with therapeutic potential.
    Keywords:  antiviral; drug discovery; endoplasmic reticulum stress; integrated stress response; optogenetics; phenotypic screening; proteostasis; small molecules; synthetic biology; unfolded protein response
    DOI:  https://doi.org/10.1016/j.cell.2025.06.024
  6. Nat Rev Mol Cell Biol. 2025 Jul 14.
    Degrons: defining the rules of protein degradation.
    Zhiqian Zhang, Elijah L Mena, Richard T Timms, Itay Koren, Stephen J Elledge.
      Degrons are pivotal components of the ubiquitin-proteasome system, serving as the recognition determinants through which E3 ubiquitin ligases identify their substrates. Degrons have central roles in both protein quality control and intracellular signalling pathways, and mutations that dysregulate degron activity are associated with a wide range of diseases, including cancer, immunological disorders and neurodegeneration. The number of well-defined degrons remains sparse relative to the ~600 E3 ubiquitin ligases encoded in the human genome. Recent advances in high-throughput degron discovery technologies have accelerated progress in this area, expanding the number of N- and C-terminal degrons, internal degrons and ubiquitin-independent degrons defined experimentally at high resolution. In this Review, we discuss the latest insights into the molecular mechanisms through which degrons act, their functional importance and their relevance in human disease, and consider how bifunctional molecules harness degrons to enable targeted protein degradation for therapeutic benefit.
    DOI:  https://doi.org/10.1038/s41580-025-00870-z
  7. Nat Commun. 2025 Jul 16. 16(1): 6548
    Elaboration of molecular glues that target TRIM21 into TRIMTACs that degrade protein aggregates.
    Marc A Scemama de Gialluly, Anthony R Allen, Elijah H Hayes, Patrick Zhuang, Ralston B Goldfarb, Amanda N Farrar, Yuriy Fedorov, Drew J Adams.
      Approaches for the discovery of molecular glues remain limited. Here we report a phenotypic screening approach in which cytotoxins whose mechanisms require ubiquitination show a gain of viability following pharmacological inhibition of the Ubiquitin-like modifier activating enzyme (UBA1/UAE). This approach reveals PRLX-93936 and BMS-214662 as molecular glues that directly target the E3 ligase TRIM21 to induce degradation of nucleoporin proteins and inhibit nuclear trafficking. The cytotoxicity of these agents correlates strongly with TRIM21 expression, suggesting re-evaluation of these clinically tested agents in patients with TRIM21-high cancers. Relative to recently disclosed TRIM21-targeting glues, PRLX-93936 and newly-synthesized analogs represent a distinct structural series, lack known cellular off-targets, and offer greatly enhanced potency. Additionally, we elaborate PRLX-93936 to a heterobifunctional degrader that uses wild-type TRIM21 to degrade a multimeric protein. Together, our work creates opportunities for targeted protein degradation and enables the design of additional TRIM21-targeting glues and Proteolysis-Targeting Chimeras (PROTACs).
    DOI:  https://doi.org/10.1038/s41467-025-61818-7
  8. Trends Mol Med. 2025 Jul 10. pii: S1471-4914(25)00145-5. [Epub ahead of print]
    The endoplasmic reticulum proteostasis network and bone disease.
    Hesso Farhan, Muhammad Zahoor, Josephine T Tauer, Wolfgang Högler.
      Bone homeostasis relies on the coordinated activity of osteoblasts and osteoclasts that balances bone formation and resorption, and of osteocytes for biomechanical sensing and hormone secretion. A key factor in the function of these cells is proteostasis, where the endoplasmic reticulum (ER) oversees protein synthesis, quality control, folding, and the secretion of proteins such as collagen type I. Emerging research links ER proteostasis defects to skeletal disorders caused by impaired bone development and mass. We explore the mechanisms of ER proteostasis, including the unfolded protein response (UPR), and discuss how genetic, metabolic, and environmental factors disrupt these pathways and contribute to bone pathology. We also highlight the need for further mechanistic insights which could pave the way for novel therapies that target ER-Golgi traffic and inhibit ER stress in bone diseases.
    Keywords:  COPII; endoplasmic reticulum; osteogenesis imperfecta; proteostasis; skeletal dysplasia; unfolded protein response
    DOI:  https://doi.org/10.1016/j.molmed.2025.06.005
  9. bioRxiv. 2025 Jun 15. pii: 2025.06.13.659581. [Epub ahead of print]
    Functional rescue of a fatal ERAD mutation via alternative splicing.
    Huilun Helen Wang, Zhihong Wang, Liangguang Leo Lin, Sunil K Verma, Weronika Gniadzik, Hui Wang, Zexin Jason Li, Lulu Jiang, Muge N Kuyumcu-Martinez, Shengyi Sun, Ling Qi.
      Endoplasmic reticulum (ER)-associated degradation (ERAD) is essential for cellular proteostasis, with the SEL1L-HRD1 protein complex targeting misfolded proteins in the ER for proteasomal degradation. Disruption of this pathway underlies a recently identified infant-onset neurodevelopmental disorder (ENDI syndrome), characterized by profound developmental delay, microcephaly, and immune deficiency. Its most severe form, ENDI with agammaglobulinemia (ENDI-A), is driven by a bi-allelic SEL1L Cys141Tyr (C141Y) mutation within the fibronectin II (FNII) domain, for which no treatment currently exists. Here, we serendipitously uncover a striking mechanism of intrinsic rescue in knock-in mouse models of the C141Y mutation: enhanced usage of an alternative splice donor site within exon 4 bypasses the mutant FNII-encoding region, restoring ERAD activity and rescuing key disease phenotypes including perinatal lethality, growth retardation, B cell deficiency, and neurodevelopmental defects. Building on this discovery, we demonstrate that antisense oligonucleotide (ASO)-mediated exon skipping in patient-derived fibroblasts generates a truncated yet functional SEL1L protein, fully rescuing ERAD function and ER proteostasis. These results establish RNA splicing modulation as a viable therapeutic strategy for ERAD deficiency and extend the clinical potential of exon-skipping therapy to diseases of protein misfolding.
    ONE-SENTENCE SUMMARY: This study reports the discovery of using antisense oligonucleotides (ASOs) to rescue the biallelic SEL1L C141Y variant, offering a potential therapeutic strategy.
    DOI:  https://doi.org/10.1101/2025.06.13.659581
  10. bioRxiv. 2025 Jul 08. pii: 2025.04.14.648540. [Epub ahead of print]
    Nuclear and cytosolic J-domain proteins provide synergistic control of Hsf1 at distinct phases of the heat shock response.
    Carmen Ruger-Herreros, Lucia Svoboda, Gurranna Male, Aseem Shrivastava, Markus Höpfler, Jiri Koubek, Günter Kramer, Fabian den Brave, Axel Mogk, David S Gross, Bernd Bukau.
      The heat shock response (HSR) is the major defense mechanism against proteotoxic stress in the cytosol and nucleus of eukaryotic cells. Initiation and attenuation of the response are mediated by stress-dependent regulation of heat shock transcription factors (HSFs). Saccharomyces cerevisiae encodes a single HSF (Hsf1), facilitating the analysis of HSR regulation. Hsf1 is repressed by Hsp70 chaperones under non-stress conditions, and becomes activated under proteotoxic stress, directly linking protein damage and its repair to the HSR. J-domain proteins (JDPs) are essential for targeting of Hsp70s to their substrates, yet the specific JDP(s) regulating Hsf1 and connecting protein damage to HSR activation remain unclear. Here we show that the yeast nuclear JDP Apj1 primarily controls the attenuation phase of the HSR by promoting Hsf1's displacement from heat shock elements in target DNA. In apj1Δ cells, HSR attenuation is significantly impaired. Additionally, yeast cells lacking both Apj1 and the major JDP Ydj1 exhibit increased HSR activation even in non-stress conditions, indicating their distinct regulatory roles. Apj1's role in both nuclear protein quality control and Hsf1 regulation underscores its role in directly linking nuclear proteostasis to HSR regulation. Together these findings establish the nucleus as key stress-sensing signaling hub.
    DOI:  https://doi.org/10.1101/2025.04.14.648540
  11. Mol Cell. 2025 Jul 08. pii: S1097-2765(25)00512-X. [Epub ahead of print]
    FMRP drives mRNP targets into translationally silenced complexes.
    Tatsuaki Kurosaki, Hana Cho, Elizabeth T Abshire, Christoph Pröschel, Shuhei Mitsutomi, Hanae Sato, Eric A J Simko, Christopher S Fraser, Hitomi Sakano, Lynne E Maquat.
      Fragile X syndrome (FXS) results from a deficiency of the ubiquitously expressed RNA-binding protein fragile X protein (FMRP). While FMRP-mediated translational repression has been attributed primarily to ribosome stalling, using immunoprecipitations and polysome profiling of non-polar- and polar-cell lysates and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses, we show that mammalian FMRP largely represses translation initiation by associating with granule constituents to preclude 40S ribosomal subunit binding. We demonstrate that FMRP associates with its target mRNAs by binding directly to eukaryotic translation initiation factor 4E (eIF4E) at the 5' cap in competition with eIF4G1 and that ataxin-2-like promotes FMRP binding to the transcribed body. The KH1 + KH2 domains of FMRP are critical for the co-immunoprecipitation of eIF4E, mRNA targets, ataxin-2-like, and PABPC1. Our findings supplement FMRP-mediated ribosome-stalling data, suggesting that FMRP largely mediates the sequestration of its mRNA targets from translation initiation and degradation in a network of FMRP molecules that simultaneously associate with cap-bound eIF4E, GC-rich mRNA regions, and poly(A)-bound PABPC1.
