bims-proteo 2025-07-27 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2025–07–27
forty-two papers selected by
Eric Chevet, INSERM

Silencing stress: Structural insights into ISR termination by the SIFI ubiquitin ligase.
Arrest Peptide Profiling resolves co-translational folding pathways and chaperone interactions in vivo.
PERK and IRE1α promote exosome secretion via blocking lysosomal degradation of multiple vesicular body.
Unveiling the hidden interactome of CRBN molecular glues.
Ubiquitin chain variability directs substrates of the Tul1 ubiquitin ligase complex to different degradation pathways.
19S proteasome loss regulates mitotic spindle assembly through a ubiquitin-independent degradation mechanism.
TM9SF3 is a mammalian Golgiphagy receptor that safeguards Golgi integrity and glycosylation fidelity.
The dual ubiquitin binding mode of SPRTN secures rapid spatiotemporal proteolysis of DNA-protein crosslinks.
Generating Surprisingly Powerful Pharmacology from Chemically Induced Protein Interactions.
Stress-induced translocation of the endoplasmic reticulum chaperone GRP78/BiP and its impact on human disease and therapy.
Structural host-virus interactome profiling of intact infected cells.
SPIDR enables multiplexed mapping of RNA-protein interactions and uncovers a mechanism for selective translational suppression upon cell stress.
Secretory autophagy mediates lysosomal and autophagic degradation for α-synuclein proteostasis.
The ubiquitin ligase Triad1 influences myeloid leukemogenesis by regulating the integrated stress response.
Endosome-ER contact sites in phagophore formation.
m6A-induced ribosome stalling and collision trigger mRNA decay.
Endoplasmic Reticulum Stress Amplifies Cytokine Responses in Astrocytes via a PERK/eIF2α/JAK1 Signaling Axis.
Mechanistic insights into the iron-sulfur cluster-dependent interaction of the autophagy receptor NCOA4 with the E3 ligase HERC2.
Design of high-specificity binders for peptide-MHC-I complexes.
Protocol to distinguish pre-mRNA from mRNA in RNA-protein interaction studies.
The rapidly expanding role of LC3-interacting regions in autophagy.
How does protein aggregate structure affect mechanisms of disaggregation?
Allosteric activation of the SPRTN protease by ubiquitin maintains genome stability.
High-resolution spatial mapping of cell state and lineage dynamics in vivo with PEtracer.
Protein Kinase C promotes peroxisome biogenesis and peroxisome-endoplasmic reticulum interaction.
LONP1 regulation of mitochondrial protein folding provides insight into beta cell failure in type 2 diabetes.
ATG8ylation-mediated tonoplast invagination mitigates vacuole damage.
A UFD1 variant encoding a microprotein modulates UFD1f and IPMK ubiquitination to play pivotal roles in anti-stress responses.
Tks5 interactome reveals endoplasmic-reticulum-associated translation machinery in invadosomes.
Parp7 generates an ADP-ribosyl degron that controls negative feedback of androgen signaling.
Spatially constrained disulfide bond shuffling delays insulin aggregation and enhances neurotoxicity.
Npl3 is required for efficient autophagosome-vacuole fusion.
eIF4A controls translation of estrogen receptor alpha and is a therapeutic target in advanced breast cancer.
UPR-induced intracellular C5aR1 promotes adaptation to the hypoxic tumour microenvironment.
RPL22 links ribosome biogenesis, RNA splicing, and sensitivity to RNA polymerase I inhibition.
PEAK1 maintains tight junctions in intestinal epithelial cells and resists colitis by inhibiting autophagy-mediated ZO-1 degradation.
TRAF2 binds to TIFA via a novel motif and contributes to its autophagic degradation.
Engineering a cell-based orthogonal ubiquitin transfer cascade for profiling the substrates of RBR E3 Parkin.
The E3 ubiquitin ligase DTX3L and the deubiquitinase USP28 fine-tune DNA repair through mutual regulation of their protein levels.
FMO5 plays a sex-specific role in goblet cell maturation and mucus barrier formation.
A chaperonin complex regulates organelle proteostasis in malaria parasites.
Natural variation of an E3 ubiquitin ligase encoding gene Chalk9 regulates grain chalkiness in rice.

Cell Chem Biol. 2025 Jul 17. pii: S2451-9456(25)00202-8. [Epub ahead of print]32(7): 905-907

Silencing stress: Structural insights into ISR termination by the SIFI ubiquitin ligase.

Zeba Rizvi, Gabriel C Lander.

The E3 ligase complex SIFI silences the integrated stress response (ISR) by targeting stress-induced proteins for degradation. In the May 6th issue of Nature, Yang et al.1 revealed how this megadalton complex recognizes diverse substrates and coordinates ubiquitin chain formation. Their insights into the ISR shutdown mechanism suggest new avenues for modulating stress responses in neurodegenerative disease.

DOI: https://doi.org/10.1016/j.chembiol.2025.06.007
Nat Commun. 2025 Jul 24. 16(1): 6833

Arrest Peptide Profiling resolves co-translational folding pathways and chaperone interactions in vivo.

Xiuqi Chen, Vincent J Hilser, Christian M Kaiser.

Cytosolic proteins begin to fold co-translationally as soon as they emerge from the ribosome during translation. These early co-translational steps are crucial for overall folding and are guided by an intricate network of interactions with molecular chaperones. Because cellular co-translational folding is challenging to detect, its timing and progression remain largely elusive. To quantitatively define co-translational folding in live cells, we developed a high-throughput method that we term "Arrest Peptide Profiling" (AP Profiling). Combining AP Profiling with single-molecule experiments, we delineate co-translational folding for a set of GTPase domains with similar structures, defining how topology shapes folding pathways. Genetic ablation of nascent chain-binding chaperones results in discrete and localized folding changes, highlighting how functional redundancy among chaperones is achieved by distinct engagement with the nascent protein. Our work provides a window into cellular folding pathways of structurally intricate proteins and paves the way for systematic studies of nascent protein folding at exceptional resolution and throughput.

DOI: https://doi.org/10.1038/s41467-025-61398-6
J Biol Chem. 2025 Jul 21. pii: S0021-9258(25)02354-3. [Epub ahead of print] 110504

PERK and IRE1α promote exosome secretion via blocking lysosomal degradation of multiple vesicular body.

Shixin Zhou, Zihan Zhou, Si Chen, Ming Wang, Likun Wang.

  The unfolded protein response (UPR) initiated under endoplasmic reticulum (ER) stress can not only maintain the ER homeostasis, but also modulate the secretion of proteins and lipids that transmit ER stress signals among cells. Exosomes are multivesicular body (MVB)-derived extracellular vesicles, constituting the unconventional protein secretion pathway. Whether and how the secretion of exosomes is regulated by the UPR remains largely unknown. Here, we reported that ER stress induces exosome secretion in an UPR-dependent way. Activation of PERK and IRE1α, two of the UPR branches, represses the acidification and catabolic activity of lysosomes. This blocked MVB-lysosome fusion, re-directing MVBs from lysosomal degradation to plasma membrane fusion, resulting in exosome release. Calcium-mediated activation of PERK, in the absence of ER stress, is sufficient to suppress lysosomal degradation and augment exosome secretion, partly through its downstream factor ATF4. Our study revealed a function of PERK and IRE1α in modulating lysosome activity and dictating the fate of MVBs, facilitating cell-cell communication via exosomes.

