bims-proteo Biomed News
on Proteostasis
Issue of 2025–02–02
forty-five papers selected by
Eric Chevet, INSERM
  1. Functionally diversified BiP orthologs control body growth, reproduction, stress resistance, aging, and ER-Phagy in Caenorhabditis elegans.
  2. The ubiquitin-conjugating enzyme UBE2D maintains a youthful proteome and ensures protein quality control during aging by sustaining proteasome activity.
  3. Convergence of orphan quality control pathways at a ubiquitin chain-elongating ligase.
  4. Stalled disomes marked by Hel2-dependent ubiquitin chains undergo Ubp2/Ubp3-mediated deubiquitination upon translational run-off.
  5. The 40S ribosomal subunit recycling complex modulates mitochondrial dynamics and endoplasmic reticulum - mitochondria tethering at mitochondrial fission/fusion hotspots.
  6. Localized K63 ubiquitin signaling is regulated by VCP/p97 during oxidative stress.
  7. Implications of frequent hitter E3 ligases in targeted protein degradation screens.
  8. Serine phosphorylation facilitates protein degradation by the human mitochondrial ClpXP protease.
  9. C-terminal amides mark proteins for degradation via SCF-FBXO31.
  10. Interplay of p23 with FKBP51 and their chaperone complex in regulating tau aggregation.
  11. PEBP1 amplifies mitochondrial dysfunction-induced integrated stress response.
  12. Mechanism of chaperone recruitment and retention on mitochondrial precursors.
  13. Discovery of DCAF16 Binders for Targeted Protein Degradation.
  14. G3BP1 ribonucleoprotein complexes regulate focal adhesion protein mobility and cell migration.
  15. Activation of lysophagy by a TBK1-SCFFBXO3-TMEM192-TAX1BP1 axis in response to lysosomal damage.
  16. Signs of damage that drive protein degradation.
  17. Development of a versatile system for evaluating the target protein degradation activity of novel ubiquitin ligases utilizing existing PROTACs.
  18. Pellino 3 E3 ligase promotes starvation-induced autophagy to prevent hepatic steatosis.
  19. Proximity proteomics provides a new resource for exploring the function of Afadin and the complexity of cell-cell adherens junctions.
  20. eIF3 engages with 3'-UTR termini of highly translated mRNAs.
  21. Cytoplasmic mRNA decay controlling inflammatory gene expression is determined by pre-mRNA fate decision.
  22. Lipid-Modulated, Graduated Inhibition of N-Glycosylation Pathway Priming Suggests Wide Tolerance of ER Proteostasis to Stress.
  23. TRIM9 Controls Growth Cone Responses to Netrin Through DCC and UNC5C.
  24. Recombinant Antibodies Inhibit Enzymatic Activity of the E3 Ubiquitin Ligase CHIP via Multiple Mechanisms.
  25. Hsp90, DnaK, and ClpB collaborate in protein reactivation.
  26. Genetically-encoded targeted protein degradation technology to remove endogenous condensation-prone proteins and improve crop performance.
  27. Pupylation-based proximity labeling reveals regulatory factors in cellulose biosynthesis in Arabidopsis.
  28. Development of Potent and Selective CK1α Molecular Glue Degraders.
  29. Positively charged cytoplasmic residues in corin prevent signal peptidase cleavage and endoplasmic reticulum retention.
  30. Exploration of degrons and their ability to mediate targeted protein degradation.
  31. Disease-associated Kv1.3 variants are energy compromised with impaired nascent chain folding.
  32. Deacetylated SNAP47 recruits HOPS to facilitate autophagosome-lysosome fusion independent of STX17.
  33. Splicing accuracy varies across human introns, tissues, age and disease.
  34. The endoplasmic reticulum degradation-enhancing α-mannosidase-like protein 3 attenuates the unfolded protein response and has pro-survival and pro-viral roles in hepatoma cells and hepatocellular carcinoma patients.
  35. Extracellular thiol isomerase ERp5 regulates integrin αIIbβ3 activation by inhibition of fibrinogen binding.
  36. Small molecule modulators of B56-PP2A restore 4E-BP function to suppress eIF4E-dependent translation in cancer cells.
  37. Discovery of an on-pathway protein folding intermediate illuminates the kinetic competition between folding and misfolding.
  38. Undocking of an extensive ciliary network induces proteostasis and cell fate switching resulting in severe primary ciliary dyskinesia.
  39. Overcoming Fluorescence Loss in mEOS-based AAA+ Unfoldase Reporters Through Covalent Linkage.
  40. Modulation of Protein-Protein Interactions with Molecular Glues in a Synthetic Condensate Platform.
  41. Genome-wide profiling of tRNA modifications by Induro-tRNAseq reveals coordinated changes.
  42. Cytoplasmic mRNA decay and quality control machineries in eukaryotes.
  43. The MAP kinase scaffold MORG1 shapes cell death in unresolved ER stress in Arabidopsis.
  44. SEC-MX: an approach to systematically study the interplay between protein assembly states and phosphorylation.
  45. A multiscale functional map of somatic mutations in cancer integrating protein structure and network topology.
  1. bioRxiv. 2025 Jan 19. pii: 2025.01.14.633073. [Epub ahead of print]
    Functionally diversified BiP orthologs control body growth, reproduction, stress resistance, aging, and ER-Phagy in Caenorhabditis elegans.
    Nicholas D Urban, Shannon M Lacy, Kate M Van Pelt, Benedict Abdon, Zachary Mattiola, Adam Klaiss, Sarah Tabler, Matthias C Truttmann.
      Cellular systems that govern protein folding rely on a delicate balance of functional redundancy and diversification to maintain protein homeostasis (proteostasis). Here, we use Caenorhabditis elegans to demonstrate how both overlapping and divergent activities of two homologous endoplasmic reticulum (ER)-resident HSP70 family chaperones, HSP-3 and HSP-4, orchestrate ER proteostasis and contribute to organismal physiology. We identify tissue-, age-, and stress-specific protein expression patterns and find both redundant and distinct functions for HSP-3 and HSP-4 in ER stress resistance, reproduction, and body size regulation. We show that only HSP-3 overexpression is sufficient to improve longevity and that loss of HSP-3 or HSP-4 during distinct stages of the worm cycle or specific tissues have opposing effects on worm lifespan. Furthermore, we find that loss of HSP-4, but not HSP-3, improves tolerance to protein aggregation induced-stress by activating ER-Phagy through the engagement of IRE-1 and the putative ER-Phagy receptor, C18E9.2. Mechanistically, we show that de-repression of IRE-1 via HSP-4 dissociation allows for direct inhibition of C18E9.2- mediated ER-Phagy and demonstrate that a conserved orthologous mechanism involving the respective human orthologs, BiP, Sec-62, and IRE-1, contributes to ER proteostasis regulation in human cells. Taken as a whole, our study demonstrates that functional diversification of orthologous proteins within a single organelle is an efficient mechanism to maximize stress resilience while also defining a novel link between ER- phagy and proteostasis regulation.
    DOI:  https://doi.org/10.1101/2025.01.14.633073
  2. PLoS Biol. 2025 Jan;23(1): e3002998
    The ubiquitin-conjugating enzyme UBE2D maintains a youthful proteome and ensures protein quality control during aging by sustaining proteasome activity.
    Liam C Hunt, Michelle Curley, Kudzai Nyamkondiwa, Anna Stephan, Jianqin Jiao, Kanisha Kavdia, Vishwajeeth R Pagala, Junmin Peng, Fabio Demontis.
      Ubiquitin-conjugating enzymes (E2s) are key for protein turnover and quality control via ubiquitination. Some E2s also physically interact with the proteasome, but it remains undetermined which E2s maintain proteostasis during aging. Here, we find that E2s have diverse roles in handling a model aggregation-prone protein (huntingtin-polyQ) in the Drosophila retina: while some E2s mediate aggregate assembly, UBE2D/effete (eff) and other E2s are required for huntingtin-polyQ degradation. UBE2D/eff is key for proteostasis also in skeletal muscle: eff protein levels decline with aging, and muscle-specific eff knockdown causes an accelerated buildup in insoluble poly-ubiquitinated proteins (which progressively accumulate with aging) and shortens lifespan. Mechanistically, UBE2D/eff is necessary to maintain optimal proteasome function: UBE2D/eff knockdown reduces the proteolytic activity of the proteasome, and this is rescued by transgenic expression of human UBE2D2, an eff homolog. Likewise, human UBE2D2 partially rescues the lifespan and proteostasis deficits caused by muscle-specific effRNAi and re-establishes the physiological levels of effRNAi-regulated proteins. Interestingly, UBE2D/eff knockdown in young age reproduces part of the proteomic changes that normally occur in old muscles, suggesting that the decrease in UBE2D/eff protein levels that occurs with aging contributes to reshaping the composition of the muscle proteome. However, some of the proteins that are concertedly up-regulated by aging and effRNAi are proteostasis regulators (e.g., chaperones and Pomp) that are transcriptionally induced presumably as part of an adaptive stress response to the loss of proteostasis. Altogether, these findings indicate that UBE2D/eff is a key E2 ubiquitin-conjugating enzyme that ensures protein quality control and helps maintain a youthful proteome composition during aging.
    DOI:  https://doi.org/10.1371/journal.pbio.3002998
  3. Mol Cell. 2025 Jan 23. pii: S1097-2765(25)00002-4. [Epub ahead of print]
    Convergence of orphan quality control pathways at a ubiquitin chain-elongating ligase.
    Sara Carrillo Roas, Yuichi Yagita, Paul Murphy, Robert Kurzbauer, Tim Clausen, Eszter Zavodszky, Ramanujan S Hegde.