    Keywords:  FMRP; Fragile X syndrome; LC-MS/MS; PABPC1; ataxin-2-like; eIF4E; embryonic human cortical neurons; mRNP; post-natal mouse cortex; translational repression
    DOI:  https://doi.org/10.1016/j.molcel.2025.06.012
  12. Nat Struct Mol Biol. 2025 Jul 14.
    Conformational plasticity of a BiP-GRP94 chaperone complex.
    Joel Cyrille Brenner, Linda Charlotte Zirden, Lana Buzuk, Yasser Almeida-Hernandez, Lea Radzuweit, Joao Diamantino, Farnusch Kaschani, Markus Kaiser, Elsa Sanchez-Garcia, Simon Poepsel, Doris Hellerschmied.
      Hsp70 and Hsp90 chaperones and their regulatory cochaperones are critical for maintaining protein homeostasis. Glucose-regulated protein 94 (GRP94), the sole Hsp90 chaperone in the secretory pathway of mammalian cells, is essential for the maturation of important secretory and transmembrane proteins. Without the requirement of cochaperones, the Hsp70 protein BiP controls regulatory conformational changes of GRP94, the structural basis of which has remained elusive. Here we biochemically and structurally characterize the formation of a BiP-GRP94 chaperone complex and its transition to a conformation expected to support the loading of substrate proteins from BiP onto GRP94. BiP initially binds to the open GRP94 dimer through an interaction interface that is conserved among Hsp70 and Hsp90 paralogs. Subsequently, binding of a second BiP protein stabilizes a semiclosed GRP94 dimer, thereby advancing the chaperone cycle. Our findings highlight a fundamental mechanism of direct Hsp70-Hsp90 cooperation, independent of cochaperones.
    DOI:  https://doi.org/10.1038/s41594-025-01619-0
  13. bioRxiv. 2025 Jun 15. pii: 2025.06.14.659625. [Epub ahead of print]
    Synergistic Control of Mitochondrial Dynamics and Function by ERAD and Autophagy in Brown Adipocytes.
    Xinxin Chen, Siwen Wang, Mauricio Torres, Sijie Hao, Shengyi Sun, Ling Qi.
      Mitochondrial quality control is essential for maintaining cellular energy homeostasis, particularly in brown adipocytes where dynamic mitochondrial remodeling supports thermogenesis. Although the SEL1L-HRD1 endoplasmic reticulum (ER)-associated degradation (ERAD) pathway and autophagy are two major proteostatic systems, how these pathways intersect to regulate mitochondrial integrity in metabolically active tissues remains poorly understood. Here, using adipocyte-specific genetic mouse models combined with high-resolution 2D and 3D ultrastructural imaging technologies, we reveal an unexpected synergy between SEL1L-HRD1 ERAD and autophagy in maintaining mitochondrial structure and function in brown adipocytes. Loss of ERAD alone triggers compensatory autophagy, whereas combined deletion of both pathways (double knockout, DKO) results in severe mitochondrial abnormalities, including the accumulation of hyperfused megamitochondria penetrated by ER tubules, even under basal room temperature conditions. These phenotypes are absent in mice lacking either pathway individually or in SEL1L-IRE1α DKO, highlighting the pathway-specific coordination between ERAD and autophagy. Mechanistically, dual loss of ERAD and autophagy induces ER expansion, excessive ER-mitochondria contact, upregulation of mitochondria-associated membrane (MAM) tethering proteins, impaired calcium transfer, and defective mitochondrial turnover. As a result, DKO adipocytes accumulate dysfunctional mitochondria, exhibit respiratory deficits, and fail to sustain thermogenesis. Collectively, our study uncovers a cooperative and previously unrecognized mechanism of mitochondrial surveillance, emphasizing the critical role of ERAD-autophagy crosstalk in preserving mitochondrial integrity and thermogenic capacity in brown fat.
    One-sentence summary: Our study uncovers a previously unrecognized synergy between SEL1L-HRD1 ERAD and autophagy that is essential for preserving mitochondrial integrity and thermogenic capacity in brown adipocytes, revealing new opportunities for targeting mitochondrial dysfunction in metabolic disease.
    DOI:  https://doi.org/10.1101/2025.06.14.659625
  14. Science. 2025 Jul 17. 389(6757): eadr8063
    Design of intrinsically disordered region binding proteins.
    Kejia Wu, Hanlun Jiang, Derrick R Hicks, Caixuan Liu, Edin Muratspahić, Theresa A Ramelot, Yuexuan Liu, Kerrie McNally, Sebastian Kenny, Andrei Mihut, Amit Gaur, Brian Coventry, Wei Chen, Asim K Bera, Alex Kang, Stacey Gerben, Mila Ya-Lan Lamb, Analisa Murray, Xinting Li, Madison A Kennedy, Wei Yang, Zihao Song, Gudrun Schober, Stuart M Brierley, John O'Neill, Michael H Gelb, Gaetano T Montelione, Emmanuel Derivery, David Baker.
      Intrinsically disordered proteins and peptides play key roles in biology, but a lack of defined structures and high variability in sequence and conformational preferences have made targeting such systems challenging. We describe a general approach for designing proteins that bind intrinsically disordered protein regions in diverse extended conformations with side chains fitting into complementary binding pockets. We used the approach to design binders for 39 highly diverse unstructured targets, including polar targets, and obtained designs with 100-picomolar to 100-nanomolar affinities in 34 cases, testing ~22 designs per target. The designs function in cells and as detection reagents and are specific for their intended targets in all-by-all binding experiments. Our approach is a major step toward a general solution to the intrinsically disordered protein and peptide recognition problem.
    DOI:  https://doi.org/10.1126/science.adr8063
  15. bioRxiv. 2025 Jun 18. pii: 2025.06.16.659938. [Epub ahead of print]
    Neuronal SEL1L-HRD1 ERAD regulates one-carbon metabolism and is essential for motor function and survival.
    Mauricio Torres, You Lu, Brent Pederson, Hui Wang, Anna Gretzinger, Liangguang Leo Lin, Jiwon Hwang, Alan Rupp, Abigail Tomlinson, Andrew J Scott, Zhen Zhao, Daniel R Wahl, Martin Myers, Costas A Lyssiotis, Ling Qi.
      Hypomorphic variants in the SEL1L-HRD1 ER-associated degradation (ERAD) complex have been linked to severe neurological syndromes in children, including neurodevelopmental delay, intellectual disability, motor dysfunction, and early death. Despite this association, its physiological importance and underlying mechanisms in neurons remain poorly understood. Here, we show that neuronal SEL1L-HRD1 ERAD is essential for maintaining one-carbon metabolism, motor function, and overall viability. Neuron-specific deletion of Sel1L in mice ( Sel1L SynCre ) resulted in growth retardation, severe motor impairments, and early mortality by 9 weeks of age-mirroring core clinical features observed in affected patients-despite preserved neuronal numbers and only modest ER stress. Multi-omics analyses, including single-nucleus RNA sequencing and metabolomics, revealed significant dysregulation of one-carbon metabolism in ERAD-deficient brains. This included activation of the serine, folate, and methionine pathways, accompanied by elevated levels of S-adenosylmethionine and related metabolites, likely resulted from induction of the integrated stress response (ISR). Together, these findings uncover a previously unappreciated role for neuronal SEL1L-HRD1 ERAD in coordinating ER protein quality control with metabolic adaptation, providing new insight into the molecular basis of ERAD-related neurodevelopmental disease.
    Summary: Using a neuron-specific Sel1L knockout mouse model, we demonstrate that Sel1L deficiency activates integrated stress responses, rewires one-carbon metabolism, and impairs motor function and survival.
    DOI:  https://doi.org/10.1101/2025.06.16.659938
  16. Mol Cell Proteomics. 2025 Jul 14. pii: S1535-9476(25)00135-5. [Epub ahead of print] 101036
    Dissecting Branch-Specific Unfolded Protein Response Activation in Drug-Tolerant BRAF-Mutant Melanoma using Data-Independent Acquisition Mass Spectrometry.
    Lea A Barny, Jake N Hermanson, Sarah K Garcia, Philip E Stauffer, Lars Plate.