Keywords:  IRE1α; PERK; extracellular vesicle; lysosome; unfolded protein response

DOI:  https://doi.org/10.1016/j.jbc.2025.110504
Nat Commun. 2025 Jul 24. 16(1): 6831

Unveiling the hidden interactome of CRBN molecular glues.

Kheewoong Baek, Rebecca J Metivier, Shourya S Roy Burman, Jonathan W Bushman, Hojong Yoon, Ryan J Lumpkin, Julia K Ryan, Dinah M Abeja, Megha Lakshminarayan, Hong Yue, Samuel Ojeda, Yuan Xiong, Jianwei Che, Alyssa L Verano, Anna M Schmoker, Nathanael S Gray, Katherine A Donovan, Eric S Fischer.

Induced proximity by molecular glues refers to strategies that leverage the recruitment of proteins to facilitate their modification, regulation or degradation. As prospective design of molecular glues remains challenging, unbiased discovery methods are necessary to discover new chemical targets. Here we establish a high throughput affinity proteomics workflow leveraging E3 ligase activity-impaired CRBN-DDB1ΔB in cell lysates for the unbiased identification of molecular glue targets. By mapping the interaction landscape of CRBN-binding molecular glues, we unveil 298 protein targets and demonstrate the utility of enrichment methods for identifying targets overlooked by established methods. We use a computational workflow to estimate target confidence and perform biochemical and structural validation of uncharacterized neo-substrates. We further identify a lead compound for the previously untargeted non-zinc finger PPIL4 through a biochemical screen. Our study provides a comprehensive inventory of targets chemically recruited to CRBN and delivers a robust and scalable workflow for identifying drug-induced protein interactions in cell lysates.

DOI: https://doi.org/10.1038/s41467-025-62099-w
J Cell Biol. 2025 Sep 01. pii: e202312133. [Epub ahead of print]224(9):

Ubiquitin chain variability directs substrates of the Tul1 ubiquitin ligase complex to different degradation pathways.

Devon D Dennison, Ryan D Baldridge.

Cellular protein quality control consists of multiple, networked systems that survey and maintain a healthy eukaryotic proteome. In Saccharomyces cerevisiae, the transmembrane ubiquitin ligase 1 (Tul1) complex is an integral membrane protein quality control system that functions within the Golgi-endosomal system. Golgi-localized Tul1 complexes target proteins for degradation by either the cytosolic proteasome or the vacuole. To understand how the complex directs substrates for degradation, we developed high-throughput functional assays for deep mutational scanning analysis of the Tul1 ubiquitin ligase. We identified mutations that disrupted Tul1 interactions with the complex or altered complex specificity by disrupting substrate polyubiquitination. This work demonstrates that Tul1 plays an important role in directing substrate degradation by influencing polyubiquitin chain length and provides tools for future study of the complex.

DOI: https://doi.org/10.1083/jcb.202312133
Cell Rep. 2025 Jul 23. pii: S2211-1247(25)00812-5. [Epub ahead of print]44(8): 116041

19S proteasome loss regulates mitotic spindle assembly through a ubiquitin-independent degradation mechanism.

Océane Marescal, Iain M Cheeseman.

  During regulated protein degradation, the 26S proteasome recognizes ubiquitinated substrates through its 19S particle and then degrades them in its 20S enzymatic core. Despite this close interdependency between proteasome subunits, we demonstrate that knockouts from different proteasome subcomplexes result in distinct cellular phenotypes. In particular, depletion of 19S PSMD lid proteins, but not that of other proteasome subunits, prevents bipolar spindle assembly during mitosis. Despite decreased ubiquitin-mediated protein degradation in PSMD knockouts, we find that the monopolar spindle phenotype is instead caused by the aberrant degradation of the kinesin motor protein KIF11. We show that KIF11 degradation occurs through the 20S proteasome in a ubiquitin-independent manner upon loss of 19S proteins and that the resulting alterations in spindle forces lead to the unique monopolar phenotype. Thus, the presence of the 19S particle ensures proper spindle formation by restraining ubiquitin-independent degradation.

Keywords:  CP: Cell biology; degradation; kinesin; mitosis; proteasome; spindle; ubiquitin

DOI:  https://doi.org/10.1016/j.celrep.2025.116041
Autophagy. 2025 Jul 25.

TM9SF3 is a mammalian Golgiphagy receptor that safeguards Golgi integrity and glycosylation fidelity.

Jiejie Yang, Yixian Cui.

  Selective autophagy of the Golgi apparatus, or Golgiphagy, depends on receptor proteins that recognize and deliver fragmented Golgi membranes into phagophores for lysosomal degradation. We recently identified TM9SF3, a Golgi-resident transmembrane protein, as a receptor mediating this process under nutrient stress and various Golgi stress conditions. TM9SF3 binds to all six mammalian Atg8 (ATG8) proteins via multiple N-terminal LC3-interacting regions (LIRs). Knockout of TM9SF3 inhibits nutrient stress-induced Golgi fragmentation, reduces autophagic delivery of Golgi components, and hinders Golgi protein degradation. In addition to nutrient stress, TM9SF3 is essential for Golgiphagy induced by monensin, brefeldin A, and glycosylation perturbations. Knockout or LIR mutation of TM9SF3 disrupts protein glycosylation, whereas its overexpression promotes the degradation of aberrantly glycosylated proteins. Notably, TM9SF3 promotes breast cancer cell proliferation, and its high expression correlates with poor patient prognosis. Our findings establish TM9SF3 as a Golgiphagy receptor essential for maintaining Golgi integrity and glycosylation fidelity, and implicate its role in supporting cancer progression.

Keywords:  Atg8 (ATG8); TM9SF3; breast cancer; golgiphagy; protein glycosylation; receptor

DOI:  https://doi.org/10.1080/15548627.2025.2539928
Nucleic Acids Res. 2025 Jul 08. pii: gkaf638. [Epub ahead of print]53(13):

The dual ubiquitin binding mode of SPRTN secures rapid spatiotemporal proteolysis of DNA-protein crosslinks.

Wei Song, Yichen Zhao, Annamaria Ruggiano, Christina Redfield, Joseph A Newman, Xiaosheng Zhu, Marta García-Flores, Abimael Cruz-Migoni, Rebecca Roddan, Anna Pérez-Ràfols, Peter J McHugh, Paul R Elliott, Kristijan Ramadan.

DNA-protein crosslinks (DPCs) are endogenous and chemotherapy-induced genotoxic DNA lesions and, if not repaired, lead to embryonic lethality, neurodegeneration, premature ageing, and cancer. DPCs are heavily polyubiquitinated, and the SPRTN protease and 26S proteasome emerged as two central enzymes for DPC proteolysis. The proteasome recognizes its substrates by their ubiquitination status. How SPRTN protease, an essential enzyme for DPC proteolysis, achieves specificity for DPCs is still not entirely clear. We found that the N-terminal SPRTN catalytic region (SprT) possesses a ubiquitin-binding domain that we named the Ubiquitin Interface of SprT Domain (USD). Using multiple biochemical, biophysical, and structural approaches, we reveal that USD binds ubiquitin chains in an avidity manner. SPRTN binding to ubiquitin chains via USD leads to ∼67-fold higher activation of SPRTN proteolysis towards polyubiquitinated DPCs than the unmodified DPCs. In contrast, the constitutive components of the replisome during unperturbed or translesional DNA synthesis, namely proliferating cell nuclear antigen (PCNA) or monoUb-PCNA, respectively, were poorly degraded, if at all, by SPRTN. This study reveals that the poly-ubiquitination of DPCs serves as the key signal for SPRTN's rapid proteolysis and determines its substrate specificity towards DPCs, rather than the replisome.