      Unassembled and partially assembled subunits of multi-protein complexes have emerged as major quality control clients, particularly under conditions of imbalanced gene expression such as stress, aging, and aneuploidy. The factors and mechanisms that eliminate such orphan subunits to maintain protein homeostasis are incompletely defined. Here, we show that the UBR4-KCMF1 ubiquitin ligase complex is required for the efficient degradation of multiple unrelated orphan subunits from the chaperonin, proteasome cap, proteasome core, and a protein targeting complex. Epistasis analysis in cells and reconstitution studies in vitro show that the UBR4-KCMF1 complex acts downstream of a priming ubiquitin ligase that first mono-ubiquitinates orphans. UBR4 recognizes both the orphan and its mono-ubiquitin and builds a K48-linked poly-ubiquitin degradation signal. The discovery of a convergence point for multiple quality control pathways may explain why aneuploid cells are especially sensitive to loss of UBR4 or KCMF1 and identifies a potential vulnerability across many cancers.
    Keywords:  E3 ligase; KCMF1; UBR4; protein quality control; proteostasis; ubiquitination
    DOI:  https://doi.org/10.1016/j.molcel.2025.01.002
  4. Commun Biol. 2025 Jan 28. 8(1): 132
    Stalled disomes marked by Hel2-dependent ubiquitin chains undergo Ubp2/Ubp3-mediated deubiquitination upon translational run-off.
    Mario Scazzari, Ying Zhang, Anna Moddemann, Sabine Rospert.
      Stalled ribosomes cause collisions, impair protein synthesis, and generate potentially harmful truncated polypeptides. Eukaryotic cells utilize the ribosome-associated quality control (RQC) and no-go mRNA decay (NGD) pathways to resolve these problems. In yeast, the E3 ubiquitin ligase Hel2 recognizes and polyubiquitinates disomes and trisomes at the 40S ribosomal protein Rps20/uS10, thereby priming ribosomes for further steps in the RQC/NGD pathways. Recent studies have revealed high concentrations of disomes and trisomes in unstressed cells, raising the question of whether and how Hel2 selects long-term stalled disomes and trisomes. This study presents quantitative analysis of in vivo-formed Hel2•ribosome complexes and the dynamics of Hel2-dependent Rps20 ubiquitination and Ubp2/Ubp3-dependent deubiquitination. Our findings show that Hel2 occupancy progressively increases from translating monosomes to disomes and trisomes. We demonstrate that disomes and trisomes with mono- or di-ubiquitinated Rps20 resolve independently of the RQC component Slh1, while those with tri- and tetra-ubiquitinated Rps20 do not. Based on the results, we propose a model in which Hel2 translates the duration of ribosome stalling into polyubiquitin chain length. This mechanism allows for the distinction between transient and long-term stalling, providing the RQC machinery with a means to select fatally stalled ribosomes over transiently stalled ones.
    DOI:  https://doi.org/10.1038/s42003-025-07569-z
  5. Nat Commun. 2025 Jan 25. 16(1): 1021
    The 40S ribosomal subunit recycling complex modulates mitochondrial dynamics and endoplasmic reticulum - mitochondria tethering at mitochondrial fission/fusion hotspots.
    Foozhan Tahmasebinia, Yinglu Tang, Rushi Tang, Yi Zhang, Will Bonderer, Maisa de Oliveira, Bretton Laboret, Songjie Chen, Ruiqi Jian, Lihua Jiang, Michael Snyder, Chun-Hong Chen, Yawei Shen, Qing Liu, Boxiang Liu, Zhihao Wu.
      The 40S ribosomal subunit recycling pathway is an integral link in the cellular quality control network, occurring after translational errors have been corrected by the ribosome-associated quality control (RQC) machinery. Despite our understanding of its role, the impact of translation quality control on cellular metabolism remains poorly understood. Here, we reveal a conserved role of the 40S ribosomal subunit recycling (USP10-G3BP1) complex in regulating mitochondrial dynamics and function. The complex binds to fission-fusion proteins located at mitochondrial hotspots, regulating the functional assembly of endoplasmic reticulum-mitochondria contact sites (ERMCSs). Furthermore, it alters the activity of mTORC1/2 pathways, suggesting a link between quality control and energy fluctuations. Effective communication is essential for resolving proteostasis-related stresses. Our study illustrates that the USP10-G3BP1 complex acts as a hub that interacts with various pathways to adapt to environmental stimuli promptly. It advances our molecular understanding of RQC regulation and helps explain the pathogenesis of human proteostasis and mitochondrial dysfunction diseases.
    DOI:  https://doi.org/10.1038/s41467-025-56346-3
  6. Mol Cell Proteomics. 2025 Jan 27. pii: S1535-9476(25)00018-0. [Epub ahead of print] 100920
    Localized K63 ubiquitin signaling is regulated by VCP/p97 during oxidative stress.
    Austin O Maduka, Sandhya Manohar, Matthew W Foster, Gustavo M Silva.
      Under stress conditions, cells reprogram their molecular machineries to mitigate damage and promote survival. Ubiquitin signaling is globally increased during oxidative stress, controlling protein fate and supporting stress defenses at several subcellular compartments. However, the rules driving subcellular ubiquitin localization to promote concerted response mechanisms remain understudied. Here, we show that K63-linked polyubiquitin chains, known to promote proteasome-independent pathways, accumulate primarily in non-cytosolic compartments during oxidative stress induced by sodium arsenite in mammalian cells. Our subcellular ubiquitin proteomic analyses of non-cytosolic compartments expanded 2.5-fold the pool of proteins (2,494) and provided a comprehensive number of sites (10,157) known to be ubiquitinated during arsenite stress, suggesting their involvement in a myriad of cellular pathways. Moreover, subcellular proteome analyses revealed proteins that are recruited to non-cytosolic compartments under stress, including a significant enrichment of helper ubiquitin-binding adaptors of the ATPase VCP that processes ubiquitinated substrates for downstream signaling. We further show that VCP recruitment to non-cytosolic compartments under arsenite stress occurs in a ubiquitin-dependent manner mediated by its adaptor NPLOC4. Additionally, we show that VCP and NPLOC4 activities are critical to sustain low levels of non-cytosolic K63-linked ubiquitin chains, supporting a cyclical model of ubiquitin conjugation and removal that is disrupted by reactive oxygen species. This work deepens our understanding of the role of localized ubiquitin and VCP signaling in the basic mechanisms of stress response and highlights new pathways and molecular players that are essential to reshape the composition and function of the human subcellular proteome under dynamic environments.
    Keywords:  K63 ubiquitin; VCP; human cells; oxidative stress; subcellular localization
    DOI:  https://doi.org/10.1016/j.mcpro.2025.100920
  7. Nat Chem Biol. 2025 Jan 27.
    Implications of frequent hitter E3 ligases in targeted protein degradation screens.
    Xiaoyu Zhang, Gabriel M Simon, Benjamin F Cravatt.
      Targeted protein degradation (TPD) offers a promising approach for chemical probe and drug discovery that uses small molecules or biologics to direct proteins to the cellular machinery for destruction. Among the >600 human E3 ligases, CRBN and VHL have served as workhorses for ubiquitin-proteasome system-dependent TPD. Identification of additional E3 ligases capable of supporting TPD would unlock the full potential of this mechanism for both research and pharmaceutical applications. This perspective discusses recent strategies to expand the scope of TPD and the surprising convergence of these diverse screening efforts on a handful of E3 ligases, specifically DCAF16, DCAF11 and FBXO22. We speculate that a combination of properties, including superficial ligandability, potential for promiscuous substrate interactions and high occupancy in Cullin-RING complexes, may position these E3 ligases as 'low-hanging fruit' in TPD screens. We also discuss complementary approaches that might further expand the E3 ligase landscape supporting TPD.
    DOI:  https://doi.org/10.1038/s41589-024-01821-z
  8. Proc Natl Acad Sci U S A. 2025 Feb 04. 122(5): e2422447122
    Serine phosphorylation facilitates protein degradation by the human mitochondrial ClpXP protease.
    Yue Feng, Monica M Goncalves, Yulia Jitkova, Alexander F A Keszei, Yongran Yan, Chaitra Sarathy, Jonathan St-Germain, Tristan M G Kenney, Matthew Tcheng, Vincent Trudel, Ross S Mancini, Rahul Upadhyay, Rose Hurren, Marcela Gronda, Matthew Schultz, Kaylen Soriano, Kaitlin Lees, Neil C Pomroy, S Quinn W Currie, Gilbert G Privé, Mark A Reed, Andrei K Yudin, Linda Z Penn, Cheryl H Arrowsmith, Brian Raught, Mohammad T Mazhab-Jafari, Siavash Vahidi, Aaron D Schimmer.
      ClpXP is a two-component mitochondrial matrix protease. The caseinolytic mitochondrial matrix peptidase chaperone subunit X (ClpX) recognizes and translocates protein substrates into the degradation chamber of the caseinolytic protease P (ClpP) for proteolysis. ClpXP degrades damaged respiratory chain proteins and is necessary for cancer cell survival. Despite the critical role of ClpXP in mitochondrial protein quality control, the specific degrons, or modifications that tag substrate proteins for degradation by human ClpXP, are still unknown. We demonstrated that phosphorylated serine (pSer) targets substrates to ClpX and facilitates their degradation by ClpXP in biochemical assays. In contrast, ClpP hyperactivated by the small-molecule drug ONC201 lost the preference for phosphorylated substrates. Hydrogen deuterium exchange mass spectrometry combined with biochemical assays showed that pSer binds the RKL loop of ClpX. ClpX variants with substitutions in the RKL loop failed to recognize phosphorylated substrates. In intact cells, ClpXP also preferentially degraded substrates with pSer. Moreover, ClpX substrates with the pSer were selectively found in aggregated mitochondrial proteins. Our work uncovers a mechanism for substrate recognition by ClpXP, with implications for targeting acute myeloid leukemia and other disorders involving ClpXP dysfunction.
    Keywords:  AAA+ proteases; degron; mitochondrial proteostasis; phosphorylation; protein degradation
    DOI:  https://doi.org/10.1073/pnas.2422447122
  9. Nature. 2025 Jan 29.
    C-terminal amides mark proteins for degradation via SCF-FBXO31.