      Cells rely on the Unfolded Protein Response (UPR) to maintain ER protein homeostasis (proteostasis) when faced with elevated levels of misfolded and aggregated proteins. The UPR is comprised of three main branches-ATF6, IRE1, and PERK-that coordinate the synthesis of proteins involved in folding, trafficking, and degradation of nascent proteins to restore ER function. Dysregulation of the UPR is linked to numerous diseases, including neurodegenerative disorders, cancer, and diabetes. Despite its importance, identifying UPR targets has been challenging due to their heterogeneous induction, which varies by cell type and tissue. Additionally, defining the magnitude and range of UPR-regulated genes is difficult because of intricate temporal regulation, feedback between UPR branches, and extensive cross-talk with other stress-signaling pathways. To comprehensively identify UPR-regulated proteins and determine their branch specificity, we developed a data-independent acquisition (DIA) liquid-chromatography mass spectrometry (LC-MS) pipeline. Our optimized workflow improved identifications of low-abundant UPR proteins and leveraged an automated SP3-based protocol on the Biomek i5 liquid handler for label-free peptide preparation. Using engineered stable cell lines that enable selective pharmacological activation of each UPR branch without triggering global UPR activation, we identified branch-specific UPR proteomic targets. These targets were subsequently applied to investigate proteomic changes in multiple BRAF-mutant melanoma cell lines treated with a BRAF inhibitor (PLX4720, i.e., vemurafenib). Our findings revealed differential regulation of the XBP1s branch of the UPR in the BRAF-mutant melanoma cell lines after PLX4720 treatment, likely due to calcium activation, suggesting that the UPR plays a role as a non-genetic mechanism of drug tolerance in melanoma. In conclusion, the validated branch-specific UPR proteomic targets identified in this study provide a robust framework for investigating this pathway across different cell types, drug treatments, and disease conditions in a high-throughput manner.
    Keywords:  Activating Transcription Factor 6; Data-Independent Acquisition (DIA); Inositol requiring enzyme 1; Protein Kinase R-like ER Kinase; Proteomics automation; Unfolded Protein Response
    DOI:  https://doi.org/10.1016/j.mcpro.2025.101036
  17. Sci Adv. 2025 Jul 18. 11(29): eadu6354
    Endogenous retrovirus-like proteins recruit UBQLN2 to stress granules and shape their functional biology.
    Harihar M Mohan, Martin G Fernandez, Camellia Huang, Rita Lin, Jaimie H Ryou, Donald Seyfried, Nikolas Grotewold, Anna J Barget, Alexandra M Whiteley, Venkatesha Basrur, Shyamal Mosalaganti, Henry L Paulson, Lisa M Sharkey.
      The human genome is replete with sequences derived from foreign elements including endogenous retrovirus-like proteins of unknown function. Here, we show that UBQLN2, a ubiquitin-proteasome shuttle factor implicated in neurodegenerative diseases, is regulated by the linked actions of two retrovirus-like proteins, retrotransposon gag-like 8 (RTL8) and paternally expressed gene 10 (PEG10). RTL8 confers on UBQLN2 the ability to complex with and regulate PEG10. PEG10, a core component of stress granules, drives the recruitment of UBQLN2 to stress granules under various stress conditions but can only do so when RTL8 is present. Changes in UBQLN2, RTL8, or PEG10 levels further remodel the kinetics of stress granule disassembly and translation recovery. PEG10 also alters overall stress granule composition by incorporating select extracellular vesicle proteins. Within stress granules, PEG10 forms virus-like particles, underscoring the structural heterogeneity of this class of biomolecular condensates. Together, these results reveal an unexpected link between pathways of cellular proteostasis and endogenous retrovirus-like proteins.
    DOI:  https://doi.org/10.1126/sciadv.adu6354
  18. Trends Biochem Sci. 2025 Jul 10. pii: S0968-0004(25)00139-2. [Epub ahead of print]
    The ribosome ubiquitination code: fine-tuning translation under stress.
    Haleigh C Wooters, Neil C Nimmagadda, Alicia M Darnell, Gustavo M Silva.
      It has become evident that a complex code of ribosome ubiquitination regulates protein synthesis, particularly in stress conditions. Ubiquitin is known largely for its role in protein stability; however, new high-throughput screening and advances in proteomics are underscoring its novel role as a master regulator of ribosome function. Still, much remains to be discovered about how this code acts and supports translation reprogramming in a context-specific manner. Here we discuss the nature of this code, the dynamics of site-specific ribosome ubiquitination, and the unique roles that multiple enzymes play in defining the translatome and cotranslational quality control pathways. We also provide insights on the importance of unraveling this code to understand the physiological impact of modified ribosome subpopulations in cellular stress and human disease.
    Keywords:  elongation control; quality control; stress response; translation reprogramming; ubiquitination
    DOI:  https://doi.org/10.1016/j.tibs.2025.06.009
  19. bioRxiv. 2025 Jun 16. pii: 2025.06.11.659168. [Epub ahead of print]
    Stress testing reveals selective vulnerabilities in protein homeostasis.
    Berent Aldikacti, Hidayet Putun, Vishal Sarsani, Rilee Zeinert, Patrick Flaherty, Peter Chien.
      Protein quality control (PQC) systems are essential for cellular resilience to proteotoxic stress. Despite intensive study for decades, functional redundancies in the system obscure the contributions of the collectively important individual genes. Here, we leverage transposon sequencing across bacteria strains lacking key chaperones and proteases to reveal hidden determinants of stress response in protein homeostasis. By profiling fitness under multiple proteotoxic stresses, we uncover stress-specific vulnerabilities and reveal how major players of PQC mask correlations between transcriptomic responses and gene fitness. As an illustration of unexpected connections, we identify a heat-specific synthetic lethality between the disaggregase ClpB and DNA Polymerase I (PolA) mediated by persistent aggregation of the RecA recombinase and toxic persistence of the heat shock regulon. Our findings reveal that stress-induced aggregation is not broadly toxic. Rather, it becomes lethal in specific genetic or environmental contexts due to the depletion of components only needed in those specific circumstances. This work presents a framework to reveal normally hidden fragility in stress responses using gene fitness scores adaptable to a variety of systems.
    DOI:  https://doi.org/10.1101/2025.06.11.659168
  20. bioRxiv. 2025 May 10. pii: 2025.05.10.653282. [Epub ahead of print]
    Scouring the human Hsp70 network uncovers diverse chaperone safeguards buffering TDP-43 toxicity.
    Edward M Barbieri, Miriam Linsenmeier, Katherine R Whiteman, Yan Cheng, Sylvanne Braganza, Katie E Copley, Paola Miranda-Castrodad, Brennen Lewis, Kevin Villafañe, Defne A Amado, Beverly L Davidson, James Shorter.
      Cytoplasmic aggregation and concomitant dysfunction of the prion-like, RNA-binding protein TDP-43 underpin several fatal neurodegenerative diseases, including amyotrophic lateral sclerosis. To elucidate endogenous defenses, we systematically scoured the entire human Hsp70 network for buffers of TDP-43 toxicity. We identify 30 J-domain proteins (2 DNAJAs, 10 DNAJBs, 18 DNAJCs), 6 Hsp70s, and 5 nucleotide-exchange factors that mitigate TDP-43 toxicity. Specific chaperones reduce TDP-43 aggregate burden and detoxify diverse synthetic or disease-linked TDP-43 variants. Sequence-activity mapping unveiled unexpected, modular mechanisms of chaperone-mediated protection. Typically, DNAJBs collaborate with Hsp70 to suppress TDP-43 toxicity, whereas DNAJCs act independently. In human cells, specific chaperones increase TDP-43 solubility and enhance viability under proteotoxic stress. Strikingly, spliceosome-associated DNAJC8 and DNAJC17 retain TDP-43 in the nucleus and promote liquid-phase behavior. Thus, we disambiguate a diverse chaperone arsenal embedded in the human proteostasis network that counters TDP-43 toxicity and illuminate mechanistic gateways for therapeutic intervention in TDP-43 proteinopathies.
    DOI:  https://doi.org/10.1101/2025.05.10.653282
  21. bioRxiv. 2025 Jun 26. pii: 2025.06.23.660251. [Epub ahead of print]
    Drug Repurposing Screen Identifies an HRI Activating Compound that Promotes Adaptive Mitochondrial Remodeling in MFN2-deficient Cells.
    Prerona Bora, Mashiat Zaman, Samantha Oviedo, Sergei Kutseikin, Nicole Madrazo, Prakhyat Mathur, Meera Pannikkat, Sophia Krasny, Alan Chu, Kristen A Johnson, Danielle A Grotjahn, Timothy E Shutt, R Luke Wiseman.
      Pathogenic variants in the mitochondrial outer membrane GTPase MFN2 cause the peripheral neuropathy Charcot-Marie-Tooth Type 2A (CMT2A). These mutations disrupt MFN2-dependent regulation of diverse aspects of mitochondrial biology including organelle morphology, motility, mitochondrial-endoplasmic reticulum (ER) contacts (MERCs), and respiratory chain activity. However, no therapies currently exist to mitigate the mitochondrial dysfunction linked to genetic deficiencies in MFN2. Herein, we performed a drug repurposing screen to identify compounds that selectively activate the integrated stress response (ISR) - the predominant stress-responsive signaling pathway responsible for regulating mitochondrial morphology and function. This screen identified the compounds parogrelil and MBX-2982 as potent and selective activators of the ISR through the OMA1-DELE1-HRI signaling axis. We show that treatment with these compounds promotes adaptive, ISR-dependent remodeling of mitochondrial morphology and protects mitochondria against genetic and chemical insults. Moreover, we show that pharmacologic ISR activation afforded by parogrelil restores mitochondrial tubular morphology, promotes mitochondrial motility, rescues MERCs, and enhances mitochondrial respiration in MFN2 -deficient cells. These results demonstrate the potential for pharmacologic HRI activation as a viable strategy to mitigate mitochondrial dysfunction in CMT2A and other pathologies associated with MFN2 deficiency.