DOI: https://doi.org/10.1093/nar/gkaf638
Acc Chem Res. 2025 Jul 24.

Generating Surprisingly Powerful Pharmacology from Chemically Induced Protein Interactions.

Stephen M Hinshaw, Steven M Banik, Nathanael S Gray.

ConspectusSmall molecules that induce proximity between proteins have transformed our ability to manipulate and study cellular processes. Beyond this, proximity-inducing small molecules and biologics are now a clinical reality with increasing reach over different targets and disease indications, benefiting from our rapidly expanding abilities to exploit diverse biochemical mechanisms and being powered by emerging design principles. Targeted protein degradation has become a predominant proximity-dependent therapeutic mechanism. We contend that there are many yet-unexplored pharmacologically useful mechanisms that can be triggered by chemically induced protein interactions. We discuss the general principles of proximity pharmacology and highlight two areas we believe are ripe for innovation.

DOI: https://doi.org/10.1021/acs.accounts.5c00225
Proc Natl Acad Sci U S A. 2025 Jul 29. 122(30): e2412246122

Stress-induced translocation of the endoplasmic reticulum chaperone GRP78/BiP and its impact on human disease and therapy.

Amy S Lee.

  Since their discoveries in the 1960s as a family of proteins produced by cells in response to stress, molecular chaperones are increasingly recognized as major regulators of cellular homeostasis in health and disease. Among the heat shock protein 70 family, the 78-kDa glucose-regulated protein (GRP78), also referred to as BiP and encoded by the HSPA5 gene, contains a signal peptide targeting it into the endoplasmic reticulum (ER). Through its interaction with the transmembrane ER stress sensors, GRP78 acts as a master regulator of the Unfolded Protein Response (UPR) which allows cells to adapt to stress observed in many human diseases. The discovery that ER stress not only upregulates GRP78 to cope with ER protein quality control but also actively promotes its relocation to other cellular compartments where they vastly expand its functional repertoire beyond the ER represents a paradigm shift. This Perspective describes the origin and linkage of GRP78 to the UPR and the mechanisms whereby ER stress actively promotes export of GRP78 from the ER, as exemplified by its translocation to the cell surface where it acts as a multifaceted receptor and a conduit for drug and viral entry, as well as its translocation into the nucleus, where it assumes the surprising role of a transcriptional regulator whereby reprogramming the cell's transcriptome. Furthermore, this Perspective addresses how these and other atypical localizations of GRP78 impact human disease, with emphasis on cancer and COVID-19, and the exciting prospect that drugs targeting GRP78 could dually suppress tumorigenesis and viral infections.

Keywords:  ER stress; GRP78/BiP; cancer; chaperone; translocation

DOI:  https://doi.org/10.1073/pnas.2412246122
Nat Commun. 2025 Jul 21. 16(1): 6713

Structural host-virus interactome profiling of intact infected cells.

Boris Bogdanow, Lars Mühlberg, Iris Gruska, Barbara Vetter, Julia Ruta, Arne Elofsson, Lüder Wiebusch, Fan Liu.

Virus-host protein-protein interactions (PPIs) are fundamental to viral infections, yet high-resolution identification of their structural and molecular determinants within the native context of intact infected cells has remained an unsolved challenge. Here, we provide detailed insights into the structural interactome of herpes simplex virus 1-infected human cells by combining in-cell cross-linking mass spectrometry with the selective enrichment of newly synthesized viral proteins. In productively infected cells, we obtain 739 PPIs based on 6,194 cross-links found across intracellular compartments and at the intact host endomembrane system. These structural host-virus interactome profiling (SHVIP) data resolve PPIs to the protein domain level and augment AlphaFold-based structural modeling, facilitating detailed predictions of PPI sites within structured and intrinsically disordered regions. Importantly, SHVIP captures parts of the virus-host PPI space that are elusive to traditional interaction proteomics approaches. Validation by molecular genetics confirms that these new SHVIP identifications are genuine virus-host PPIs occurring in the complex environment of intact infected cells.

DOI: https://doi.org/10.1038/s41467-025-61618-z
Cell. 2025 Jul 21. pii: S0092-8674(25)00743-3. [Epub ahead of print]

SPIDR enables multiplexed mapping of RNA-protein interactions and uncovers a mechanism for selective translational suppression upon cell stress.

Erica Wolin, Jimmy K Guo, Mario R Blanco, Isabel N Goronzy, Darvesh Gorhe, Wenzhao Dong, Andrew A Perez, Abdurrahman Keskin, Elizabeth Valenzuela, Ahmed A Abdou, Carl R Urbinati, Ross Kaufhold, H Tomas Rube, Jailson Brito Querido, Mitchell Guttman, Marko Jovanovic.

  RNA-binding proteins (RBPs) regulate all stages of the mRNA life cycle, yet current methods generally map RNA targets of RBPs one protein at a time. To overcome this limitation, we developed SPIDR (split-and-pool identification of RBP targets), a highly multiplexed split-pool method that profiles the binding sites of dozens of RBPs simultaneously. SPIDR identifies precise, single-nucleotide binding sites for diverse classes of RBPs. Using SPIDR, we uncovered an interaction between LARP1 and the 18S rRNA and resolved this interaction to the mRNA entry channel of the 40S ribosome using cryoelectron microscopy (cryo-EM), providing a potential mechanistic explanation for LARP1's role in translational suppression. We explored changes in RBP binding upon mTOR inhibition and identified that 4EBP1 preferentially associates with translationally repressed mRNAs upon mTOR inhibition. SPIDR has the potential to significantly advance our understanding of RNA biology by enabling rapid, de novo discovery of RNA-protein interactions at an unprecedented scale.

Keywords:  CLIP; LARP1; RNA; RNA-binding proteins; mTOR; ribosome; translation

DOI:  https://doi.org/10.1016/j.cell.2025.06.042
J Biol Chem. 2025 Jul 17. pii: S0021-9258(25)02324-5. [Epub ahead of print] 110474

Secretory autophagy mediates lysosomal and autophagic degradation for α-synuclein proteostasis.

Taiki Sawai, Yoshitsugu Nakamura, Shigeki Arawaka.