    Matthias F Muhar, Jakob Farnung, Martina Cernakova, Raphael Hofmann, Lukas T Henneberg, Moritz M Pfleiderer, Annina Denoth-Lippuner, Filip Kalčic, Ajse S Nievergelt, Marwa Peters Al-Bayati, Nikolaos D Sidiropoulos, Viola Beier, Matthias Mann, Sebastian Jessberger, Martin Jinek, Brenda A Schulman, Jeffrey W Bode, Jacob E Corn.
      During normal cellular homeostasis, unfolded and mislocalized proteins are recognized and removed, preventing the build-up of toxic byproducts1. When protein homeostasis is perturbed during ageing, neurodegeneration or cellular stress, proteins can accumulate several forms of chemical damage through reactive metabolites2,3. Such modifications have been proposed to trigger the selective removal of chemically marked proteins3-6; however, identifying modifications that are sufficient to induce protein degradation has remained challenging. Here, using a semi-synthetic chemical biology approach coupled to cellular assays, we found that C-terminal amide-bearing proteins (CTAPs) are rapidly cleared from human cells. A CRISPR screen identified FBXO31 as a reader of C-terminal amides. FBXO31 is a substrate receptor for the SKP1-CUL1-F-box protein (SCF) ubiquitin ligase SCF-FBXO31, which ubiquitylates CTAPs for subsequent proteasomal degradation. A conserved binding pocket enables FBXO31 to bind to almost any C-terminal peptide bearing an amide while retaining exquisite selectivity over non-modified clients. This mechanism facilitates binding and turnover of endogenous CTAPs that are formed after oxidative stress. A dominant human mutation found in neurodevelopmental disorders reverses CTAP recognition, such that non-amidated neosubstrates are now degraded and FBXO31 becomes markedly toxic. We propose that CTAPs may represent the vanguard of a largely unexplored class of modified amino acid degrons that could provide a general strategy for selective yet broad surveillance of chemically damaged proteins.
    DOI:  https://doi.org/10.1038/s41586-024-08475-w
  10. Nat Commun. 2025 Jan 14. 16(1): 669
    Interplay of p23 with FKBP51 and their chaperone complex in regulating tau aggregation.
    Pijush Chakraborty, Markus Zweckstetter.
      The pathological deposition of tau and amyloid-beta into insoluble amyloid fibrils are pathological hallmarks of Alzheimer's disease. Molecular chaperones are important cellular factors contributing to the regulation of tau misfolding and aggregation. Here we reveal an Hsp90-independent mechanism by which the co-chaperone p23 as well as a molecular complex formed by two co-chaperones, p23 and FKBP51, modulates tau aggregation. Integrating NMR spectroscopy, SAXS, molecular docking, and site-directed mutagenesis we reveal the structural basis of the p23-FKBP51 complex. We show that p23 specifically recognizes the TPR domain of FKBP51 and interacts with tau through its C-terminal disordered tail. We further show that the p23-FKBP51 complex binds tau to form a dynamic p23-FKBP51-tau trimeric complex that delays tau aggregation and thus may counteract Hsp90-FKBP51 mediated toxicity. Taken together, our findings reveal a co-chaperone mediated Hsp90-independent chaperoning of tau protein.
    DOI:  https://doi.org/10.1038/s41467-025-56028-0
  11. Elife. 2025 Jan 29. pii: RP102852. [Epub ahead of print]13
    PEBP1 amplifies mitochondrial dysfunction-induced integrated stress response.
    Ling Cheng, Ian Meliala, Yidi Kong, Jingyuan Chen, Christopher G Proud, Mikael Björklund.
      Mitochondrial dysfunction is involved in numerous diseases and the aging process. The integrated stress response (ISR) serves as a critical adaptation mechanism to a variety of stresses, including those originating from mitochondria. By utilizing mass spectrometry-based cellular thermal shift assay (MS-CETSA), we uncovered that phosphatidylethanolamine-binding protein 1 (PEBP1), also known as Raf kinase inhibitory protein (RKIP), is thermally stabilized by stresses which induce mitochondrial ISR. Depletion of PEBP1 impaired mitochondrial ISR activation by reducing eukaryotic translation initiation factor 2α (eIF2α) phosphorylation and subsequent ISR gene expression, which was independent of PEBP1's role in inhibiting the RAF/MEK/ERK pathway. Consistently, overexpression of PEBP1 potentiated ISR activation by heme-regulated inhibitor (HRI) kinase, the principal eIF2α kinase in the mitochondrial ISR pathway. Real-time interaction analysis using luminescence complementation in live cells revealed an interaction between PEBP1 and eIF2α, which was disrupted by eIF2α S51 phosphorylation. These findings suggest a role for PEBP1 in amplifying mitochondrial stress signals, thereby facilitating an effective cellular response to mitochondrial dysfunction. Therefore, PEBP1 may be a potential therapeutic target for diseases associated with mitochondrial dysfunction.
    Keywords:  PEBP1; cell biology; human; integrated stress response; mitochondrial dysfunction
    DOI:  https://doi.org/10.7554/eLife.102852
  12. Mol Biol Cell. 2025 Jan 29. mbcE25010035
    Mechanism of chaperone recruitment and retention on mitochondrial precursors.
    Szymon Juszkiewicz, Sew-Yeu Peak-Chew, Ramanujan S Hegde.
      Nearly all mitochondrial proteins are imported into mitochondria from the cytosol. How nascent mitochondrial precursors acquire and sustain import-competence in the cytosol under normal and stress conditions is incompletely understood. Here, we show that under normal conditions, the Hsc70 and Hsp90 systems interact with and redundantly minimize precursor degradation. During acute import stress, Hsp90 buffers precursor degradation, preserving proteins in an import-competent state until stress resolution. Unexpectedly, buffering by Hsp90 relies critically on a mitochondrial targeting signal (MTS), the absence of which greatly decreases precursor-Hsp90 interaction. Site-specific photo-crosslinking and biochemical reconstitution showed how the MTS directly engages co-chaperones of Hsc70 (St13 and Stip1) and Hsp90 (p23 and Cdc37) to facilitate chaperone retention on the mature domain. Thus, the MTS has a previously unappreciated role in regulating chaperone dynamics on mitochondrial precursors to buffer their degradation and maintain import competence, functions that may facilitate restoration of mitochondrial homeostasis after acute import stress.
    DOI:  https://doi.org/10.1091/mbc.E25-01-0035
  13. ACS Chem Biol. 2025 Jan 30.
    Discovery of DCAF16 Binders for Targeted Protein Degradation.
    Miguel A Campos, Isabella A Riha, Chenlu Zhang, Chen Mozes, Karl A Scheidt, Xiaoyu Zhang.
      Conventional small-molecule drugs primarily operate by inhibiting protein function, but this approach is limited when proteins lack well-defined ligand-binding pockets. Targeted protein degradation (TPD) offers an alternative approach by harnessing cellular degradation pathways to eliminate specific proteins. Recent studies have expanded the potential of TPD by identifying additional E3 ligases, with DCAF16 emerging as a promising candidate for facilitating protein degradation through both proteolysis-targeting chimera (PROTAC) and molecular glue mechanisms. In this study, we revisited a previously reported compound and discovered that it covalently binds to DCAF16. We further optimized it into a FKBP12-targeting PROTAC, MC-25B. This PROTAC engages DCAF16 at cysteines C177-179, leading to the degradation of nuclear-localized FKBP12. We further demonstrated the versatility of this DCAF16 recruiter by degrading additional endogenous proteins. Compared to the first-generation DCAF16-based PROTAC, which was derived from a fragment electrophile, this DCAF16 recruiter-based PROTAC exhibits improved proteome-wide selectivity.
    DOI:  https://doi.org/10.1021/acschembio.4c00799
  14. Cell Rep. 2025 Jan 29. pii: S2211-1247(25)00008-7. [Epub ahead of print]44(2): 115237
    G3BP1 ribonucleoprotein complexes regulate focal adhesion protein mobility and cell migration.
    Liana C Boraas, Mengwei Hu, Pieter Martino, Lauren Thornton, Charles E Vejnar, Gang Zhen, Longhui Zeng, Dylan M Parker, Andy L Cox, Antonio J Giraldez, Xiaolei Su, Christine Mayr, Siyuan Wang, Stefania Nicoli.
      The subcellular localization of mRNAs plays a pivotal role in biological processes, including cell migration. For instance, β-actin mRNA and its associated RNA-binding protein (RBP), ZBP1/IGF2BP1, are recruited to focal adhesions (FAs) to support localized β-actin synthesis, crucial for cell migration. However, whether other mRNAs and RBPs also localize at FAs remains unclear. Here, we identify hundreds of mRNAs that are enriched at FAs (FA-mRNAs). FA-mRNAs share characteristics with stress granule (SG) mRNAs and are found in ribonucleoprotein (RNP) complexes with the SG RBP. Mechanistically, G3BP1 binds to FA proteins in an RNA-dependent manner, and its RNA-binding and dimerization domains, essential for G3BP1 to form RNPs in SG, are required for FA localization and cell migration. We find that G3BP1 RNPs promote cell speed by enhancing FA protein mobility and FA size. These findings suggest a previously unappreciated role for G3BP1 RNPs in regulating FA function under non-stress conditions.
    Keywords:  CP: Cell biology; CP: Molecular biology; G3BP1; RNA localization; cell migration; focal adhesions; protein mobility; ribonucleoprotein complexes
    DOI:  https://doi.org/10.1016/j.celrep.2025.115237
  15. Nat Commun. 2025 Jan 28. 16(1): 1109
    Activation of lysophagy by a TBK1-SCFFBXO3-TMEM192-TAX1BP1 axis in response to lysosomal damage.
    Na Yeon Park, Doo Sin Jo, Jae-Yoon Yang, Ji-Eun Bae, Joon Bum Kim, Yong Hwan Kim, Seong Hyun Kim, Pansoo Kim, Dong-Seok Lee, Tamotsu Yoshimori, Eun-Kyeong Jo, Eunbyul Yeom, Dong-Hyung Cho.