    DOI:  https://doi.org/10.1101/2025.06.23.660251
  22. PLoS Biol. 2025 Jul 16. 23(7): e3002958
    Influenza A virus induces PI4P production at the endoplasmic reticulum in an ATG16L1-dependent manner to promote the egress of viral ribonucleoproteins.
    Carla Alemany, Juliane Da Graça, Quentin Giai Gianetto, Maud Dupont, Sylvain Paisant, Thibaut Douché, Catherine Isel, Cédric Delevoye, Lydia Danglot, Mariette Matondo, Etienne Morel, Jean-Baptiste Brault, Nadia Naffakh.
      The genomic RNAs of influenza A viruses (IAVs) are replicated in the nucleus of infected cells in the form of viral ribonucleoproteins (vRNPs) before being exported to the cytoplasm. The small GTPase RAB11A is involved in the transport of vRNPs to the sites of viral assembly at the plasma membrane, but the molecular mechanisms involved remain largely unknown. Here we show that IAV infection remodels the architecture of the endoplasmic reticulum (ER) sheets, where vRNPs tend to accumulate in the absence of RAB11A. To decipher the interplay between RAB11A, vRNPs, and the ER, we investigated viral-induced perturbations of RAB11A proximity interactome. To this end, we generated cells stably expressing a TurboID-RAB11A fusion protein and performed biotin-based proximity labeling upon viral infection. We found that cellular regulators of phophatidylinositol-4-phosphate (PI4P) homeostasis, including the autophagic and stress response protein ATG16L1, are significantly enriched at the vicinity of RAB11A in infected cells. Infection induces an increase in cellular PI4P levels in an ATG16L1-dependent manner, while ATG16L1 relocalizes to ER membranes upon infection. Depletion of ATG16L1 decreases the co-distribution of vRNPs with PI4P punctae on ER membranes, and reduces the accumulation of vRNPs at the plasma membrane as well as the production of IAV infectious particles. Our data extend to IAVs the notion that viruses can modulate the metabolism and localization of phosphoinositides to control host membrane dynamics and point to the ER as an essential platform for vRNP transport. They provide evidence for a pivotal role of ATG16L1 in regulating the identity of endomembranes and coordinating RAB11A and PI4P-enriched membranes to ensure delivery of vRNPs to the plasma membrane.
    DOI:  https://doi.org/10.1371/journal.pbio.3002958
  23. J Comput Aided Mol Des. 2025 Jul 15. 39(1): 51
    Mechanistic insights into PROTAC-mediated degradation through an integrated framework of molecular dynamics, free energy landscapes, and quantum mechanics: A case study on kinase degraders.
    Ashis Nandy, Kiran Boppana, Samiron Phukan.
      Targeted protein degradation by proteolysis-targeting chimeras (PROTAC) is dependent on formation and plasticity of ternary complexes enabling ubiquitination. In this study, we employed long-timescale molecular dynamics (MD) simulations, free energy landscape analysis, and quantum mechanical (QM) calculations to investigate the molecular determinants of PROTAC efficacy. Using three model systems (FAK-VHL, BTK-CRBN, and TTK-CRBN), each with three PROTACs of varying potencies, we analyzed a total of nine ternary complexes over 500 ns MD simulations each. Simulation events analysis revealed that potent PROTACs maintain stable and important interactions between the protein of interest (POI) and the E3 ligase, while weaker PROTACs exhibit diminished or no interactions. Conformational dynamics and changes in the interaction patterns between the POI-E3-ligase complexes highlighted the importance of ternary complex plasticity in degradation efficiency. These findings were also supported by the distribution of free energy landscape during simulations. Distributions of the free energy landscape offer insights into the stability of population states and open new avenues for understanding their degradation potential. Additionally, to overcome the limitation of conventional docking models, we highlight the importance of QM and DFT based methods to predict the impact of binding at the E3-ligase site which corelates with the degradation potentials of PROTACs. These insights provide a new computational framework for rational PROTAC design.
    Keywords:  BTK; FAK; Free energy landscape; MD simulation; PROTACs; QM calculations; TTK
    DOI:  https://doi.org/10.1007/s10822-025-00630-3
  24. RNA. 2025 Jul 16. pii: rna.080652.125. [Epub ahead of print]
    Molecular basis for the interactions of eIF2β with eIF5, eIF2B, and 5MP1 and their regulation by CK2.
    Paul A Wagner, Meimei Song, Paul Doran, Aysenur Seker, Ralf Ficner, Bernhard Kuhle, Assen Marintchev.
      The heterotrimeric GTPase eukaryotic translation initiation factor 2 (eIF2) delivers the initiator Met-tRNAi to the ribosomal translation preinitiation complex (PIC). eIF2β has three lysine-rich repeats (K-boxes), important for binding to the GTPase-activating protein eIF5, the guanine nucleotide exchange factor eIF2B, and the regulator eIF5-mimic protein (5MP). Here, we combine X-ray crystallography with NMR to understand the molecular basis and dynamics of these interactions. The crystal structure of yeast eIF5-CTD in complex with eIF2β K-box 3 reveals an extended binding site on eIF2β, far beyond the K-box. We show that eIF2β contains three distinct binding sites, centered on each of the K-boxes, and that human eIF5, eIF2Bε, and 5MP1 can bind to all three sites. Our results reveal how eIF2B speeds up the dissociation of eIF5 from eIF2-GDP to promote nucleotide exchange; and how 5MP1 can destabilize eIF5 binding to eIF2 and the PIC, to promote stringent start codon selection. All these affinities are increased by CK2 phosphomimetic mutations, highlighting the role of CK2 in both remodeling and stabilizing the translation apparatus.
    Keywords:  NMR; X-ray crystallography; integrated stress response (ISR); protein structure; translation initiation
    DOI:  https://doi.org/10.1261/rna.080652.125
  25. bioRxiv. 2025 Jun 14. pii: 2025.06.13.659525. [Epub ahead of print]
    Assessing the Suitability of Deubiquitylases As Substrates For Targeted Protein Degradation.
    Joel Tong, J Monty Watkins, James M Burke, Thomas Kodadek.
      Deubiquitylases (DUBs) are a family of specialized proteases that hydrolyze the isopeptide bond between a lysine and the C-terminal carboxylate of Ubiquitin. DUBs are involved in a myriad of cellular processes and many are attractive drug targets. However, it has proven extremely difficult to develop selective inhibitors due to the high degree of homology between DUB active sites. Targeted protein degradation using a proteolysis-targeting chimera (PROTAC) that recognizes the DUB in a less conserved region outside of the catalytic domain constitutes an attractive alternative strategy for selectively inhibiting a given DUB. Such ligands are unlikely to block the catalytic activity of the enzyme, raising the concern that auto-deubiqtuiylation will make DUBs inherently poor substrates for PROTACs of this type. Since drug-like ligands that engage DUBs outside of the active site are extremely rare, this issue is difficult to address in a straightforward fashion. In this study we establish a generally applicable chemical genetics workflow to evaluate the degradability of DUBs by a PROTAC. The data indicate that some DUBs are readily degradable and some are not. In particular, USP11, an attractive drug target in various cancers and Alzheimer's disease, is shown to be rapidly degradable, while its paralogs, USP4 and USP15 resist degradation through auto-deubiquitylation.
    Significance: The development of selective inhibitors of Deubiquitylase enzymes (DUBs) is difficult due to a high level of homology in the active sites of the ≈ 100 such enzymes in the human proteome. A potentially attractive strategy to achieve this goal would be to develop PROTACs or molecular glues that engage the target DUB in a less conserved region outside of the catalytic domain. However, this raises the concern that auto-deubiquitylation would make DUBs poor substrates for this modality. Here we describe a chemical genetics system to evaluate this issue. We find that some Dubs are readily degradable via the Ubiquitin-proteasome pathway and some are not. Of the latter category, some resist turnover through auto-deubiquitylation and some are simply poor proteasome substrates.
    Highlights: Some active DUBs, including USP11 are vulnerable to targeted protein degradation by a PROTAC.Other DUBs, including USP4 and USP15, resist PROTAC-mediated degradation via auto-deubiquitylation.Some DUBs are poor substrates for targeted degradation because they are processed inefficiently by the proteasome.
    DOI:  https://doi.org/10.1101/2025.06.13.659525
  26. bioRxiv. 2025 Jul 07. pii: 2025.07.03.663042. [Epub ahead of print]
    LCK-targeting molecular glues overcome resistance to inhibitor-based therapy in T-cell acute lymphoblastic leukemia.
    Jun J Yang, Gisele Nishiguchi, Satoshi Yoshimura, Marisa Actis, Justin T Seffernick, Jamie A Jarusiewicz, Anup Aggarwal, Angelina Li, Yong Li, DongGeun Lee, Lei Yang, Anand Mayasundari, Zoran Rankovic, Marcus Fischer.