  Autophagy has two distinct pathways, degradation and secretion. Autophagic degradation plays a pivotal role in proteostasis. However, the role of autophagic secretion in proteostasis maintenance is not fully understood. Here, we investigate how the blockade of autophagic secretion impairs proteostasis in SH-SY5Y cells. siRNA-mediated knockdown of a modulator for autophagosome formation, ATG5, BECN1 or FIP200 inhibited autophagic flux and secretion, causing accumulation of Triton X-100-insoluble α-synuclein, which is an aggregate-prone protein responsible for neuronal loss in Parkinson's disease. The blockade of autophagic secretion by knockdown of t-SNARE SNAP23 or STX4 increased autophagic flux for p62 degradation, but these knockdowns induced enlargement and membrane damage of lysosomes as well as lysosomal dysfunction. SNAP23 or STX4 knockdown caused accumulation of Triton X-100-insoluble α-synuclein against induction of lysophagy. GBA knockdown showed lysosomal damage with the increase in autophagic secretion. RAB8A, a small GTPase regulator of polarized sorting to the plasma membrane, knockdown blocked autophagic secretion and produced lysosomal damage. SNAP23, STX4 or RAB8A knockdown further accelerated accumulation of Triton X-100-insoluble α-synuclein caused by a lysosomal protease inhibitor cocktail. Collectively, these findings suggest that SNAP23, STX4 or RAB8A knockdown blocks autophagic secretion and upregulates autophagic flux as a compensatory response to help maintain degradation. However, these knockdowns impair α-synuclein proteostasis because of lysosomal damage that they induce, counteracting compensatory effects of autophagic degradation, including lysophagy. Autophagic secretion and degradation may collaboratively form the clearance pathway required for maintaining lysosomal function by reducing the burden of aggregate-prone protein cargo.

Keywords:  Parkinson disease; autophagy; lysosome; protein secretion; proteostasis; synuclein

DOI:  https://doi.org/10.1016/j.jbc.2025.110474
J Biol Chem. 2025 Jul 16. pii: S0021-9258(25)02334-8. [Epub ahead of print] 110484

The ubiquitin ligase Triad1 influences myeloid leukemogenesis by regulating the integrated stress response.

Hao Wang, Francis Suh, Mariafausta Fischietti, Brian Wray, Szymon K Filip, Peter A Faull, Liping Hu, Weiqi Huang, Bin Liu, Leonidas C Platanias, Elizabeth A Eklund.

  Increased expression of a set of homeodomain transcription factors, including HoxA10, characterizes an adverse prognosis subtype of acute myeloid leukemia (AML). Examples of this subtype include AML with KMT2A or MYST3/CREBBP gene rearrangements, and an AML subset with normal cytogenetics. Previously, we identified ARIH2, the gene encoding Triad1, as a HoxA10 target gene. We determined that transcriptional activation of ARIH2 by HoxA10 was necessary to terminate emergency granulopoiesis during the innate immune response, but also antagonized leukemogenesis in a murine model of KMT2A-rearranged AML. Triad1 expression progressively decreases during the latent period preceding AML in this model, and Triad1-knockdown accelerates AML development. Triad1 is an E3 ubiquitin ligase, and we found knocking down Triad1 decreased protein ubiquitination in myeloid cells. Therefore, proteins with Triad1-dependent ubiquitination might regulate leukemogenesis and/or the innate immune response. By proteomic screen, we identified Triad1-dependent ubiquitination of a set of proteins that regulate the integrated stress response (ISR), including Gcn1. The ISR prevents metabolic exhaustion during sustained inflammation by decreasing total mRNA translation and global protein synthesis, while altering the translatome to correct metabolic deficiencies and inhibit apoptosis. In cells with Triad1-knockdown, we defined a translatome consistent with ISR-activation and reversed by co-knockdown of Gcn1. Gcn1-knockdown also delayed AML development in a KMT2A-rearranged murine model, and reversed effects of Triad1-knockdown on leukemogenesis. These results suggest ISR-inhibition mediates Triad1-related leukemia suppression, and activation of the ISR enhances leukemogenesis in this adverse prognosis AML subtype.

Keywords:  E3 ubiquitin ligase; gene expression; innate immunity; leukemia; ubiquitination

DOI:  https://doi.org/10.1016/j.jbc.2025.110484
Trends Cell Biol. 2025 Jul 22. pii: S0962-8924(25)00154-0. [Epub ahead of print]

Endosome-ER contact sites in phagophore formation.

Mengnan Xu, Pingping Wang, Xian-Ping Dong.

  Autophagy is a crucial 'self-eating' mechanism used by eukaryotic cells to degrade and recycle cytosolic materials. A recent study by Da Graça et al. reports that the dynamic mobilization of endosome-endoplasmic reticulum (ER) contact sites (EERCS) in response to starvation creates a confined environment that facilitates Ca2+-dependent phagophore biogenesis.

Keywords:  autophagy; calcium; endoplasmic reticulum (ER); endosome; endosome-ER contact sites (EERCS); phagophore

DOI:  https://doi.org/10.1016/j.tcb.2025.07.002
Mol Cell. 2025 Jul 17. pii: S1097-2765(25)00553-2. [Epub ahead of print]85(14): 2630-2632

m6A-induced ribosome stalling and collision trigger mRNA decay.

Feiyan Liu, Yu Zhou.

How m6A in different transcript regions regulates mRNA decay remains an intriguing question. Three studies from the labs of König,1 Jaffrey,2 and Steinmetz3 uncovered translation-dependent CDS-m6A decay: m6A in the coding sequence (CDS) induces ribosome stalling and collision to trigger mRNA decay, while tRNA modification alleviates the effects.

DOI: https://doi.org/10.1016/j.molcel.2025.06.024
Glia. 2025 Jul 20.

Endoplasmic Reticulum Stress Amplifies Cytokine Responses in Astrocytes via a PERK/eIF2α/JAK1 Signaling Axis.

Anirudhya Lahiri, Savannah G Sims, Jessica A Herstine, Alicia Meyer, Micah J Marshall, Ishrat Jahan, Subhodip Adhicary, Allison M Bradbury, Gordon P Meares.

  Aberrant activation of multiple cellular processes and signaling pathways is a hallmark of many neurological disorders. Understanding how these processes interact is crucial for elucidating the neuropathogenesis of these diseases. Among these, endoplasmic reticulum (ER) stress, activation of the unfolded protein response (UPR), and neuroinflammation are frequently implicated. Previously, we demonstrated that ER stress synergizes with tumor necrosis factor (TNF)-α to amplify interleukin (IL)-6 and C-C motif chemokine ligand (CCL)20 production in astrocytes through a Janus kinase 1 (JAK1)-dependent mechanism. Here, we expand on this finding by defining the scope and underlying mechanisms of this phenomenon. We show that ER stress and TNF-α cooperatively enhance inflammatory gene expression in astrocytes via a signaling axis that requires both protein kinase R (PKR)-like ER kinase (PERK) and JAK1. PERK-mediated phosphorylation of eukaryotic translation initiation factor (eIF)2α suppresses protein translation, delaying the expression of negative regulators such as NF-κB inhibitor (IκB)α and suppressor of cytokine signaling (SOCS)3 following TNF-α or oncostatin M (OSM) stimulation, respectively. Pharmacological reversal of p-eIF2α-dependent translational suppression using the small molecule integrated stress response inhibitor (ISRIB) restored IκBα and SOCS3 expression and attenuated the ER stress-induced enhancement of TNF-α- or OSM-driven inflammatory responses. Notably, astrocytes harboring a vanishing white matter-associated EIF2B5 mutation revealed that translational attenuation alone is insufficient to amplify cytokine-induced gene expression. Together, these findings identify a PERK/eIF2α/JAK1 signaling axis that sensitizes astrocytes to inflammatory cytokines, providing new mechanistic insights into the interactions between ER stress and neuroinflammation.

Keywords:  IL‐6; JAK/STAT; glia; integrated stress response; neuroinflammation; unfolded protein response

DOI:  https://doi.org/10.1002/glia.70067
Proc Natl Acad Sci U S A. 2025 Jul 29. 122(30): e2510269122

Mechanistic insights into the iron-sulfur cluster-dependent interaction of the autophagy receptor NCOA4 with the E3 ligase HERC2.