      Lysophagy eliminates damaged lysosomes and is crucial to cellular homeostasis; however, its underlying mechanisms are not entirely understood. We screen a ubiquitination-related compound library and determine that the substrate recognition component of the SCF-type E3 ubiquitin ligase complex, SCFFBXO3(FBXO3), which is a critical lysophagy regulator. Inhibition of FBXO3 reduces lysophagy and lysophagic flux in response to L-leucyl-L-leucine methyl ester (LLOMe). Furthermore, FBXO3 interacts with TMEM192, leading to its ubiquitination in LLOMe-treated cells. We also identify TAX1BP1 as a critical autophagic adaptor that recognizes ubiquitinated TMEM192 during lysophagy and find that TBK1 activation is crucial for lysophagy, as it phosphorylates FBXO3 in response to lysosomal damage. Knockout of FBXO3 significantly impairs lysophagy, and its reconstitution with a loss-of-function mutant (V221I) further confirms its essential role in lysophagy regulation. Collectively, our findings highlight the significance of the TBK1-FBXO3-TMEM192-TAX1BP1 axis in lysophagy and emphasize the critical role of FBXO3 in lysosomal integrity.
    DOI:  https://doi.org/10.1038/s41467-025-56294-y
  16. Nature. 2025 Jan 29.
    Signs of damage that drive protein degradation.
    Alfred Freeberg, Michael Rapé.
      
    Keywords:  Cell biology; Proteomics
    DOI:  https://doi.org/10.1038/d41586-025-00082-7
  17. Biochem Biophys Res Commun. 2025 Jan 20. pii: S0006-291X(25)00084-1. [Epub ahead of print]749 151370
    Development of a versatile system for evaluating the target protein degradation activity of novel ubiquitin ligases utilizing existing PROTACs.
    Shinya Sato, Mei Matsukawa, Masaaki Takemoto, Takumi Okamoto, Atsushi Saito, Issei Omura, Koji Matsuhisa, Hiroaki Ikeda, Kazunori Imaizumi, Masayuki Kaneko.
      Proteolysis-Targeting Chimeras (PROTAC) are a bifunctional molecule that binds to a protein of interest (POI) and a ubiquitin ligase, thereby inducing the ubiquitination and degradation of POI. Many PROTACs currently utilize a limited number of ubiquitin ligases, such as von Hippel-Lindau (VHL) and Cereblon. Because these ubiquitin ligases are widely expressed in normal tissues, unexpected side effects can occur. Therefore, to expand the repertoire of ubiquitin ligases that can be utilized in PROTACs, we aimed to develop a versatile system to identify suitable novel ubiquitin ligases for PROTAC-mediated protein degradation using existing PROTACs. Chimeric ubiquitin ligases are constructed by fusing VHL with the ubiquitin ligase of interest that is stably expressed in cells. An existing PROTAC that binds to VHL was added to the cells, and the POI degradation activity was evaluated. In this study, we showed that epidermal growth factor receptor can be degraded by an existing PROTAC utilizing a chimeric ubiquitin ligase that fuses VHL and endoplasmic reticulum-localized ubiquitin ligase, HRD1. These results demonstrate that this novel approach can be used to identify suitable ubiquitin ligases for PROTAC-mediated degradation using existing PROTACs. Expanding the repertoire of ubiquitin ligases that can be utilized for PROTAC by using this versatile system is expected to enable the development of more effective and specific PROTACs for cancer and other diseases.
    Keywords:  HRD1; Molecular Glue; Molecular-targeted drug; PROTAC; Ubiquitin ligase; VHL
    DOI:  https://doi.org/10.1016/j.bbrc.2025.151370
  18. Sci Adv. 2025 Jan 17. 11(3): eadr2450
    Pellino 3 E3 ligase promotes starvation-induced autophagy to prevent hepatic steatosis.
    Srinivasa P Kolapalli, Carsten J Beese, Steven E Reid, Sólveig H Brynjólfsdóttir, Maria H Jørgensen, Ashish Jain, Joyceline Cuenco, Monika Lewinska, Ahmad Abdul-Al, Aida R López, Marja Jäättelä, Kei Sakamoto, Jesper B Andersen, Kenji Maeda, Tor E Rusten, Anders H Lund, Lisa B Frankel.
      Nutrient deprivation is a major trigger of autophagy, a conserved quality control and recycling process essential for cellular and tissue homeostasis. In a high-content image-based screen of the human ubiquitome, we here identify the E3 ligase Pellino 3 (PELI3) as a crucial regulator of starvation-induced autophagy. Mechanistically, PELI3 localizes to autophagic membranes, where it interacts with the ATG8 proteins through an LC3-interacting region (LIR). This facilitates PELI3-mediated ubiquitination of ULK1, driving ULK1's subsequent proteasomal degradation. PELI3 depletion leads to an aberrant accumulation and mislocalization of ULK1 and disrupts the early steps of autophagosome formation. Genetic deletion of Peli3 in mice impairs fasting-induced autophagy in the liver and enhances starvation-induced hepatic steatosis by reducing autophagy-mediated clearance of lipid droplets. Notably, PELI3 expression is decreased in the livers of patients with metabolic dysfunction-associated steatotic liver disease (MASLD), suggesting its role in hepatic steatosis development in humans. The findings suggest that PELI3-mediated control of autophagy plays a protective role in liver health.
    DOI:  https://doi.org/10.1126/sciadv.adr2450
  19. Biol Open. 2025 Feb 15. pii: bio061811. [Epub ahead of print]14(2):
    Proximity proteomics provides a new resource for exploring the function of Afadin and the complexity of cell-cell adherens junctions.
    Wangsun Choi, Dennis Goldfarb, Feng Yan, Michael B Major, Alan S Fanning, Mark Peifer.
      The network of proteins at the interface between cell-cell adherens junctions and the actomyosin cytoskeleton provides robust yet dynamic connections that facilitate cell shape change and motility. While this was initially thought to be a simple linear connection via classic cadherins and their associated catenins, we now have come to appreciate that many more proteins are involved, providing robustness and mechanosensitivity. Defining the full set of proteins in this network remains a key objective in our field. Proximity proteomics provides a means to define these networks. Mammalian Afadin and its Drosophila homolog Canoe are key parts of this protein network, facilitating diverse cell shape changes during gastrulation and other events of embryonic morphogenesis. Here we report results of several proximity proteomics screens, defining proteins in the neighborhood of both the N- and C-termini of mammalian Afadin in the premier epithelial model, MDCK cells. We compare our results with previous screens done in other cell types, and with proximity proteomics efforts with other junctional proteins. These reveal the value of multiple screens in defining the full network of neighbors and offer interesting insights into the overlap in protein composition between different epithelial cell junctions.
    Keywords:  Adherens junctions; Afadin; BioID; Canoe; Cell adhesion
    DOI:  https://doi.org/10.1242/bio.061811
  20. Elife. 2025 Jan 29. pii: RP102977. [Epub ahead of print]13
    eIF3 engages with 3'-UTR termini of highly translated mRNAs.
    Santi Mestre-Fos, Lucas Ferguson, Marena I Trinidad, Nicholas T Ingolia, Jamie H D Cate.
      Stem cell differentiation involves a global increase in protein synthesis to meet the demands of specialized cell types. However, the molecular mechanisms underlying this translational burst and the involvement of initiation factors remains largely unknown. Here, we investigate the role of eukaryotic initiation factor 3 (eIF3) in early differentiation of human pluripotent stem cell (hPSC)-derived neural progenitor cells (NPCs). Using Quick-irCLIP and alternative polyadenylation (APA) Seq, we show eIF3 crosslinks predominantly with 3' untranslated region (3'-UTR) termini of multiple mRNA isoforms, adjacent to the poly(A) tail. Furthermore, we find that eIF3 engagement at 3'-UTR ends is dependent on polyadenylation. High eIF3 crosslinking at 3'-UTR termini of mRNAs correlates with high translational activity, as determined by ribosome profiling, but not with translational efficiency. The results presented here show that eIF3 engages with 3'-UTR termini of highly translated mRNAs, likely reflecting a general rather than specific regulatory function of eIF3, and supporting a role of mRNA circularization in the mechanisms governing mRNA translation.
    Keywords:  3'-UTR; biochemistry; cell biology; chemical biology; eIF3; human; protein synthesis; quick-irCLIP; ribosome profiling
    DOI:  https://doi.org/10.7554/eLife.102977
  21. Mol Cell. 2025 Jan 23. pii: S1097-2765(25)00001-2. [Epub ahead of print]
    Cytoplasmic mRNA decay controlling inflammatory gene expression is determined by pre-mRNA fate decision.
    Annika Bestehorn, Julius von Wirén, Christina Zeiler, Jeanne Fesselet, Sebastian Didusch, Maurizio Forte, Kevin Doppelmayer, Martina Borroni, Anita Le Heron, Sara Scinicariello, WeiQiang Chen, Manuela Baccarini, Vera Pfanzagl, Gijs A Versteeg, Markus Hartl, Pavel Kovarik.
      The fidelity of immune responses depends on timely controlled and selective mRNA degradation that is largely driven by RNA-binding proteins (RBPs). It remains unclear whether stochastic or directed processes govern the selection of an individual mRNA molecule for degradation. Using human and mouse cells, we show that tristetraprolin (TTP, also known as ZFP36), an essential anti-inflammatory RBP, destabilizes target mRNAs via a hierarchical molecular assembly. The assembly formation strictly relies on the interaction of TTP with RNA. The TTP homolog ZFP36L1 exhibits similar requirements, indicating a broader relevance of this regulatory program. Unexpectedly, the assembly of the cytoplasmic mRNA-destabilization complex is licensed in the nucleus by TTP binding to pre-mRNA, which we identify as the principal TTP target rather than mRNA. Hence, the fate of an inflammation-induced mRNA is decided concomitantly with its synthesis. This mechanism prevents the translation of excessive and potentially harmful inflammation mediators, irrespective of transcription.
    Keywords:  RNA-binding proteins; Zfp36; immune responses; inflammation; mRNA decay; macrophage; posttranscriptional regulation of gene expression; tristetraprolin
    DOI:  https://doi.org/10.1016/j.molcel.2025.01.001
  22. ACS Cent Sci. 2025 Jan 22. 11(1): 107-115
    Lipid-Modulated, Graduated Inhibition of N-Glycosylation Pathway Priming Suggests Wide Tolerance of ER Proteostasis to Stress.