      Drug resistance is a major challenge in cancer therapy, especially in the context of kinase inhibitors. While targeted protein degradation (TPD) was a distinct mode of action compared to inhibition-based therapeutic targeting, the potential value of TPD in drug-resistant cancer remains unclear. Here, we report the discovery of cereblon-recruiting molecular glue degraders (MGDs) targeting LCK, an oncogenic kinase in T-cell acute lymphoblastic leukemia (T-ALL). By high-throughput screening and medicinal chemistry optimization, we developed a series of MGDs that induced CRBN-dependent degradation of LCK as well as potent cytotoxicity in T-ALL in vitro. Structure-activity relationship analysis and ternary complex modeling revealed a non-canonical degron at the LCK-CRBN interface involving the G-loop, whose mutation disrupts this interaction. Unlike inhibitors and inhibitor-based PROTACs, these MGDs engage LCK in regions distal to the ATP binding site and thus their activities in T-ALL are not affected by gate-keeper LCK mutations that drive resistance to inhibitor-based therapeutics. Taken together, our data underscore the potential of LCK-targeting MGDs as a strategy to overcome kinase inhibitor resistance in T-ALL, highlighting a potentially generalizable strategy in cancer therapy.
    DOI:  https://doi.org/10.1101/2025.07.03.663042
  27. JCI Insight. 2025 Jul 15. pii: e188459. [Epub ahead of print]
    Chronic integrated stress response causes dysregulated cholesterol synthesis in white matter disease.
    Karin Lin, Nina Ly, Rejani B Kunjamma, Ngoc Vu, Bryan King, Holly M Robb, Eric G Mohler, Janani Sridar, Qi Hao, José Zavala-Solorio, Chunlian Zhang, Varahram Shahryari, Nick van Bruggen, Caitlin F Connelly, Bryson D Bennett, James J Lee, Carmela Sidrauski.
      Maladaptive integrated stress response (ISR) activation is observed in human diseases of the brain. Genetic mutations of eIF2B, a critical mediator of protein synthesis, cause chronic pathway activation resulting in a leukodystrophy but the precise mechanism is unknown. We generated N208Y eIF2Bα mice and found that this metabolite binding mutation leads to destabilization of eIF2Bα, a systemic ISR, and neonatal lethality. 2BAct, an eIF2B activator, rescued lethality and significantly extended the lifespan of this severe model, underscoring its therapeutic potential in pediatric disease. Continuous treatment was required for survival, as withdrawal led to ISR induction in all tissues and rapid deterioration, thereby providing a model to assess the impact of the ISR in vivo by tuning drug availability. Single nuclei RNA-sequencing of the CNS identified astrocytes, oligodendrocytes, and ependymal cells as the cell types most susceptible to eIF2B dysfunction and revealed dysfunctional maturation of oligodendrocytes. Moreover, ISR activation decreased cholesterol biosynthesis, a process critical for myelin formation and maintenance. As such, persistent ISR engagement may contribute to pathology in other demyelinating diseases.
    Keywords:  Cell biology; Cholesterol; Demyelinating disorders; Metabolism; Mouse models; Neuroscience
    DOI:  https://doi.org/10.1172/jci.insight.188459
  28. bioRxiv. 2025 Jun 12. pii: 2025.06.09.658393. [Epub ahead of print]
    Integrating structural homology with deep learning to achieve highly accurate protein-protein interface prediction for the human interactome.
    Dapeng Xiong, Mateo Torres, Diana Murray, Le Li, Aniket C Naravane, Robert Fragoza, Barry Honig, Haiyuan Yu.
      A significant portion of disease-causing mutations occur at protein-protein interfaces however, the number of structurally resolved multi-protein complexes is extremely small. Here we present a computational pipeline, PIONEER2.0, that integrates 3D structural similarity with geometric deep learning to accurately predict protein binding partner-specific interfacial residues for all experimentally observed human binary protein-protein interactions. We estimate that AlphaFold3 fails to produce high-quality structural models for about half of the human interactome; for these challenging cases, PIONEER2.0 significantly outperforms AlphaFold3 in predicting their interface residues, making PIONEER2.0 an excellent alternative and complementary tool in real-world applications. We further systematically validated PIONEER2.0 predictions experimentally by generating 1,866 mutations and testing their impact on 5,010 mutation-interaction pairs, confirming PIONEER-predicted interfaces are comparable in accuracy as experimentally determined interfaces using PDB co-complex structures. We then used PIONEER2.0 to create a comprehensive multiscale structurally informed human interactome encompassing all 352,124 experimentally determined binary human protein interactions in the literature. We find that PIONEER2.0-predicted interfaces are instrumental in prioritizing disease-associated mutations and thus provide insight into their underlying molecular mechanisms. Overall, our PIONEER2.0 framework offers researchers a valuable tool at an unprecedented scale for studying disease etiology and advancing personalized medicine.
    DOI:  https://doi.org/10.1101/2025.06.09.658393
  29. Nat Commun. 2025 Jul 12. 16(1): 6452
    Intracellular pathogen effector reprograms host gene expression by inhibiting mRNA decay.
    Yevgen Levdansky, Justin C Deme, David J Turner, Claire T Piczak, Filip Pekovic, Anna L Valkov, Sergey G Tarasov, Susan M Lea, Eugene Valkov.
      Legionella pneumophila, an intracellular bacterial pathogen, injects effector proteins into host cells to manipulate cellular processes and promote its survival and proliferation. Here, we reveal a unique mechanism by which the Legionella effector PieF perturbs host mRNA decay by targeting the human CCR4-NOT deadenylase complex. High-resolution cryo-electron microscopy structures and biochemical analyses reveal that PieF binds with nanomolar affinity to the NOT7 and NOT8 catalytic subunits of CCR4-NOT, obstructing RNA access and displacing a catalytic Mg²⁺ ion from the active site. Additionally, PieF prevents NOT7/8 from associating with their partner deadenylases NOT6/6L, inhibiting the assembly of a functional deadenylase complex. Consequently, PieF robustly blocks mRNA poly(A) tail shortening and degradation with striking potency and selectivity for NOT7/8. This inhibition of deadenylation by PieF impedes cell cycle progression in human cells, revealing a novel bacterial strategy to modulate host gene expression.
    DOI:  https://doi.org/10.1038/s41467-025-61194-2
  30. Cell. 2025 Jul 08. pii: S0092-8674(25)00728-7. [Epub ahead of print]
    Glucose restriction shapes pre-metastatic innate immune landscapes in the lung through exosomal TRAIL.
    Cai-Yuan Wu, Chun-Xiang Huang, Xiang-Ming Lao, Zi-Wen Zhou, Jia-Hong Jian, Zheng-Xi Li, Yong-Yi Wu, Zheng-Yu Liu, Lei Chen, Lianxin Liu, Limin Zheng, Yuan Wei, Dong-Ming Kuang.
      Targeting glucose metabolism has emerged as a promising strategy for inhibiting tumor growth. However, we herein uncover an unexpected paradox: while glucose deprivation through a low-carbohydrate diet or impaired in situ metabolism suppresses primary tumor growth, it simultaneously promotes lung metastasis by depleting natural killer (NK) cells via lung macrophages. Mechanistically, glucose deprivation induces endoplasmic reticulum (ER) stress, activating HMG-CoA reductase degradation protein 1 (HRD1) to catalyze K63-linked ubiquitination of TRAIL, which is then packaged into exosomes via the endosomal sorting complex required for transport (ESCRT) complex. These exosomal TRAIL molecules polarize PVR+ macrophages, triggering NK cell exhaustion and establishing a pre-metastatic niche. Notably, TIGIT blockade not only prevents metastasis induced by glucose deprivation but also enhances its anti-tumor effects. Clinically, low glucose metabolism correlates with higher 2-year postoperative recurrence across 15 cancer types. Furthermore, plasma exosomal TRAIL outperforms traditional markers, such as α-fetoprotein (AFP) and tumor size, in predicting early postoperative lung metastasis, revealing both the risks and therapeutic potential of targeting glucose metabolism.
    Keywords:  NK cell; exosomal TRAIL; low-carbohydrate diet; lung metastasis; macrophage
    DOI:  https://doi.org/10.1016/j.cell.2025.06.027
  31. J Biol Chem. 2025 Jul 10. pii: S0021-9258(25)02319-1. [Epub ahead of print] 110469
    Interpreting ribosome dynamics during mRNA translation.
    Saori Uematsu, Shu-Bing Qian.
      Translation takes a central position in gene expression, its swift response to environmental stress is evolutionarily conserved. Upon chemical damage to the messenger RNA (mRNA) or the lack of building blocks, the ribosome stalls during elongation and halts the production line. Even under normal growth conditions, the translation machinery encounters constant hinderances such as varied codon composition or nascent chains with distinct features. However, it is challenging to define these kinetics experimentally partly due to the inherent variations of ribosome behavior during mRNA translation. To ensure the flow of ribosomal traffic, cells employ several mechanisms to circumvent the traffic jam. When the roadblock is not resolved timely, trailing ribosomes can collide with stalled ribosomes. However, the boundary between physiological queuing and pathological collision is often blurred, representing a fundamental gap in our understanding of ribosome dynamics. To cope with translational barriers, several signaling pathways are activated to adjust the rate of global translation and rescue the local stalled ribosome. Deficiencies of cellular response to translational stress have been associated with a wide array of human diseases. In this review, we focus on fundamental aspects of the ribosome dynamics during mRNA translation. We provide an overview of causes, outcomes, and cellular responses to ribosome stalling and collision on mRNA. We highlight questions that may clarify the biological roles of distinct ribosome behavior during mRNA translation and emphasize the mechanistic connection between altered ribosome dynamics and human diseases.