Haobo Liu, Liqiang Shen, Xinyu Gong, Xindi Zhou, Yichao Huang, Yuqian Zhou, Zhenpeng Guo, Hanbo Guo, Shichao Wang, Lifeng Pan.

  NCOA4, a dedicated autophagy receptor for mediating selective autophagy of ferritin (ferritinophagy), plays a vital role in maintaining cellular iron homeostasis. The cellular abundance of NCOA4 is regulated by the E3 ligase HERC2 that can specifically target NCOA4 for proteasomal degradation under iron-replete conditions. However, the detailed molecular mechanism governing the iron-dependent recognition of NCOA4 by HERC2 remains elusive. Here, using multidisciplinary approaches, we systematically characterize the HERC2-binding domain (HBD) of NCOA4 and its interaction with HERC2. We uncover that NCOA4 HBD harbors a [2Fe-2S] cluster and can exist in two different states, the apo-form state and the [2Fe-2S] cluster-bound state. Moreover, we unravel that HERC2 can effectively recognize the [2Fe-2S] cluster-bound NCOA4 HBD through its Cullin-7-PARC-HERC2 (CPH) domain and iron-sulfur cluster-dependent NCOA4-binding domain (INBD) with a synergistic binding mode. The determined crystal structures of HERC2(2540-2700) and its complex with the [2Fe-2S] cluster-bound NCOA4 HBD together with relevant biochemical and cellular results not only elucidate how NCOA4 HBD specifically senses cellular iron level by binding a [2Fe-2S] cluster but also reveal the molecular basis underlying the specific interaction of HERC2 with the [2Fe-2S] cluster-bound NCOA4 HBD. In summary, our findings provide mechanistic insights into the iron-dependent turnover of NCOA4 by HERC2 and expand our understanding of the regulatory mechanism of NCOA4-mediated ferritinophagy.

Keywords:  HERC2; NCOA4; autophagy; ferritinophagy; ubiquitination

DOI:  https://doi.org/10.1073/pnas.2510269122
Science. 2025 Jul 24. 389(6758): 386-391

Design of high-specificity binders for peptide-MHC-I complexes.

Bingxu Liu, Nathan F Greenwood, Julia E Bonzanini, Amir Motmaen, Jeremy Meyerberg, Tao Dao, Xinyu Xiang, Russell Ault, Jazmin Sharp, Chunyu Wang, Gian Marco Visani, Dionne K Vafeados, Nicole Roullier, Armita Nourmohammad, David A Scheinberg, K Christopher Garcia, David Baker.

Class I major histocompatibility complex (MHC-I) molecules present peptides derived from intracellular antigens on the cell surface for immune surveillance. Proteins that recognize peptide-MHC-I (pMHCI) complexes with specificity for diseased cells could have considerable therapeutic utility. Specificity requires recognition of outward-facing amino acid residues within the disease-associated peptide as well as avoidance of extensive contacts with ubiquitously expressed MHC. We used RFdiffusion to design pMHCI-binding proteins that make extensive contacts with the peptide and identified specific binders for 11 target pMHCs starting from either experimental or predicted pMHCI structures. Upon incorporation into chimeric antigen receptors, designs for eight targets conferred peptide-specific T cell activation. Our approach should have broad utility for both protein- and cell-based pMHCI targeting.

DOI: https://doi.org/10.1126/science.adv0185
STAR Protoc. 2025 Jul 22. pii: S2666-1667(25)00373-9. [Epub ahead of print]6(3): 103967

Protocol to distinguish pre-mRNA from mRNA in RNA-protein interaction studies.

Christina Zeiler, Annika Bestehorn, Pavel Kovarik.

  Transcriptome-wide studies on interactions between RNA-binding proteins (RBPs) and protein-coding RNAs in general preclude interpretations regarding RBP preference for binding to the more abundant mRNA over the less abundant pre-mRNA. Here, we present a protocol to determine the binding preference of the RBP tristetraprolin (TTP, Zfp36) for pre-mRNA versus mRNA. We describe steps for the identification and quantitation of intronic and exonic fragments in RNA bound to TTP. This protocol can potentially be applied to any RBP. For complete details on the use and execution of this protocol, please refer to Bestehorn et al.1.

Keywords:  Bioinformatics; Gene Expression; Genomics; Molecular Biology; RNA-seq; Sequence analysis; Sequencing

DOI:  https://doi.org/10.1016/j.xpro.2025.103967
J Cell Biol. 2025 Aug 04. pii: e202504076. [Epub ahead of print]224(8):

The rapidly expanding role of LC3-interacting regions in autophagy.

Brian J North, Dorotea Fracchiolla, Michael J Ragusa, Sascha Martens, Christopher J Shoemaker.

LC3-interacting regions (LIRs), or Atg8-interacting motifs (AIMs), are short linear motifs found in unstructured loops or intrinsically disordered regions of many autophagy-related proteins. LIRs were initially identified for their role in binding to Atg8 family proteins on autophagosomal membranes. However, emerging evidence suggests that LIRs and their surrounding residues mediate interactions with a wide array of proteins beyond Atg8s. This broadens the biological significance of LIRs in autophagy, rendering them an organizing principle of the autophagy machinery. In this perspective, we explore recent advances highlighting the multifunctional roles of LIRs, including their capacity to mediate binding with diverse factors. We discuss insights into the mechanisms underlying LIR-mediated interactions and propose an updated model to explain Atg8 diversification in higher eukaryotes. We conclude by addressing key challenges and outlining future directions for understanding LIR biology and its broader implications for cellular homeostasis.

DOI: https://doi.org/10.1083/jcb.202504076
Biochem Soc Trans. 2025 Jul 23. pii: BST20253077. [Epub ahead of print]

How does protein aggregate structure affect mechanisms of disaggregation?

YuChen Yang, Hays S Rye.

  Protein misfolding and aggregation underpin numerous pathological conditions, including Alzheimer's, Parkinson's, and Huntington's diseases. Within cells, the competition between protein folding and misfolding- driven aggregation necessitates intricate quality control systems known collectively as the proteostasis network, with molecular chaperones playing central roles. Critical gaps remain in our understanding of why certain protein aggregates are amenable to efficient chaperone-mediated disassembly, while others resist such intervention. Aggregates can be most broadly categorized into structurally ordered amyloid fibrils and more irregular amorphous clusters. Amyloid fibrils are characterized by a highly structured, cross-β-sheet architecture, and they generally display nucleation-driven growth kinetics. In contrast, amorphous aggregates form through heterogeneous interactions among partially unfolded proteins, which typically lack ordered and repeating structure but still display poorly understood, specific assembly constraints. Importantly, amorphous aggregation and amyloid formation are often linked to one another, with several different types of aggregate structures forming at the same time. The ability of molecular chaperones to remodel and disassemble aggregates is affected by aggregate size, internal structure, surface dynamics, and exposure of chaperone-binding sites. However, despite these insights, the mechanistic complexity, aggregate heterogeneity, and dynamic properties present substantial experimental and theoretical challenges. Addressing these challenges will require innovative approaches combining single-molecule biophysics, structural biology, and computational modeling to unveil universal principles governing protein aggregation and disaggregation within cellular environments.