    Andrew M Giltrap, Niamh Morris, Yin Yao Dong, Stephen A Cochrane, Thomas Krulle, Steven Hoekman, Martin Semmelroth, Carina Wollnik, Timea Palmai-Pallag, Elisabeth P Carpenter, Jonathan Hollick, Alastair Parkes, York Rudhard, Benjamin G Davis.
      Protein N-glycosylation is a cotranslational modification that takes place in the endoplasmic reticulum (ER). Disruption of this process can result in accumulation of misfolded proteins, known as ER stress. In response, the unfolded protein response (UPR) restores proteostasis or responds by controlling cellular fate, including increased expression of activating transcription factor 4 (ATF4) that can lead to apoptosis. The ability to control and manipulate such a stress pathway could find use in relevant therapeutic areas, such as in treating cancerous states in which the native ER stress response is often already perturbed. The first committed step in the N-glycosylation pathway is therefore a target for potential ER stress modulation. Here, using structure-based design, the scaffold of the natural product tunicamycin allows construction of a panel capable of graduated inhibition of DPAGT1 through lipid-substituent-modulated interaction. The development of a quantitative, high-content, cellular immunofluorescence assay allowed precise determination of downstream mechanistic consequences (through the nuclear localization of key proxy transcription factor ATF4 as a readout of resulting ER stress). Only the most potent inhibition of DPAGT1 generates an ER stress response. This suggests that even low-level "background" biosynthetic flux toward protein glycosylation is sufficient to prevent response to ER stress. "Tuned" inhibitors of DPAGT1 also now seemingly successfully decouple protein glycosylation from apoptotic response to ER stress, thereby potentially allowing access to cellular states that operate at the extremes of normal ER stress.
    DOI:  https://doi.org/10.1021/acscentsci.4c01506
  23. J Neurochem. 2025 Jan;169(1): e70002
    TRIM9 Controls Growth Cone Responses to Netrin Through DCC and UNC5C.
    Sampada P Mutalik, Chris T Ho, Ellen C O'Shaughnessy, Anca G Frasineanu, Aneri B Shah, Stephanie L Gupton.
      The guidance cue netrin-1 promotes both growth cone attraction and growth cone repulsion. How netrin-1 elicits diverse axonal responses, beyond engaging the netrin receptor DCC and UNC5 family members, remains elusive. Here, we demonstrate that murine netrin-1 induces biphasic axonal responses in cortical neurons: Attraction at lower concentrations and repulsion at higher concentrations using both a microfluidic-based netrin-1 gradient and bath application of netrin-1. We find that repulsive turning in a netrin gradient is blocked by knockdown of UNC5C, whereas attractive turning is impaired by knockdown of DCC. TRIM9 is a brain-enriched E3 ubiquitin ligase previously shown to bind and cluster the attractive receptor DCC at the plasma membrane and regulate netrin-dependent attractive responses. However, whether TRIM9 also regulated repulsive responses to netrin-1 remained to be seen. In this study, we show that TRIM9 localizes and interacts with both the attractive netrin receptor DCC and the repulsive netrin receptor, UNC5C. We find that deletion of murine Trim9 alters both attractive and repulsive axon turning and changes in growth cones size in response to murine netrin-1. TRIM9 was required for netrin-1-dependent changes in the surface levels of DCC and UNC5C in the growth cone during morphogenesis. We demonstrate that DCC at the membrane regulates the growth cone area and show that TRIM9 negatively regulates FAK activity in the absence of both repulsive and attractive concentrations of netrin-1. Together, our work demonstrates that TRIM9 interacts with and regulates both DCC and UNC5C during attractive and repulsive axonal responses to netrin-1.
    DOI:  https://doi.org/10.1111/jnc.70002
  24. J Biol Chem. 2025 Jan 23. pii: S0021-9258(25)00067-5. [Epub ahead of print] 108220
    Recombinant Antibodies Inhibit Enzymatic Activity of the E3 Ubiquitin Ligase CHIP via Multiple Mechanisms.
    Dong Hee Chung, Emily J Connelly, Aparna Unnikrishnan, Shih-Wei Chuo, Kristin Wucherer, Corey M Nadel, Jason E Gestwicki, Daniel R Southworth, Charles S Craik.
      Carboxyl-terminus of Hsp70-Interacting Protein (CHIP) is an E3 ubiquitin ligase that marks misfolded substrates for degradation. Hyper-activation of CHIP has been implicated in multiple diseases, including cystic fibrosis and cancer, suggesting that it may be a potential drug target. However, there are few tools available for exploring this possibility. Moreover, the best ways of inhibiting CHIP's function are not obvious, as this complex protein is composed of a tetratricopeptide repeat (TPR) domain, a U-box domain, and a coiled-coil domain that mediates homodimerization. To probe the structure and function of CHIP, we report an antibody panning campaign that yielded six recombinant Fabs with affinity for CHIP. Interestingly, these antibodies varied in their binding site(s) and impact on CHIP function, such as inhibiting TPR interactions, autoubiquitination, and/or substrate ubiquitination. Of particular interest, antibody 2F1 nearly eliminated substrate binding (IC50 = 2.7 μM) and limited ubiquitination and autoubiquitination. Cryo-electron microscopy of the 2F1:CHIP complex revealed a 2:1 binding mode (Fab:CHIP dimer), with 2F1 bound to the U-box domain and simultaneously displacing the TPR domain. Together, these studies provide insight into ways of inhibiting CHIP's activity and provide a series of new probes for exploring the function of this important E3 ubiquitin ligase.
    Keywords:  Autoubiquitination; Carboxyl-terminus of Hsp70-Interacting Protein (CHIP); Cryo‐electron microscopy; E3 ubiquitin ligase; Protein-protein interactions; Recombinant antibody; Ubiquitination
    DOI:  https://doi.org/10.1016/j.jbc.2025.108220
  25. Proc Natl Acad Sci U S A. 2025 Feb 04. 122(5): e2422640122
    Hsp90, DnaK, and ClpB collaborate in protein reactivation.
    Joel R Hoskins, Anushka C Wickramaratne, Connor P Jewell, Lisa M Jenkins, Sue Wickner.
      Hsp70, Hsp90, and ClpB/Hsp100 are molecular chaperones that help regulate proteostasis. Bacterial and yeast Hsp70s and their cochaperones function synergistically with Hsp90s to reactivate inactive and aggregated proteins by a mechanism that requires a direct interaction between Hsp90 and Hsp70 both in vitro and in vivo. Escherichia coli and yeast Hsp70s also collaborate in bichaperone systems with ClpB and Hsp104, respectively, to disaggregate and reactivate aggregated proteins and amyloids such as prions. These collaborations are dependent on direct interactions between ClpB/Hsp104 and Hsp70. We explored the possibility that E. coli homologs of Hsp70, Hsp90, and ClpB, referred to as DnaK, Hsp90Ec, and ClpB, respectively, in combination with two DnaK cochaperones, DnaJ and GrpE, could promote protein disaggregation and reactivation under conditions where bichaperone systems are ineffective. Our results show that Hsp90Ec is able to overcome the inhibition of protein disaggregation and reactivation observed when the concentration of DnaK is approaching physiological concentrations. We found that ATP hydrolysis and substrate binding by all three chaperones are essential for the collaborative function. The work further shows that ClpB acts early in protein reactivation with DnaK and its cochaperones; E. coli Hsp90 acts at a later stage after ClpB. The results highlight the collaboration among chaperones to regulate and maintain proteostasis.
    Keywords:  DnaJ; Hsp100; Hsp70; HtpG; molecular chaperones
    DOI:  https://doi.org/10.1073/pnas.2422640122
  26. Nat Commun. 2025 Jan 29. 16(1): 1159
    Genetically-encoded targeted protein degradation technology to remove endogenous condensation-prone proteins and improve crop performance.
    Ming Luo, Sitao Zhu, Hua Dang, Qing Wen, Ruixia Niu, Jiawei Long, Zhao Wang, Yongjia Tong, Yuese Ning, Meng Yuan, Guoyong Xu.
      Effective modulation of gene expression in plants is achievable through tools like CRISPR and RNA interference, yet methods for directly modifying endogenous proteins remain lacking. Here, we identify the E3 ubiquitin ligase E3TCD1 and develope a Targeted Condensation-prone-protein Degradation (TCD) strategy. The X-E3TCD1 fusion protein acts as a genetically engineered degrader, selectively targeting endogenous proteins prone to condensation. For example, a transgenic E3TCD1 fusion with Teosinte branched 1 (TB1) degrades the native TB1 protein, resulting in increased tiller numbers in rice. Additionally, conditional degradation of the negative defense regulator Early Flowering 3 via a pathogen-responsive ProTBF1-uORFsTBF1 cassette enhances rice blast resistance without affecting flowering time in the absence of pathogen. Unlike prevailing targeted protein degradation strategies, the TCD system does not rely on small molecules, antibodies, or genetic knock-in fusion tags, demonstrating its promise as a transgene-based approach for optimizing crop performance.
    DOI:  https://doi.org/10.1038/s41467-025-56570-x
  27. Nat Commun. 2025 Jan 20. 16(1): 872
    Pupylation-based proximity labeling reveals regulatory factors in cellulose biosynthesis in Arabidopsis.
    Shuai Zheng, Lise C Noack, Ouda Khammy, Andreas De Meyer, Ghazanfar Abbas Khan, Nancy De Winne, Dominique Eeckhout, Daniël Van Damme, Staffan Persson.