    DOI:  https://doi.org/10.1016/j.jbc.2025.110469
  32. Am J Physiol Renal Physiol. 2025 Jul 15.
    Valosin-Containing Protein (VCP) in Cilia Morphology: a novel target in ADPKD.
    Carlotta Pioppini, Rishi Bhardwaj, Ria Schönauer, Jan Halbritter, Fatima Hassan, Kai-Uwe Eckardt, Sorin Fedeles, Duygu Elif Yilmaz, Matteus Krappitz.
      Autosomal dominant polycystic kidney disease (ADPKD) is a hereditary disorder leading to kidney cyst formation and loss of kidney function. The major causative genes Pkd1 and Pkd2 encode for the ciliary proteins Polycystin-1 (PC1) and Polycystin-2 (PC2), respectively, which are involved in ciliary functions. Within PKD1-defective cells, the accumulation of misfolded PC1 proteins triggers the unfolded protein response (UPR). Among the pathways activated, the ER-associated degradation (ERAD), mediated by proteins such as valosin-containing protein (VCP), aims to alleviate the unfolded or misfolded protein burden. Our study investigates the genetic relationship between VCP and PC1-dependent cystogenesis. We found that the pharmacological inhibition of VCP ameliorates the cystic phenotype in Pkd1-knockout mice. This effect is associated with increased ER stress-dependent apoptosis in PC1-deficient cells. Additionally, we discovered that VCP is localized in the primary cilia and its inhibition affects cilia assembly and reduces the cilia length.
    Keywords:  ADPKD; UPR; VCP; ciliopathy
    DOI:  https://doi.org/10.1152/ajprenal.00032.2025
  33. Nat Commun. 2025 Jul 14. 16(1): 6467
    Scaffold-hopping for molecular glues targeting the 14-3-3/ERα complex.
    Markella Konstantinidou, Marios Zingiridis, Marloes A M Pennings, Michael Fragkiadakis, Johanna M Virta, Jezrael L Revalde, Emira J Visser, Christian Ottmann, Luc Brunsveld, Constantinos G Neochoritis, Michelle R Arkin.
      Molecular glues, small molecules that bind cooperatively at a protein-protein interface, have emerged as powerful modalities for the modulation of protein-protein interactions (PPIs) and "undruggable" targets. The systematic identification of new chemical matter with a molecular glue mechanism of action remains a significant challenge in drug discovery. Here, we present a scaffold hopping approach, using as a starting point our previously developed molecular glues for the native 14-3-3/estrogen receptor alpha (ERα) complex. The novel, computationally designed scaffold is based on the Groebke-Blackburn-Bienaymé multi-component reaction (MCR), leading to drug-like analogs with multiple points of variation, thus enabling the rapid derivatization and optimization of the scaffold. Structure-activity relationships (SAR) are developed using orthogonal biophysical assays, such as intact mass spectrometry, TR-FRET and SPR. Rational structure-guided optimization is facilitated by multiple crystal structures of ternary complexes with the glues, 14-3-3 and phospho-peptides mimicking the highly disordered C-terminus of ERα. Cellular stabilization of 14-3-3/ERα for the most potent analogs is confirmed using a NanoBRET assay with full-length proteins in live cells. Our approach highlights the potential of MCR chemistry, combined with scaffold hopping, to drive the development and optimization of unprecedented molecular glue scaffolds.
    DOI:  https://doi.org/10.1038/s41467-025-61176-4
  34. Bioinformatics. 2025 Jul 01. 41(Supplement_1): i342-i351
    Accurate PROTAC-targeted degradation prediction with DegradeMaster.
    Jie Liu, Michael J Roy, Luke Isbel, Fuyi Li.
       MOTIVATION: Proteolysis-targeting chimeras (PROTACs) are heterobifunctional molecules that can degrade "undruggable" protein of interest by recruiting E3 ligases and hijacking the ubiquitin-proteasome system. Some efforts have been made to develop deep learning-based approaches to predict the degradation ability of a given PROTAC. However, existing deep learning methods either simplify proteins and PROTACs as 2D graphs by disregarding crucial 3D spatial information or exclusively rely on limited labels for supervised learning without considering the abundant information from unlabeled data. Nevertheless, considering the potential to accelerate drug discovery, it is critical to develop more accurate computational methods for PROTAC-targeted protein degradation prediction.
    RESULTS: This study proposes DegradeMaster, a semisupervised E(3)-equivariant graph neural network-based predictor for targeted degradation prediction of PROTACs. DegradeMaster leverages an E(3)-equivariant graph encoder to incorporate 3D geometric constraints into the molecular representations and utilizes a memory-based pseudolabeling strategy to enrich annotated data during training. A mutual attention pooling module is also designed for interpretable graph representation. Experiments on both supervised and semisupervised PROTAC datasets demonstrate that DegradeMaster outperforms state-of-the-art baselines, with substantial improvement of AUROC by 10.5%. Case studies show DegradeMaster achieves 88.33% and 77.78% accuracy in predicting the degradability of VZ185 candidates on BRD9 and ACBI3 on KRAS mutants. Visualization of attention weights on 3D molecule graph demonstrates that DegradeMaster recognizes linking and binding regions of warhead and E3 ligands and emphasizes the importance of structural information in these areas for degradation prediction. Together, this shows the potential for cutting-edge tools to highlight functional PROTAC components, thereby accelerating novel compound generation.
    AVAILABILITY AND IMPLEMENTATION: The source code and datasets are available at https://github.com/ABILiLab/DegradeMaster and https://zenodo.org/records/14715718.
    DOI:  https://doi.org/10.1093/bioinformatics/btaf191
  35. Nat Commun. 2025 Jul 17. 16(1): 6598
    De novo designed protein guiding targeted protein degradation.
    Zhendong Li, Gan Qiao, Xianghe Wang, Ming Wang, Jinyu Cheng, Guipeng Hu, Xiaomin Li, Jing Wu, Jia Liu, Cong Gao, Liming Liu.
      Targeted protein degradation is a powerful tool for biological research, cell therapy, and synthetic biology. However, conventional methods often depend on pre-fused degrons or chemical degraders, limiting their wider applications. Here we develop a guided protein labeling and degradation system (GPlad) in Escherichia coli, using de novo designed guide proteins and arginine kinase (McsB) for precise degradation of various proteins, including fluorescent proteins, metabolic enzymes, and human proteins. We expand GPlad into versatile tools such as antiGPlad, OptoGPlad, and GPTAC, enabling reversible inhibition, optogenetic regulation, and biological chimerization. The combination of GPlad and antiGPlad allows for programmable circuit construction, including ON/OFF switches, signal amplifiers, and oscillators. OptoGPlad-mediated degradation of MutH accelerates E. coli evolution under protocatechuic acid stress, reducing the required generations from 220 to 100. GPTAC-mediated degradation of AroE enhanced the titer of 3-dehydroshikimic acid to 92.6 g/L, a 23.8% improvement over the conventional CRISPR interference method. We provide a tunable, plug-and-play strategy for straightforward protein degradation without the need for pre-fusion, with substantial implications for synthetic biology and metabolic engineering.
    DOI:  https://doi.org/10.1038/s41467-025-62050-z
  36. bioRxiv. 2025 Jun 28. pii: 2025.06.25.661590. [Epub ahead of print]
    Chaperone saturation mediates translation and protein folding efficiency.
    Andrew T Martens, Vincent J Hilser.
      Whether the emergence of a nascent protein from the ribosome and the formation of structural elements are synchronized has been a longstanding question (Chaney and Clark, 2015; Deane and Saunders, 2011; Levinthal, 1968; Marin, 2008; Sauna and Kimchi-Sarfaty, 2011; Spencer and Barral, 2012; Tsai et al., 2008; Zhang and Ignatova, 2011). Paradoxically, kinetically efficient translation can induce mis-folding and aggregation despite the presence of molecular chaperones (Siller et al., 2010; Spencer et al., 2012), which in Escherichia coli are induced by unfolded protein (Parsell and Sauer, 1989) via σ 32 (Craig and Gross, 1991). The molecular mechanisms mediating translation efficiency and protein folding efficiency remain poorly understood. Using ribosome profiling (Ingolia et al., 2009) and protein quantitation, we show that synonymous changes to Firefly Luciferase ( Luc ) mRNA have a direct effect on its translation efficiency. These changes alone cause up to a 70-fold difference in Luc protein levels. However, increased Luc protein is met with at most a ∼2-fold increase in chaperone levels, revealing that the σ 32 transcriptional response has saturable properties. This response is found to be poised near its midpoint (where it is most sensitive to perturbation) when Luc mRNA has an intermediate translation efficiency. These results suggest not only that chaperone saturation limits the ability of cells to maintain protein folding homeostasis when challenged with highly efficient translation, but that translation efficiency and protein folding efficiency evolved for mutual sensitivity.