Keywords:  amyloid; molecular chaperones; protein aggregation; protein misfolding; proteostasis

DOI:  https://doi.org/10.1042/BST20253077
Nat Commun. 2025 Jul 21. 16(1): 5422

Allosteric activation of the SPRTN protease by ubiquitin maintains genome stability.

Sophie Dürauer, Hyun-Seo Kang, Christian Wiebeler, Yuka Machida, Dina S Schnapka, Denitsa Yaneva, Christian Renz, Maximilian J Götz, Pedro Weickert, Abigail C Major, Aldwin S Rahmanto, Sophie M Gutenthaler-Tietze, Lena J Daumann, Petra Beli, Helle D Ulrich, Michael Sattler, Yuichi J Machida, Nadine Schwierz, Julian Stingele.

The DNA-dependent protease SPRTN maintains genome stability by degrading toxic DNA-protein crosslinks (DPCs). To understand how SPRTN's promiscuous protease activity is confined to cleavage of crosslinked proteins, we reconstitute the repair of DPCs including their modification with SUMO and ubiquitin chains in vitro. We discover that DPC ubiquitylation strongly activates SPRTN independently of SPRTN's known ubiquitin-binding domains. Using protein structure prediction, MD simulations and NMR spectroscopy we reveal that ubiquitin binds to SPRTN's protease domain, promoting an open, active conformation. Replacing key interfacial residues prevents allosteric activation of SPRTN by ubiquitin, leading to genomic instability and cell cycle defects in cells expressing truncated SPRTN variants that cause premature aging and liver cancer in Ruijs-Aalfs syndrome patients. Collectively, our results reveal a ubiquitin-dependent regulatory mechanism that ensures SPRTN activity is deployed precisely when and where it is needed.

DOI: https://doi.org/10.1038/s41467-025-61224-z
Science. 2025 Jul 24. eadx3800

High-resolution spatial mapping of cell state and lineage dynamics in vivo with PEtracer.

Luke W Koblan, Kathryn E Yost, Pu Zheng, William N Colgan, Matthew G Jones, Dian Yang, Arhan Kumar, Jaspreet Sandhu, Alexandra Schnell, Dawei Sun, Can Ergen, Reuben A Saunders, Xiaowei Zhuang, William E Allen, Nir Yosef, Jonathan S Weissman.

Charting the spatiotemporal dynamics of cell fate determination in development and disease is a long-standing objective in biology. Here we present the design, development, and extensive validation of PEtracer, a prime editing-based, evolving lineage tracing technology compatible with both single-cell sequencing and multimodal imaging methodologies to jointly profile cell state and lineage in dissociated cells or while preserving cellular context in tissues with high spatial resolution. Using PEtracer coupled with MERFISH spatial transcriptomic profiling in a syngeneic mouse model of tumor metastasis, we reconstruct the growth of individually-seeded tumors in vivo and uncover distinct modules of cell-intrinsic and cell-extrinsic factors that coordinate tumor growth. More generally, PEtracer enables systematic characterization of cell state and lineage relationships in intact tissues over biologically-relevant temporal and spatial scales.

DOI: https://doi.org/10.1126/science.adx3800
J Cell Biol. 2025 Sep 01. pii: e202505040. [Epub ahead of print]224(9):

Protein Kinase C promotes peroxisome biogenesis and peroxisome-endoplasmic reticulum interaction.

Anya Borisyuk, Charlotte Howman, Sundararaghavan Pattabiraman, Daniel Kaganovich, Triana Amen.

Peroxisomes carry out a diverse set of metabolic functions, including oxidation of very long-chain fatty acids, degradation of D-amino acids and hydrogen peroxide, and bile acid production. Many of these functions are upregulated on demand; therefore, cells control peroxisome abundance, and by extension peroxisome function, in response to environmental and developmental cues. The mechanisms upregulating peroxisomes in mammalian cells have remained unclear. Here, we identify a signaling regulatory network that coordinates cellular demand for peroxisomes and peroxisome abundance by regulating peroxisome proliferation and interaction with ER. We show that PKC promotes peroxisome PEX11b-dependent formation. PKC activation leads to an increase in peroxisome-ER contact site formation through inactivation of GSK3β. We show that removal of VAPA and VAPB impairs peroxisome biogenesis and PKC regulation. During neuronal differentiation, active PKC leads to a significant increase in peroxisome formation. We propose that peroxisomal regulation by transient PKC activation enables fine-tuned responses to the need for peroxisomal activity.

DOI: https://doi.org/10.1083/jcb.202505040
Nat Metab. 2025 Jul 21.

LONP1 regulation of mitochondrial protein folding provides insight into beta cell failure in type 2 diabetes.

Jin Li, Yamei Deng, Marie Gasser, Jie Zhu, Emily M Walker, Vaibhav Sidarala, Emma C Reck, Dre L Hubers, Mabelle B Pasmooij, Chun-Shik Shin, Khushdeep Bandesh, Eftyhmios Motakis, Siddhi Nargund, Romy Kursawe, Venkatesha Basrur, Alexey I Nesvizhskii, Michael L Stitzel, David C Chan, Guy A Rutter, Scott A Soleimanpour.

Protein misfolding is a contributor to the development of type 2 diabetes (T2D), but the specific role of impaired proteostasis is unclear. Here we show a robust accumulation of misfolded proteins in the mitochondria of human pancreatic islets from patients with T2D and elucidate its impact on β cell viability through the mitochondrial matrix protease LONP1. Quantitative proteomics studies of protein aggregates reveal that islets from donors with T2D have a signature resembling mitochondrial rather than endoplasmic reticulum protein misfolding. Loss of LONP1, a vital component of the mitochondrial proteostatic machinery, with reduced expression in the β cells of donors with T2D, yields mitochondrial protein misfolding and reduced respiratory function, leading to β cell apoptosis and hyperglycaemia. LONP1 gain of function ameliorates mitochondrial protein misfolding and restores human β cell survival after glucolipotoxicity via a protease-independent effect requiring LONP1-mitochondrial HSP70 chaperone activity. Thus, LONP1 promotes β cell survival and prevents hyperglycaemia by facilitating mitochondrial protein folding. These observations provide insights into the nature of proteotoxicity that promotes β cell loss during the pathogenesis of T2D, which could be considered as future therapeutic targets.

DOI: https://doi.org/10.1038/s42255-025-01333-7
Nat Commun. 2025 Jul 18. 16(1): 6621

ATG8ylation-mediated tonoplast invagination mitigates vacuole damage.

Xuanang Zheng, Juncai Ma, Jing Li, Siyu Chen, Jun Luo, Jianxiong Wu, Kaiyan Zhang, Changlian Peng, Yonglun Zeng, Faqiang Li, Byung-Ho Kang, Caiji Gao, Jun Zhou.

While ATG8ylation, the lipidation of ATG8-family proteins, is canonically linked to double-membrane autophagosome formation, emerging studies demonstrate its non-canonical association with single-membrane organelles. The functional significance of ATG8ylation in these compartments, however, remains unclear. Here, we demonstrate that ionophores rapidly trigger ATG8 conjugation to the vacuolar membrane (tonoplast), a process reliant on the ATG conjugation system rather than the upstream autophagic regulators. Inhibiting reactive oxygen species (ROS) generation or V-ATPase function greatly impedes the targeting of ATG8 to the tonoplast. Intriguingly, the attachment of ATG8 to the tonoplast enhances its invagination and fosters the formation of intraluminal vesicles within vacuoles, which is achieved independently of the ESCRT machinery or cytoskeletal components. The emergence of ATG8-positive vesicles may facilitate the restoration of vacuolar acidification by redirecting proton flow from the vacuole-to-cytoplasm to an intravacuolar direction, which aids in the rapid recovery of plant growth after removal of monensin. Furthermore, under alkaline stress, ATG8 targets the tonoplast and induces vacuolar membrane invagination via a regulatory mechanism similar to that of monensin, indicating that ATG8ylation-mediated vacuolar remodeling represents an adaptive mechanism against environmental alkalinization in plants.