      Knowledge about how and where proteins interact provides a pillar for cell biology. Protein proximity-labeling has emerged as an important tool to detect protein interactions. Biotin-related proximity labeling approaches are by far the most commonly used but may have labeling-related drawbacks. Here, we use pupylation-based proximity labeling (PUP-IT) as a tool for protein interaction detection in plants. We show that PUP-IT readily confirmed protein interactions for several known protein complexes across different types of plant hosts and that the approach increased detection of specific interactions as compared to biotin-based proximity labeling systems. To further demonstrate the power of PUP-IT, we used the system to identify protein interactions of the protein complex that underpin cellulose synthesis in plants. Apart from known complex components, we identified the ARF-GEF BEN1 (BFA-VISUALIZED ENDOCYTIC TRAFFICKING DEFECTIVE1). We show that BEN1 contributes to cellulose synthesis by regulating both clathrin-dependent and -independent endocytosis of the cellulose synthesis protein complex from the plasma membrane. Our results highlight PUP-IT as a powerful proximity labeling system to identify protein interactions in plant cells.
    DOI:  https://doi.org/10.1038/s41467-025-56192-3
  28. J Med Chem. 2025 Jan 28.
    Development of Potent and Selective CK1α Molecular Glue Degraders.
    Qixiang Geng, Zixuan Jiang, Woong Sub Byun, Katherine A Donovan, Zhe Zhuang, Fen Jiang, Hannah M Jones, Hlib Razumkov, Michelle T Tang, Roman C Sarott, Eric S Fischer, Steven M Corsello, Stephen M Hinshaw, Nathanael S Gray.
      Molecular glue degraders (MGDs) are small molecules that facilitate proximity between a target protein and an E3 ubiquitin ligase, thereby inducing target protein degradation. Glutarimide-containing compounds are MGDs that bind cereblon (CRBN) and recruit neosubstrates. Through explorative synthesis of a glutarimide-based library, we discovered a series of molecules that induce casein kinase 1 alpha (CK1α) degradation. By scaffold hopping and rational modification of the chemical scaffold, we identified an imidazo[1,2-a]pyrimidine compound that induces potent and selective CK1α degradation. A structure-activity relationship study of the lead compound, QXG-6442, identified the chemical features that contribute to degradation potency and selectivity compared to other frequently observed neosubstrates. The glutarimide library screening and structure-activity relationship medicinal chemistry approach we employed is generally useful for developing new molecular glue degraders toward new targets of interest.
    DOI:  https://doi.org/10.1021/acs.jmedchem.4c02415
  29. Commun Biol. 2025 Jan 20. 8(1): 89
    Positively charged cytoplasmic residues in corin prevent signal peptidase cleavage and endoplasmic reticulum retention.
    Hui Li, Shijin Sun, Wenjun Guo, Lina Wang, Zihao Zhang, Yue Zhang, Ce Zhang, Meng Liu, Shengnan Zhang, Yayan Niu, Ningzheng Dong, Qingyu Wu.
      Positively charged residues are commonly located near the cytoplasm-membrane interface, which is known as the positive-inside rule in membrane topology. The mechanism underlying the function of these charged residues remains poorly understood. Herein, we studied the function of cytoplasmic residues in corin, a type II transmembrane serine protease in cardiovascular biology. We found that the positively charged residue at the cytoplasm-membrane interface of corin was not a primary determinant in membrane topology but probably served as a charge-repulsion mechanism in the endoplasmic reticulum (ER) to prevent interactions with proteins in the ER, including the signal peptidase. Substitution of the positively charged residue with a neutral or acidic residue resulted in corin secretion likely due to signal peptidase cleavage. In signal peptidase-deficient cells, the mutant corin proteins were not secreted but retained in the ER. Similar results were found in the low-density lipoprotein receptor and matriptase-2 that have positively charged residues at and near the cytoplasm-membrane interface. These results provide important insights into the role of the positively charged cytoplasmic residues in mammalian single-pass transmembrane proteins.
    DOI:  https://doi.org/10.1038/s42003-025-07545-7
  30. RSC Med Chem. 2025 Jan 01.
    Exploration of degrons and their ability to mediate targeted protein degradation.
    Timothy J Harris, Darci J Trader.
      Degrons are short amino acid sequences that can facilitate the degradation of protein substrates. They can be classified as either ubiquitin-dependent or -independent based on their interactions with the ubiquitin proteasome system (UPS). These amino acid sequences are often found in exposed regions of proteins serving as either a tethering point for an interaction with an E3 ligase or initiating signaling for the direct degradation of the protein. Recent advancements in the protein degradation field have shown the therapeutic potential of both classes of degrons through leveraging their degradative effects to engage specific protein targets. This review explores what targeted protein degradation applications degrons can be used in and how they have inspired new degrader technology to target a wide variety of protein substrates.
    DOI:  https://doi.org/10.1039/d4md00787e
  31. bioRxiv. 2025 Jan 18. pii: 2025.01.17.631970. [Epub ahead of print]
    Disease-associated Kv1.3 variants are energy compromised with impaired nascent chain folding.
    Aaron Sykes, Lannawill Caruth, Shefali Setia Verma, Toshinori Hoshi, Carol Deutsch.
      Human Kv1.3, encoded by KCNA3 , is expressed in neuronal and immune cells. Its impaired expression or function produces chronic inflammatory disease and autoimmune disorders, the severity of which correlates with Kv1.3 protein expression. The intersubunit recognition domain, T1, at the cytosolic N-terminus of Kv1.3, acquires secondary, tertiary, and quaternary structures during early biogenesis while the nascent protein is attached to the ribosome and/or the ER membrane. In this study, we ask whether native KCNA3 gene variants in T1 are associated with human disease and whether they manifest early-stage folding defects, energetic instabilities, and conformational distortion of subunits. We use three approaches: first, the unbiased "genome-first" approach to determine phenotype associations of specific KCNA3 rare variants. Second, we use biochemical assays to assess early-stage tertiary and quaternary folding and membrane association of these variants during early biogenesis. Third, we use all-atom molecular dynamics simulations of the T1 tetramer to assess structural macroscopic and energetic stability differences between wildtype (WT) Kv1.3 and a single-point variant, R114G. Measured folding probabilities and membrane associations are dramatically reduced in several of the native variants compared to WT. Simulations strikingly show that the R114G variant produces more energetically unstable and dynamic T1 domains, concomitant with tertiary unwinding and impaired formation of symmetrical tetramers. Our findings identify molecular mechanisms by which rare variants influence channel assembly, potentially contributing to diverse clinical phenotypes underlying human disease.
    DOI:  https://doi.org/10.1101/2025.01.17.631970
  32. Nat Commun. 2025 Jan 09. 16(1): 543
    Deacetylated SNAP47 recruits HOPS to facilitate autophagosome-lysosome fusion independent of STX17.
    Fenglei Jian, Shen Wang, Wenmin Tian, Yang Chen, Shixuan Wang, Yan Li, Cong Ma, Yueguang Rong.
      Autophagy, a conserved catabolic process implicated in a diverse array of human diseases, requires efficient fusion between autophagosomes and lysosomes to function effectively. Recently, SNAP47 has been identified as a key component of the dual-purpose SNARE complex mediating autophagosome-lysosome fusion in both bulk and selective autophagy. However, the spatiotemporal regulatory mechanisms of this SNARE complex remain unknown. In this study, we found that SNAP47 undergoes acetylation followed by deacetylation during bulk autophagy and mitophagy. The acetylation status of SNAP47 is regulated by the acetyltransferase CBP and the deacetylase HDAC2. Notably, the spatiotemporal regulatory dynamics of SNAP47 acetylation differ between bulk autophagy and mitophagy due to distinct regulation on the activity of acetyltransferase and deacetylase. Acetylated SNAP47 inhibits autophagosome-lysosome fusion by indirectly impeding SNARE complex assembly. Mechanistically, deacetylated SNAP47 recruits HOPS components to autophagic vacuoles independently of STX17 and STX17-SNAP47 interaction, while acetylated SNAP47 inhibits this recruitment, consequently leading to the failure of SNARE complex assembly. Taken together, our study uncovers a SNAP47 acetylation-dependent regulatory mechanism governing autophagosome-lysosome fusion by modulating the recruitment of HOPS to autophagic vacuoles without involving STX17, SNAP47-STX17 interaction and ternary SNARE complex formation.
    DOI:  https://doi.org/10.1038/s41467-025-55906-x
  33. Nat Commun. 2025 Jan 27. 16(1): 1068
    Splicing accuracy varies across human introns, tissues, age and disease.
    S García-Ruiz, D Zhang, E K Gustavsson, G Rocamora-Perez, M Grant-Peters, A Fairbrother-Browne, R H Reynolds, J W Brenton, A L Gil-Martínez, Z Chen, D C Rio, J A Botia, S Guelfi, L Collado-Torres, M Ryten.
      Alternative splicing impacts most multi-exonic human genes. Inaccuracies during this process may have an important role in ageing and disease. Here, we investigate splicing accuracy using RNA-sequencing data from >14k control samples and 40 human body sites, focusing on split reads partially mapping to known transcripts in annotation. We show that splicing inaccuracies occur at different rates across introns and tissues and are affected by the abundance of core components of the spliceosome assembly and its regulators. We find that age is positively correlated with a global decline in splicing fidelity, mostly affecting genes implicated in neurodegenerative diseases. We find support for the latter by observing a genome-wide increase in splicing inaccuracies in samples affected with Alzheimer's disease as compared to neurologically normal individuals. In this work, we provide an in-depth characterisation of splicing accuracy, with implications for our understanding of the role of inaccuracies in ageing and neurodegenerative disorders.
    DOI:  https://doi.org/10.1038/s41467-024-55607-x
  34. J Biomed Sci. 2025 Jan 22. 32(1): 11
    The endoplasmic reticulum degradation-enhancing α-mannosidase-like protein 3 attenuates the unfolded protein response and has pro-survival and pro-viral roles in hepatoma cells and hepatocellular carcinoma patients.
    Alina-Veronica Ghionescu, Mihaela Uta, Andrei Sorop, Catalin Lazar, Petruta R Flintoaca-Alexandru, Gabriela Chiritoiu, Livia Sima, Stefana-Maria Petrescu, Simona Olimpia Dima, Norica Branza-Nichita.