    DOI:  https://doi.org/10.1101/2025.06.25.661590
  37. FEBS J. 2025 Jul 14.
    Emerging roles for integrated stress response signaling in homeostasis.
    Shyama Nandakumar, Lydia Grmai, Deepika Vasudevan.
      Across phyla, organisms have evolved signaling mechanisms to cope with cell-intrinsic and -extrinsic stressors. The integrated stress response (ISR) is a prime example of such a mechanism and has well-defined roles from yeast to humans in dealing with stress burdens imposed by nutrient deprivation, protein misfolding, infectious agents, and oxidative stress. As with many fundamental cellular processes, the complexity of ISR signaling increases with evolutionary complexity. While single-celled organisms have been reported to utilize ISR signaling in the context of stress, multicellular organisms also rely on ISR signaling components for a number of homeostatic functions. The role of ISR signaling in the absence of obvious stressors is less well-studied, though useful insights into this can be extrapolated from prior studies using loss-of-function mutants in model organisms. This review summarizes the known (and inferred) homeostatic roles for ISR signaling components and speculates on cellular functions and principles that might require stress-adaptive mechanisms such as ISR signaling to maintain homeostasis.
    Keywords:  ATF4; ISR; homeostasis; stress
    DOI:  https://doi.org/10.1111/febs.70166
  38. bioRxiv. 2025 Jun 09. pii: 2025.06.08.658532. [Epub ahead of print]
    DDX3X acts as a selective dual switch regulator of mRNA translation in acute ER stress.
    Abd-El Monsif A Shawky, Allison Scarboro, Josef Mick, Mahmoud Dondeti, Kenneth Avanzino, Constantine A Simintiras, Maria Hatzoglou, Anastasios Vourekas.
      In eukaryotes, regulation of mRNA translation initiation greatly impacts gene expression, and is critical for cellular stress responses. DDX3X is a ubiquitous DEAD-box RNA helicase whose precise role in 5' UTR scanning and start codon decoding in non-stressed and stressed cells is still elusive. Here we show that DDX3X engages with thousands of mRNAs as part of the eIF4F-mediated 48S scanning complex, simultaneously acting to promote or suppress translation of select mRNAs in non-stressed conditions, and switches this regulation in opposite directions in acute ER stress. We find distinct DDX3X binding patterns of differentially regulated mRNAs, which lead us to identify N4-acetylation of cytidines surrounding the start codon as an accompanying feature of mRNAs subject to DDX3X-mediated selective dual regulation. Our findings illuminate the role of DDX3X in stress response and highlight a novel connection between an RNA helicase and a post-transcriptional modification in regulating mRNA translation.
    DOI:  https://doi.org/10.1101/2025.06.08.658532
  39. N Biotechnol. 2025 Jul 10. pii: S1871-6784(25)00069-X. [Epub ahead of print]89 82-90
    Hac1p-based inverse secretory pathway engineering (Hi-SPE) of Pichia pastoris for improved glucose oxidase production.
    Xinyu Zhao, Jiayu Fang, Sijie Yu, Guoxia Liu, Yanping Zhang, Yin Li, Taicheng Zhu.
      Secretion and folding are common bottlenecks in protein expression using eukaryotic systems, and engineering the secretory pathway to enhance host cell capabilities is a key strategy for improving protein secretion. However, secretion is a very complex process, making the identification of likely targets for engineering a formidable task. In this study, using glucose oxidase (GOX) expression in Pichia pastoris (Komagataella phaffii) as a model, we introduce a strategy called Hac1p-based inverse secretory pathway engineering (Hi-SPE). This strategy leverages Hac1p, the actuator of the unfolded protein response, which is a naturally evolved mechanism to cope with protein overload in endoplasmic reticulum (ER) of eukaryotic cells. When combined with comparative transcriptomics, Hi-SPE narrows down the target from several hundred genes in traditional approaches to 20 secretion-related protein genes. Results showed that overexpression of six out of seven selected genes improved GOX secretion, including the co-chaperone, JEM1, which increased GOX expression per OD600 by 147.6 %. Further optimization through combinatorial expression of secretion-related proteins led to a strain co-expressing JEM1, KAR2, and CNE1, achieving a GOX titer of 1903.2 U/mL in 1-L fed-batch fermentation. Additionally, transcriptomic analysis revealed the physiological effects of JEM1 overexpression on P. pastoris. This study highlights Hi-SPE as a powerful strategy for improving protein secretion in eukaryotic systems.
    Keywords:  Hac1p; Pichia pastoris (Komagataella phaffii); comparative transcriptome; glucose oxidase; secretory pathway engineering
    DOI:  https://doi.org/10.1016/j.nbt.2025.07.002
  40. J Crohns Colitis. 2025 Jul 03. pii: jjaf102. [Epub ahead of print]19(7):
    Susceptibility to inflammatory bowel diseases promotes invasive carcinomas in a murine model of ATF6-driven colon cancer.
    Janine Kövilein, Adam Sorbie, Sevana Khaloian, Vanessa Küntzel, Miriam von Stern, Mohamed Ahmed, Sebastian Jarosch, Marianne Remke, Amira Metwaly, Elena M Reuss, Dirk H Busch, Matthieu Allez, Katja Steiger, Barbara Schraml, Olivia I Coleman, Dirk Haller.
       BACKGROUND AND AIMS: Chronic inflammation in inflammatory bowel disease (IBD) patients represents a risk factor for developing colitis-associated cancer (CAC). We previously linked the endoplasmic reticulum unfolded protein response (UPRER) signal transducer activating transcription factor 6 (ATF6) with spontaneous microbiota-dependent colonic adenoma development in mice expressing epithelial-specific activated ATF6 (nATF6IEC).
    METHODS: To investigate IBD-related risk factors in ATF6-mediated tumorigenesis, we crossed tumor-free monoallelic (tg/wt) nATF6IEC mice with interleukin-10 deficient mice (Il10-/-). We characterized our newly generated murine model under germ-free (GF) and specific pathogen-free (SPF) conditions, including tumor phenotype and immune cell characterizations, as well as complex human stool and minimal consortium colonizations.
    RESULTS: IL-10 deficiency initiated tumor susceptibility, with 77% of 12-week tg/wt;Il10-/- mice developing colonic adenomas and invasive carcinomas in this novel CAC mouse model. Tumor formation correlated with mucosal immune cell infiltration, characterized by CD11b+ granulocytes and monocytes, and mucosa-associated dysbiosis. Colonization of germ-free nATF6IEC;Il10-/- mice with minimal biosynthetic consortia and IBD stool re-established CAC, confirming microbiota-dependent ATF6-driven tumorigenesis. Increased ATF6 expression in IBD patients during active disease highlights human relevance.
    CONCLUSION: Our findings show that IBD susceptibility heightens the risk for ATF6-driven tumorigenesis.
    Keywords:  ER stress; IBD-relevant minimal consortium; activating transcription factor 6; colitis-associated cancer; human microbiota associations
    DOI:  https://doi.org/10.1093/ecco-jcc/jjaf102
  41. Structure. 2025 Jul 08. pii: S0969-2126(25)00223-0. [Epub ahead of print]
    CryoEM-enabled visual proteomics reveals de novo structures of oligomeric protein complexes.
    Yuanbo Shen, Ailiena O Maggiolo, Tianzheng Zhang, Rebeccah A Warmack.
      Single particle cryoelectron microscopy (cryoEM) and cryoelectron tomography (cryoET) are powerful methods for unveiling unique and functionally relevant structural states. Aided by mass spectrometry and machine learning, they promise to facilitate the visual exploration of proteomes. Leveraging visual proteomics, we interrogate structures isolated from a complex cellular milieu by cryoEM to identify and classify molecular structures and complexes de novo. By comparing three automated model building programs, CryoID, DeepTracer, and ModelAngelo, we determine the identity of six distinct oligomeric protein complexes from partially purified extracts of the nitrogen-fixing bacterium Azotobacter vinelandii using both anaerobic and aerobic cryoEM, including two original oligomeric structures. Overall, by allowing the study of near-native oligomeric protein states, cryoEM-enabled visual proteomics reveals unique structures that correspond to relevant species observed in situ.
    Keywords:  anaerobic; cryoEM; cryoET; metabolism; metalloproteins; method development; nitrogen fixation; protein filaments; structural biology; visual proteomics
    DOI:  https://doi.org/10.1016/j.str.2025.06.007
  42. bioRxiv. 2025 May 05. pii: 2025.05.05.652304. [Epub ahead of print]
    Targeting the canonical ER stress IRE1α/XBP1 pathway counteracts pancreatic cancer-induced skeletal muscle wasting.
    Aniket S Joshi, Meiricris Tomaz da Silva, Anh Tuan Vuong, Bowen Xu, Ravi K Singh, Ashok Kumar.