DOI: https://doi.org/10.1038/s41467-025-62084-3
Nat Commun. 2025 Jul 21. 16(1): 6695

A UFD1 variant encoding a microprotein modulates UFD1f and IPMK ubiquitination to play pivotal roles in anti-stress responses.

Xiuzhi Li, Xiaolin Wang, Xu Liu, Ge Shan, Liang Chen.

Eukaryotic cells make multiple efforts to cope with internal and external stresses; such mechanisms include metabolic responses and the generation of stress-responsive mRNA isoforms (SR-mRNAisos), such as the classical XBP1s. Here, we identified a mammalian conserved SR-mRNAiso, UFD1s, which encodes a microprotein with anti-stress functions. UFD1s decreased the K63-linked ubiquitination levels of UFD1 full-length protein (UFD1f) via competitive binding to the E3 ubiquitin ligase MARCH7, and therefore regulated the dynamics of protein ubiquitination. Inositol polyphosphate multikinase (IPMK) was identified as the most significantly UFD1s-regulated target in terms of changes in K48- and K11-ubiquitination. UFD1s promoted autophagy and fatty acid oxidation, and IPMK was consistently destabilized. Ufd1s-deficient male mice exhibited metabolic disorders and accelerated NASH progression. Plasmid or circRNA expressing UFD1s alleviated NASH in mice, indicating that UFD1s has therapeutic value. Our findings revealed a mammalian conserved microprotein that plays crucial roles in anti-stress regulation through the modulation of ubiquitination and metabolism.

DOI: https://doi.org/10.1038/s41467-025-62073-6
FEBS J. 2025 Jul 24.

Tks5 interactome reveals endoplasmic-reticulum-associated translation machinery in invadosomes.

Léa Normand, Benjamin Bonnard, Margaux Sala, Sylvaine Di Tommaso, Cyril Dourthe, Anne-Aurélie Raymond, Jean-William Dupuy, Luc Mercier, Jacky G Goetz, Violaine Moreau, Elodie Henriet, Frédéric Saltel.

  The ability to progress and invade through the extracellular matrix is a characteristic shared by both normal and cancer cells through the formation of structures called invadosomes, which include invadopodia and podosomes. These invadosomes are plastic and dynamic structures that can adopt different organizations-such as rosettes, dots, or linear invadosomes-depending on the cell types and the environment. In this study, we used the specific invadosome marker SH3 and PX domain-containing protein 2A (SH3PXD2A; also known as Tks5) to identify common features in these different organizations. Tks5 immunoprecipitation coupled with mass spectrometry analysis allowed us to identify common proteins in these different models. We identified elements of the translation machinery, in particular the eukaryotic translation initiation factor 4B (EIF4B) protein, but also endoplasmic reticulum (ER) proteins as part of the invadosome structure. Providing new data on invadosome molecular composition through the Tks5 interactome, we identified that ER-associated translation machinery is recruited to invadosomes and involved in their formation, persistence, and function in all types of invadosomes.

Keywords:  ER; Tks5; cancer; invadosomes; translation

DOI:  https://doi.org/10.1111/febs.70196
EMBO J. 2025 Jul 18.

Parp7 generates an ADP-ribosyl degron that controls negative feedback of androgen signaling.

Krzysztof Wierbiłowicz, Chun-Song Yang, Ahmed Almaghasilah, Patryk A Wesołowski, Philipp Pracht, Natalia M Dworak, Jack Masur, Sven Wijngaarden, Dmitri V Filippov, David J Wales, Joshua B Kelley, Aakrosh Ratan, Bryce M Paschal.

  The androgen receptor (AR) transduces the effects of circulating and tumor-derived androgens to the nucleus through ligand-induced changes in protein conformation, localization, and chromatin engagement. Defining how these events are integrated with signal transduction is critical to understand how AR drives prostate cancer and unveil pathway features that are potentially amenable to therapeutic intervention. We describe a novel post-transcriptional mechanism that controls AR levels on chromatin and gene output based on highly selective, inducible degradation. We find that the mono-ADP-ribosyltransferase PARP7 generates an ADP-ribosyl degron in the DNA-binding domain of AR, which is recognized by the ADP-ribose reader domain in the ubiquitin E3 ligase DTX2 and degraded by the proteasome. Mathematical modeling of the pathway suggested that PARP7 ADP-ribosylates chromatin-bound AR, a prediction that was validated in cells using an AR DNA-binding mutant. Non-conventional ubiquitin conjugation to ADP-ribosyl-cysteine and degradation by the proteasome forms the basis of a negative feedback loop that regulates modules of AR target genes. Our data expand the repertoire of mono-ADP-ribosyltransferases to include gene regulation via highly selective protein degradation.

Keywords:  ADP-ribosylation; AR; DTX2; RBN2397; Ubiquitin

DOI:  https://doi.org/10.1038/s44318-025-00510-4
Nat Commun. 2025 Jul 25. 16(1): 6841

Spatially constrained disulfide bond shuffling delays insulin aggregation and enhances neurotoxicity.

Weida Qin, Ruomeng Li, Juan Liu, Jun Liu, Xiaoli Wang, Binbin Hu, Zhen Zheng, Zhilin Yu, Gongyu Li.

Disulfide bond shuffling (DBS) critically influences protein aggregation and stability, yet its spatial constraints and biological implications remain poorly understood. Here, we demonstrate that insulin undergoes DBS within an extended spatial range up to ~19 Å, generating heterogeneous crosslinked oligomers that alter aggregation pathway. While DBS products initially delay aggregation by inhibiting primary nucleation and elongation steps, they ultimately promote the formation of distinct fibrillar structures with enhanced β-sheet content. Native ion mobility-mass spectrometry reveals molecular crosstalk between DBS products and native insulin via both covalent and non-covalent interactions. Notably, DBS-modified insulin fibrils exhibit significantly increased neurotoxicity in neuronal and pancreatic cells through mitochondrial apoptosis activation as supported by proteomic and biophysical analysis. Our findings underscore the importance of controlling DBS in insulin for therapeutic applications and provide insights into the role of disulfide dynamics in protein aggregation and cytotoxicity, with implications for insulin and broader protein misfolding contexts.

DOI: https://doi.org/10.1038/s41467-025-62257-0
Autophagy. 2025 Jul 23.

Npl3 is required for efficient autophagosome-vacuole fusion.

Zhangyuan Yin, Zhihai Zhang, Xu Liu, Daniel J Klionsky.