       BACKGROUND: Chronic hepatitis B virus (HBV) infection is a major risk for development of hepatocellular carcinoma (HCC), a frequent malignancy with a poor survival rate. HBV infection results in significant endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) signaling, a contributing factor to carcinogenesis. As part of the UPR, the ER-associated degradation (ERAD) pathway is responsible for removing the burden of misfolded secretory proteins, to re-establish cellular homeostasis. Emerging evidence indicates consistent upregulation of ERAD factors, including members of the ER degradation-enhancing alpha-mannosidase-like protein (EDEM) family in infection and various tumor types. However, the significance of this gene expression pattern in HBV-driven pathology is just beginning to be deciphered.
    METHODS: In this study we quantified the expression of the ERAD factor EDEM3, in a cohort of HCC patients with and without HBV infection, and validated our results by analysis of publically available transcriptomic and microarray data sets. We performed mechanistic studies in HepaRG cells with modulated EDEM3 expression to address UPR, ERAD, autophagy and apoptosis signaling, and their consequences on HBV infection.
    RESULTS: Our work revealed significantly elevated EDEM3 expression in HCC tissues irrespective of HBV infection, while the highest levels were observed in tissues from HBV-infected patients. Investigation of published transcriptomic data sets confirmed EDEM3 upregulation in independent HCC patient cohorts, associated with tumor progression, poor survival prognosis and resistance to therapy. EDEM3-overexpressing hepatic cells exhibited attenuated UPR and activated secretory autophagy, which promoted HBV production. Conversely, cell depletion of EDEM3 resulted in significant ER stress inducing pro-apoptotic mechanisms and cell death.
    CONCLUSIONS: We provide evidence of major implications of the ERAD pathway in HBV infection and HCC development and progression. Our results suggest that ERAD activation in HBV-infected cells is a protective mechanism against prolonged ER stress, potentially contributing to establishment of chronic HBV infection and promoting tumorigenesis. Developing specific inhibitors for ERAD factors may be an attractive approach to improve efficiency of current antiviral and anticancer therapies.
    Keywords:  Autophagy; Cancer; ER degradation; HBV infection
    DOI:  https://doi.org/10.1186/s12929-024-01103-9
  35. Platelets. 2025 Dec;36(1): 2455743
    Extracellular thiol isomerase ERp5 regulates integrin αIIbβ3 activation by inhibition of fibrinogen binding.
    Kaifei Sun, Yaqiong Zhang, Aizhen Yang, Yuxin Zhang, Zhenzhen Zhao, Xiaofeng Yan, Yi Lu, Yue Han, Depei Wu, Freda Passam, Jingyu Zhang, Yi Wu.
      Recent studies have shown that anti-ERp5 antibodies inhibit platelet activation and thrombus formation; Moreover, ERp5-deficient platelets exhibit enhanced platelet reactivity via regulation of endoplasmic reticulum (ER) stress. In this study, we used a new ERp5-knockout mouse model as well as recombinant ERp5 (rERp5) protein, to examine the role of ERp5 in platelet function and thrombosis. Although platelet-specific ERp5-deficient mice had decreased platelet count, the mice had shortened tail-bleeding times and enhanced platelet accumulation in FeCl3-induced mesenteric artery injury, compared with wild-type mice. Using platelet-specific ERp5-deficient mice, we found that ERp5 deficiency increased platelet aggregation, granule secretion, and integrin αIIbβ3 activation. Wild-type recombinant ERp5 protein (rERp5-wt) and inactive mutant ERp5 protein (rERp5-mut) both inhibited human platelet aggregation and the binding of fibrinogen to human platelets, indicating that ERp5 protein interferes with the interaction between integrin αIIbβ3 and its ligand fibrinogen, and its enzymatic activity is not required for this process. Consistently, wild-type mice injected with rERp5-wt or rERp5-mut protein had prolonged tail-bleeding times. Our results provide important evidence that platelet ERp5 negatively regulates platelet activation and thrombus formation, via steric hindrance interfering with integrin αIIbβ3 ligation.
    Keywords:  ERp5; integrin αIIbβ3; platelets; redox regulation; thrombosis
    DOI:  https://doi.org/10.1080/09537104.2025.2455743
  36. J Clin Invest. 2025 Jan 27. pii: e176093. [Epub ahead of print]
    Small molecule modulators of B56-PP2A restore 4E-BP function to suppress eIF4E-dependent translation in cancer cells.
    Michelle A Lum, Kayla A Jonas, Shreya Parmar, Adrian R Black, Caitlin M O'Connor, Stephanie Dobersch, Naomi Yamamoto, Tess M Robertson, Aidan Schutter, Miranda Giambi, Rita A Avelar, Analisa DiFeo, Nicholas T Woods, Sita Kugel, Goutham Narla, Jennifer D Black.
      Dysregulated eIF4E-dependent translation is a central driver of tumorigenesis and therapy resistance. eIF4E binding proteins (4E-BP1/2/3) are major negative regulators of eIF4E-dependent translation that are inactivated in tumors through inhibitory phosphorylation or downregulation. Previous studies have linked PP2A phosphatase(s) to activation of 4E-BP1. Here, we leveraged biased small molecule activators of PP2A (SMAPs) to explore the role of B56-PP2A(s) in 4E-BP regulation and the potential of B56-PP2A activation for restoring translational control in tumors. SMAP treatment promoted PP2A-dependent hypophosphorylation of 4E-BP1/2, supporting a role for B56-PP2As (e.g., B56α-PP2A) as 4E-BP phosphatases. Unexpectedly, SMAPs induced transcriptional upregulation of 4E-BP1 through a B56 PP2A→TFE3/TFEB→ATF4 axis. Cap-binding and co-immunoprecipitation assays showed that B56-PP2A(s) activation blocks assembly of the eIF4F translation initiation complex, and cap-dependent translation assays confirmed the translation inhibitory effects of SMAPs. Thus, B56-PP2A(s) orchestrate a translation repressive program involving transcriptional induction and activation of 4E-BP1. Notably, SMAPs promoted 4E-BP1-dependent apoptosis in tumor cells and potentiated 4E-BP1 function in the presence of ERK or mTOR inhibitors, agents that rely on inhibition of eIF4E-dependent translation for antitumor activity. These findings, combined with the ability of SMAPs to regulate 4E-BP1 in vivo, highlight the potential of PP2A activators for cancer therapy and overcoming therapy resistance.
    Keywords:  Cell biology; Oncology; Phosphoprotein phosphatases; Signal transduction; Translation
    DOI:  https://doi.org/10.1172/JCI176093
  37. bioRxiv. 2024 Dec 17. pii: 2024.12.14.628475. [Epub ahead of print]
    Discovery of an on-pathway protein folding intermediate illuminates the kinetic competition between folding and misfolding.
    Qing Luan, Patricia L Clark.
      Our current understanding of protein folding is based predominantly on studies of small (<150 aa) proteins that refold reversibly from a chemically denatured state. As protein length increases, the competition between off-pathway misfolding and on-pathway folding likewise increases, creating a more complex energy landscape. Little is known about how intermediates populated during the folding of larger proteins affect navigation of this more complex landscape. Previously, we reported extremely slow folding rates for the 539 aa β-helical passenger domain of pertactin (P.69T), including conditions that favor the formation of a kinetically trapped, off-pathway partially folded state (PFS). The existence of an on-pathway intermediate for P.69T folding was speculated but its characterization remained elusive. In this work, we exploited the extremely slow kinetics of PFS unfolding to develop a double-jump "denaturant challenge" assay. With this assay, we identified a transient unfolding intermediate, PFS*, that adopts a similar structure to PFS, including C-terminal folded structure and a disordered N-terminus, yet unfolds much more quickly than PFS. Additional experiments revealed that PFS* also functions as an on-pathway intermediate for P.69T folding. Collectively, these results support a two-step, C-to-N-terminal model for P.69T folding: folding initiates in the C-terminus with the rate-limiting formation of the transient on-pathway PFS* intermediate, which sits at the junction of the kinetic competition between folding and misfolding. Notably, processive folding from C-to-N-terminus also occurs during C-to-N-terminal translocation of P.69T across the bacterial outer membrane. These results illuminate the crucial role of kinetics when navigating a complex energy landscape for protein folding.
    DOI:  https://doi.org/10.1101/2024.12.14.628475
  38. Sci Transl Med. 2025 Jan 29. 17(783): eadp5173
    Undocking of an extensive ciliary network induces proteostasis and cell fate switching resulting in severe primary ciliary dyskinesia.
    Steven L Brody, Jiehong Pan, Tao Huang, Jian Xu, Huihui Xu, Jeffrey R Koenitizer, Steven K Brennan, Rashmi Nanjundappa, Thomas G Saba, Nisreen Rumman, Andrew Berical, Finn J Hawkins, Xiangli Wang, Rui Zhang, Moe R Mahjoub, Amjad Horani, Susan K Dutcher.
      Primary ciliary dyskinesia is a rare monogenic syndrome that is associated with chronic respiratory disease, infertility, and laterality defects. Although more than 50 genes causative of primary ciliary dyskinesia have been identified, variants in the genes encoding coiled-coil domain-containing 39 (CCDC39) and CCDC40 in particular cause severe disease that is not explained by loss of ciliary motility alone. Here, we sought to understand the consequences of these variants on cellular functions beyond impaired motility. We used human cells with pathogenic variants in CCDC39 and CCDC40, Chlamydomonas reinhardtii genetics, cryo-electron microscopy, and proteomics to define perturbations in ciliary assembly and cilia stability, as well as multiple motility-independent pathways. Analysis of proteomics of cilia from patient cells identified that the absence of the axonemal CCDC39/CCDC40 heterodimer resulted in the loss of a network of more than 90 ciliary structural proteins, including 14 that were defined as ciliary address recognition proteins, which provide docking for the missing structures. The absence of the network impaired microtubule architecture, activated cell quality control pathways, switched multiciliated cell fate to mucus-producing cells and resulted in a defective periciliary barrier. In CCDC39 variant cells, these phenotypes were reversed through expression of a normal CCDC39 transgene. These findings indicate that the CCDC39/CCDC40 heterodimer functions as a scaffold to support the assembly of an extensive network of ciliary proteins, whose loss results in both motility-dependent and motility-independent phenotypes that may explain the severity of disease. Gene therapy might be a potential treatment option to be explored in future studies.