      Cancer-driven cachexia is a deleterious syndrome which involves progressive loss of skeletal muscle mass with or without fat loss, fatigue, and weakness that cannot be reversed by nutritional intake. Recent studies have shown deregulation of endoplasmic reticulum (ER)-induced unfolded protein response (UPR) pathways in skeletal muscles in various catabolic conditions, including cancer growth. However, the role of individual arms of the UPR in regulation of muscle mass remains poorly understood. Here, we demonstrate that the IRE1α/XBP1 arm of the UPR stimulates the activation of ubiquitin-proteasome system, autophagy, JAK-STAT3 signaling, and fatty acid oxidation in skeletal muscle of the KPC mouse model of pancreatic cancer cachexia. Furthermore, our results show that IRE1α/XBP1 pathway is a key contributor to cachexia as targeted ablation of XBP1 transcription factor in mouse skeletal muscle inhibits KPC tumor-induced muscle wasting. Transcriptionally active XBP1 protein binds to the promoter region of multiple genes whose products are involved in skeletal muscle wasting. Treatment of KPC tumor-bearing mice with 4µ8C, a small molecule IRE1α inhibitor, reverses cachexia-induced molecular changes and improves skeletal muscle mass and strength. Altogether, our study highlights that the IRE1α/XBP1 signaling axis mediates pancreatic cancer-induced muscle wasting and inhibition of this pathway could be a potential approach to mitigate muscle wasting in pancreatic cancer patients.
    DOI:  https://doi.org/10.1101/2025.05.05.652304
  43. J Clin Invest. 2025 Jul 15. pii: e189048. [Epub ahead of print]135(14):
    Cancer-associated SPOP mutations enlarge nuclear size and facilitate nuclear envelope rupture upon farnesyltransferase inhibitor treatment.
    Zixi Wang, Lei Li, Qi Ye, Yuzeshi Lei, Mingming Lu, Leihong Ye, Jialu Kang, Wenyue Huang, Shan Xu, Ke Wang, Jing Liu, Yang Gao, Chenji Wang, Jian Ma, Lei Li.
      Nuclear size is crucial for cellular functions and often increases with malignancy. Irregular nuclei are linked to aggressive tumors, driven by genetic and epigenetic changes. However, the precise mechanisms controlling nuclear size are still not fully understood. In this study, we demonstrated that cancer-associated speckle-type POZ protein (SPOP) mutations enlarged nuclear size by reducing the protein level of lamin B2 (LMNB2), a key nuclear integrity protein. Mechanistically, SPOP bound to LMNB2 and promoted its mono-ubiquitination at lysine-484, which protected it from degradation by the E3 ubiquitin ligase WD repeat domain 26. SPOP mutations disrupted this process, leading to reduced LMNB2 levels and impaired nuclear envelope (NE) integrity. This compromised NE was more vulnerable to damage from farnesyltransferase inhibitors (FTIs), causing nuclear rupture in SPOP-mutant tumor cells. This study identified SPOP as a positive regulator of nuclear size; the findings suggest tumors with SPOP mutations may be vulnerable to FTI-based therapies.
    Keywords:  Cancer; Cell biology; Drug therapy; Molecular biology; Therapeutics
    DOI:  https://doi.org/10.1172/JCI189048
  44. Science. 2025 Jul 17. 389(6757): eado2403
    Polyglycine-mediated aggregation of FAM98B disrupts tRNA processing in GGC repeat disorders.
    Jason Yang, Yunhan Xu, David R Ziehr, Martin S Taylor, Max L Valenstein, Evgeni M Frenkel, Jack R Bush, Kate Rutter, Igor Stevanovski, Charlie Y Shi, Maheswaran Kesavan, Ricardo Mouro Pinto, Ira Deveson, David P Bartel, David M Sabatini, Raghu R Chivukula.
      Aggregation-prone polyglycine-containing proteins produced from expanded GGC repeats are implicated in an emerging family of neurodegenerative disorders. In this study, we showed that polyglycine itself forms aggregates that incorporate endogenous glycine-rich proteins, including FAM98B, a component of the transfer RNA (tRNA) ligase complex (tRNA-LC) that harbors the most glycine-rich sequence in the human proteome. Through this glycine-rich intrinsically disordered region (IDR), polyglycine sequesters and depletes the tRNA-LC, disrupting tRNA processing. Accordingly, patient tissues revealed aggregate-associated FAM98B depletion and accumulation of aberrant tRNA splicing intermediates. Furthermore, Fam98b depletion in adult mice caused progressive motor coordination deficits and hindbrain pathology. Our data suggest that the FAM98B glycine-rich IDR mechanistically links previously disparate neurodegenerative disorders of protein aggregation and tRNA processing.
    DOI:  https://doi.org/10.1126/science.ado2403
  45. Mol Ther Nucleic Acids. 2025 Sep 09. 36(3): 102602
    A proximity-labeling-based approach to directly detect mRNA delivery to specific subcellular locations.
    Alfredo D Smart, Merryn E Hughes, Angela Downie Ruiz Velasco, Naoto Hori, Snow Stolnik, Catherine L Jopling.
      Messenger RNA (mRNA) therapeutics show considerable promise but face delivery challenges, as effective cytosolic entry and subsequent translation are normally limited by endosomal entrapment. While various approaches have been used to investigate determinants of effective RNA delivery, these methods tend to be indirect, qualitative, or rely on labeled RNA. There is a need for quantitative approaches that can directly measure mRNA delivery to its functional sites within the cell. Here, we adapted the APEXseq approach for proximity biotinylation and isolation of mRNA at specific subcellular locations. We combined APEX2 labeling with reverse-transcription quantitative PCR to investigate mRNA delivery to the cytoplasm and endoplasmic reticulum, the two major sites of translation, and found it was most effective in the endoplasmic reticulum. We incorporated a biotinylated spike-in RNA to improve existing methodology by allowing normalization of data and optimization of mRNA pull-down conditions. Finally, we combined this method with protein assays to investigate the role of different signal peptides in mRNA delivery to, and translation at, the endoplasmic reticulum. This new approach shows promise as a tool for future investigation of productive delivery of therapeutic mRNA.
    Keywords:  APEX; MT: Delivery Strategies; endoplasmic reticulum; mRNA delivery; proximity biotinylation; signal peptide
    DOI:  https://doi.org/10.1016/j.omtn.2025.102602
  46. EMBO J. 2025 Jul 16.
    The proteostatic landscape of healthy human oocytes.
    Gabriele Zaffagnini, Miquel Solé, Juan Manuel Duran, Nikolaos P Polyzos, Elvan Böke.
      Oocytes, female germ cells that develop into eggs, are among the longest-lived cells in the animal body. Recent studies on mouse oocytes highlight unique adaptations in protein homeostasis (proteostasis) within these cells. However, the mechanisms of proteostasis in human oocytes remain virtually unstudied. We present the first large-scale study of proteostatic activity in human oocytes using over 100 freshly donated oocytes from 21 healthy women aged 19-34 years. We analysed the activity and distribution of lysosomes, proteasomes, and mitochondria in both immature and mature oocytes. Notably, human oocytes exhibit nearly twofold lower proteolytic activity than surrounding somatic cells, with further decreases as oocytes mature. Oocyte maturation is also coupled with lysosomal exocytosis and a decrease in mitochondrial membrane potential. We propose that reduced organelle activity preserves key cellular components critical for early embryonic development during the prolonged maturation of human oocytes. Our findings highlight the distinctive biology of human oocytes and the need to investigate human-specific reproductive biology to address challenges in female fertility.
    Keywords:  Female Fertility; Human Oocytes; Lysosomes; Mitochondria; Proteostasis
    DOI:  https://doi.org/10.1038/s44318-025-00493-2
  47. J Cell Sci. 2025 Jul 14. pii: jcs.263790. [Epub ahead of print]
    Somatic mutations in IRE1α regulate keratinocyte migration and survival by differentially activating Rho GTPases.
    Saie Mogre, Lily Robinson, Komal Sethia, Bipin Rimal, Jeongin Son, Christian Pacifico, Lorraine Santy, Andrew Patterson, Adam B Glick.
      IRE1α is an Endoplasmic Reticulum (ER) transmembrane protein with cytoplasmic kinase and endoribonuclease (RNase) domains. Under ER stress, IRE1α can splice Xbp1 mRNA enabling translation of this Unfolded Protein Response transcription factor or mediate sequence-specific degradation of mRNAs through Regulated IRE1α-Dependent Decay (RIDD). Somatic mutations in IRE1α occur in many different human cancers including non-melanoma skin cancers (NMSC). To understand their role in skin cancer pathogenesis, we generated immortalized primary mouse keratinocytes inducibly expressing multiple engineered and cancer-associated mutations, including those present in NMSC. All NMSC mutations tested were activating mutations with elevated autophosphorylation and enhanced RIDD activity relative to Xbp1 splicing. Pathway analysis of RNA-Seq data and in vitro studies showed that RNase-impaired mutations enhanced cell migration due to increased levels of active RhoA and a RIDD target, Angptl4. In contrast, activating mutations exhibited elevated Rac1 activation, enrichment of genes involved in DNA repair, increased phospho-ATR levels, and improved survival in response to UVB irradiation, a critical etiological factor for sun-exposed skin cancers. Together, these results suggest divergent roles of IRE1α mutations by mediating critical tumor-promoting events in keratinocytes.
    Keywords:  ER Stress; IRE1α; Migration; RIDD; UV-induced apoptosis
    DOI:  https://doi.org/10.1242/jcs.263790
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