    DOI:  https://doi.org/10.1126/scitranslmed.adp5173
  39. bioRxiv. 2025 Jan 15. pii: 2025.01.14.633048. [Epub ahead of print]
    Overcoming Fluorescence Loss in mEOS-based AAA+ Unfoldase Reporters Through Covalent Linkage.
    Isabella R Walter, Baylee A Smith, Dominic Castanzo, Matthew L Wohlever.
      Recent work has demonstrated that the soluble photoconvertable fluorescent protein mEOS can be a reporter for AAA+ (ATPases Associated with diverse cellular Activities) unfoldase activity. Given that many AAA+ proteins process membrane proteins, we sought to adapt mEOS for use with membrane protein substrates. However, direct genetic fusion of mEOS to a membrane protein completely abolished fluorescence, severely limiting the utility of mEOS for studying AAA+ proteins. To circumvent this challenge, we separately purified mEOS and a AAA+ degron, covalently linked them via Sortase, and photoconverted the linked construct. This innovative approach preserves fluorescence and enables functional analysis, offering a broadly applicable platform for the study of membrane associated AAA+ proteins.
    DOI:  https://doi.org/10.1101/2025.01.14.633048
  40. J Am Chem Soc. 2025 Jan 28.
    Modulation of Protein-Protein Interactions with Molecular Glues in a Synthetic Condensate Platform.
    Thijs W van Veldhuisen, Renske M J Dijkstra, Auke A Koops, Peter J Cossar, Jan C M van Hest, Luc Brunsveld.
      Misregulation of protein-protein interactions (PPIs) underlies many diseases; hence, molecules that stabilize PPIs, known as molecular glues, are promising drug candidates. Identification of novel molecular glues is highly challenging among others because classical biochemical assays in dilute aqueous conditions have limitations for evaluating weak PPIs and their stabilization by molecular glues. This hampers the systematic discovery and evaluation of molecular glues. Here, we present a synthetic condensate platform for the study of PPIs and molecular glues in a crowded macromolecular environment that more closely resembles the dense cellular milieu. With this platform, weak PPIs can be enhanced by sequestration. The condensates, based on amylose derivatives, recruit the hub protein 14-3-3 via affinity-based uptake, which results in high local protein concentrations ideal for the efficient screening of molecular glues. Clients of 14-3-3 are sequestered in the condensates based on their enhanced affinity upon treatment with molecular glues. Fine control over the condensate environment is illustrated by modulating the reactivity of dynamic covalent molecular glues by the adjustment of pH and the redox environment. General applicability of the system for screening of molecular glues is highlighted by using the nuclear receptor PPARγ, which recruits coregulators via an allosteric PPI stabilization mechanism. The condensate environment thus provides a unique dense molecular environment to enhance weak PPIs and enable subsequent evaluation of small-molecule stabilization in a molecular setting chemically en route to the cellular interior.
    DOI:  https://doi.org/10.1021/jacs.4c17567
  41. Nat Commun. 2025 Jan 26. 16(1): 1047
    Genome-wide profiling of tRNA modifications by Induro-tRNAseq reveals coordinated changes.
    Yuko Nakano, Howard Gamper, Henri McGuigan, Sunita Maharjan, Jiatong Li, Zhiyi Sun, Erbay Yigit, Sebastian Grünberg, Keerthana Krishnan, Nan-Sheng Li, Joseph A Piccirilli, Ralph Kleiner, Nicole Nichols, Brian D Gregory, Ya-Ming Hou.
      While all native tRNAs undergo extensive post-transcriptional modifications as a mechanism to regulate gene expression, mapping these modifications remains challenging. The critical barrier is the difficulty of readthrough of modifications by reverse transcriptases (RTs). Here we use Induro-a new group-II intron-encoded RT-to map and quantify genome-wide tRNA modifications in Induro-tRNAseq. We show that Induro progressively increases readthrough over time by selectively overcoming RT stops without altering the misincorporation frequency. In a parallel analysis of Induro vs. a related RT, we provide comparative datasets to facilitate the prediction of each modification. We assess tRNA modifications across five human cell lines and three mouse tissues and show that, while the landscape of modifications is highly variable throughout the tRNA sequence framework, it is stabilized for modifications that are required for reading of the genetic code. The coordinated changes have fundamental importance for development of tRNA modifications in protein homeostasis.
    DOI:  https://doi.org/10.1038/s41467-025-56348-1
  42. Nat Rev Genet. 2025 Jan 27.
    Cytoplasmic mRNA decay and quality control machineries in eukaryotes.
    Megan E Dowdle, Jens Lykke-Andersen.
      mRNA degradation pathways have key regulatory roles in gene expression. The intrinsic stability of mRNAs in the cytoplasm of eukaryotic cells varies widely in a gene- and isoform-dependent manner and can be regulated by cellular cues, such as kinase signalling, to control mRNA levels and spatiotemporal dynamics of gene expression. Moreover, specialized quality control pathways exist to rid cells of non-functional mRNAs produced by errors in mRNA processing or mRNA damage that negatively impact translation. Recent advances in structural, single-molecule and genome-wide methods have provided new insights into the central machineries that carry out mRNA turnover, the mechanisms by which mRNAs are targeted for degradation and the general principles that govern mRNA stability at a global level. This improved understanding of mRNA degradation in the cytoplasm of eukaryotic cells is finding practical applications in the design of therapeutic mRNAs.
    DOI:  https://doi.org/10.1038/s41576-024-00810-1
  43. bioRxiv. 2025 Jan 13. pii: 2025.01.08.632046. [Epub ahead of print]
    The MAP kinase scaffold MORG1 shapes cell death in unresolved ER stress in Arabidopsis.
    Joo Yong Kim, Dae Kwan Ko, Federica Brandizzi.
      Governed by the unfolded protein response (UPR), the ability to counteract endoplasmic reticulum (ER) stress is critical for maintaining cellular homeostasis under adverse conditions. Unresolved ER stress leads to cell death through mechanisms that are yet not completely known. To identify key UPR effectors involved in unresolved ER stress, we performed an ethyl methanesulfonate (EMS) suppressor screen on the Arabidopsis bzip28/60 mutant, which is impaired in activating cytoprotective UPR pathways. This screen identified MAP kinase organizer 1 (MORG1), a conserved MAP kinase scaffold protein, as a previously uncharacterized regulator of ER stress tolerance. The coffin1 mutant, which carries a mutation in MORG1 , exhibited enhanced resilience to ER stress by partially restoring UPR gene expression and promoting growth under stress conditions. Mechanistically, we found that MORG1 modulates MPK6-dependent phosphorylation of the stress-responsive transcription factor WRKY8. Loss of WRKY8 phenocopied the coffin1 mutant, highlighting WRKY8's role as a key repressor in the UPR. Together, these findings reveal a MORG1-MPK6-WRKY8 signaling axis that fine-tunes UPR gene expression, providing new insights into ER stress regulation and strategies for improving stress tolerance in multicellular eukaryotes.
    DOI:  https://doi.org/10.1101/2025.01.08.632046
  44. Nat Commun. 2025 Jan 30. 16(1): 1176
    SEC-MX: an approach to systematically study the interplay between protein assembly states and phosphorylation.
    Ella Doron-Mandel, Benjamin J Bokor, Yanzhe Ma, Lena A Street, Lauren C Tang, Ahmed A Abdou, Neel H Shah, George Rosenberger, Marko Jovanovic.
      A protein's molecular interactions and post-translational modifications (PTMs), such as phosphorylation, can be co-dependent and reciprocally co-regulate each other. Although this interplay is central for many biological processes, a systematic method to simultaneously study assembly states and PTMs from the same sample is critically missing. Here, we introduce SEC-MX (Size Exclusion Chromatography fractions MultipleXed), a global quantitative method combining Size Exclusion Chromatography and PTM-enrichment for simultaneous characterization of PTMs and assembly states. SEC-MX enhances throughput, allows phosphopeptide enrichment, and facilitates quantitative differential comparisons between biological conditions. Conducting SEC-MX on HEK293 and HCT116 cells, we generate a proof-of-concept dataset, mapping thousands of phosphopeptides and their assembly states. Our analysis reveals intricate relationships between phosphorylation events and assembly states and generates testable hypotheses for follow-up studies. Overall, we establish SEC-MX as a valuable tool for exploring protein functions and regulation beyond abundance changes.
    DOI:  https://doi.org/10.1038/s41467-025-56303-0
  45. Nat Commun. 2025 Jan 24. 16(1): 975
    A multiscale functional map of somatic mutations in cancer integrating protein structure and network topology.
    Yingying Zhang, Alden K Leung, Jin Joo Kang, Yu Sun, Guanxi Wu, Le Li, Jiayang Sun, Lily Cheng, Tian Qiu, Junke Zhang, Shayne D Wierbowski, Shagun Gupta, James G Booth, Haiyuan Yu.
      A major goal of cancer biology is to understand the mechanisms driven by somatically acquired mutations. Two distinct methodologies-one analyzing mutation clustering within protein sequences and 3D structures, the other leveraging protein-protein interaction network topology-offer complementary strengths. We present NetFlow3D, a unified, end-to-end 3D structurally-informed protein interaction network propagation framework that maps the multiscale mechanistic effects of mutations. Built upon the Human Protein Structurome, which incorporates the 3D structures of every protein and the binding interfaces of all known protein interactions, NetFlow3D integrates atomic, residue, protein and network-level information: It clusters mutations on 3D protein structures to identify driver mutations and propagates their impacts anisotropically across the protein interaction network, guided by the involved interaction interfaces, to reveal systems-level impacts. Applied to 33 cancer types, NetFlow3D identifies 2 times more 3D clusters and incorporates 8 times more proteins in significantly interconnected network modules compared to traditional methods.
    DOI:  https://doi.org/10.1038/s41467-024-54176-3
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