  Macroautophagy/autophagy is a highly conserved catabolic membrane trafficking process through which various intracellular constituents, from proteins to organelles, are targeted for vacuolar/lysosomal degradation. Autophagy is tightly regulated both temporally and in magnitude at multiple levels to prevent either excessive or insufficient activity. To date, only a few RNA-binding proteins have been characterized as regulating the expression of genes essential for autophagy, and the contribution of post-transcriptional regulation in autophagy activity remains poorly understood. Here, through a genetic screen for autophagy-defective mutants, we identified Npl3, a nucleus-cytoplasm shuttling mRNA-binding protein, as essential for both bulk and selective types of autophagy. Deletion of NPL3 does not affect autophagosome biogenesis, closure, or maturation; however, it severely impairs autophagosome-vacuole fusion and results in minimal autophagosome turnover. We further demonstrated that this regulation depends on the RNA-binding domain of Npl3 and its capability for nuclear re-import. Together, our results reveal a novel layer of post-transcriptional regulation of autophagy.

Keywords:  Autophagy; RNA-binding protein; membrane trafficking

DOI:  https://doi.org/10.1080/15548627.2025.2537559
Proc Natl Acad Sci U S A. 2025 Jul 29. 122(30): e2424286122

eIF4A controls translation of estrogen receptor alpha and is a therapeutic target in advanced breast cancer.

Jacob A Boyer, Ezra Y Rosen, Malvika Sharma, Madeline A Dorso, Nicholas Mai, Corina Amor, Jason M Reiter, Ram Kannan, Sunyana Gadal, Jianing Xu, Matthew Miele, Zhuoning Li, Xiaoping Chen, Qing Chang, Fresia Pareja, Stephan Worland, Douglas Warner, Sam Sperry, Gary G Chiang, Peggy A Thompson, Guangli Yang, Ouathek Ouerfelli, Alexander Drilon, Elisa de Stanchina, Hans-Guido Wendel, Sarat Chandarlapaty, Neal Rosen.

  Most breast cancers depend on hormone-stimulated estrogen receptor alpha (ER) activity and are sensitive to ER inhibition. Resistance can arise from activating mutations in the gene encoding ER (ESR1) or from reactivation of downstream targets. Newer ER antagonists occasionally show efficacy but are largely ineffective as single agents in the long term. Here, we show that ER translation is eIF4E/cap-independent yet sensitive to inhibitors of the translation initiation factor eIF4A. EIF4A inhibition reduces the expression of ER and cell cycle regulators such as cyclin D1. This leads to growth suppression in ligand-independent breast cancer models, including those driven by ER mutants and fusion proteins. Efficacy is enhanced by adding the ER degrader, fulvestrant. The combination further lowers ER expression and blocks tumor growth in vitro and in vivo. In an early clinical trial (NCT04092673), the eIF4A inhibitor zotatifin was combined with either fulvestrant or fulvestrant plus CDK4 inhibitor, abemaciclib, in patients with acquired resistance to these agents. Multiple clinical responses including a handful of durable regressions were observed, with little toxicity. Thus, eIF4A inhibition could be useful for treating ER+ breast cancer resistant to other modalities.

Keywords:  breast cancer; eIF4A; estrogen receptor; translation; zotatifin

DOI:  https://doi.org/10.1073/pnas.2424286122
Cell Death Dis. 2025 Jul 22. 16(1): 547

UPR-induced intracellular C5aR1 promotes adaptation to the hypoxic tumour microenvironment.

Tatsuya Suwa, Kelly Sw Lee, Ian J Chai, Heather O L Clark, David J MacLean, Nicole Machado, Gonzalo Rodriguez-Berriguete, Lolita Singh, Geoff S Higgins, Ester M Hammond, Monica M Olcina.

Dysregulation of the C5a-C5a receptor 1 (C5aR1) signalling axis underlies inflammation and immune-driven pathology. C5aR1 was traditionally thought to be primarily expressed on the cell membrane, although recent reports indicate the importance of intracellular C5aR1 expression for the inflammatory effector functions of various cell types. However, the mechanisms regulating C5aR1 expression and localisation remain unclear. In tumours with an immunosuppressive microenvironment, we recently found C5aR1 expression on malignant epithelial cells, highlighting potential tumour cell-specific functions. Here, we show that physical conditions of the tumour microenvironment leading to immunosuppression, induce C5aR1 expression and control its intracellular localisation. Mechanistically, we find that low oxygen (hypoxia) induces C5aR1 expression in an unfolded protein response (UPR)-dependent manner via enhanced endoplasmic reticulum stress. Furthermore, hypoxia drives endocytosis, relocating C5aR1 from the cell membrane to the intracellular compartment. By genetically and pharmacologically targeting the C5a/C5aR1 axis, we show that C5aR1 mediates cellular adaptation to hypoxia by regulating processes associated with cell fate, including autophagy and apoptosis. In line with hypoxia-induced intracellular C5aR1 pools, the most significant pharmacological effects on cell survival are observed with selective small molecule inhibitors of C5aR1 associated with high cell permeability. These results suggest that the dysregulated C5a/C5aR1 axis and the hypoxia-induced shift in C5aR1 localisation support tumour cell survival in the hypoxic tumour microenvironment and provide new insights into therapeutic strategies for targeting the C5a/C5aR1 axis in cancer.

DOI: https://doi.org/10.1038/s41419-025-07862-z
Cell Chem Biol. 2025 Jul 17. pii: S2451-9456(25)00174-6. [Epub ahead of print]32(7): 899-901

RPL22 links ribosome biogenesis, RNA splicing, and sensitivity to RNA polymerase I inhibition.

Kenyon Weis, Omar Abdel-Wahab.

In this issue of Cell Chemical Biology, Fan et al.1 identify that mutations in the ribosomal protein RPL22 confer sensitivity to RNA polymerase I inhibitors. RPL22 regulates MDM4 function and cell death via splicing of the MDM4 mRNA. These findings connect ribosome biogenesis with RNA splicing through RPL22.

DOI: https://doi.org/10.1016/j.chembiol.2025.06.001
Nat Commun. 2025 Jul 24. 16(1): 6777

PEAK1 maintains tight junctions in intestinal epithelial cells and resists colitis by inhibiting autophagy-mediated ZO-1 degradation.

Zaikuan Zhang, Yajun Xie, Qiying Yi, Jianing Liu, Lin Yang, Runzhi Wang, Jing Cai, Xinyi Li, Xiaosong Feng, Shixiang Yao, Zheng Pan, Magdalena Paolino, Qin Zhou.

Tight junctions are crucial for maintaining intestinal barrier homeostasis, but how organisms modulate these junctions remain unclear. Here, we show a role for PEAK1 at cell-cell contact sites, where it interacts with ZO-1 via a conserved region spanning amino acids 714-731. This interaction masks the LC3-interacting region on ZO-1, preventing autophagy-mediated ZO-1 degradation and preserving the integrity of tight junctions in intestinal epithelial cells. Src-mediated phosphorylation of PEAK1 at Y724 promotes the binding between PEAK1 and ZO-1 to stabilize ZO-1 in intestinal epithelial cells. Additionally, PEAK1 binds to CSK to positively regulate Src activity. Loss of PEAK1 in intestinal epithelial cells leads to decreased Src activity and lower ZO-1 protein levels, resulting in disrupted tight junctions, both in vitro and in vivo. In mice, Peak1 deficiency increases intestinal epithelium permeability and exacerbates inflammation in experimentally induced colitis models. Our findings reveal PEAK1's critical role in maintaining tight junction integrity and resistance to intestinal inflammation, extending its known function from promoting tumor cell proliferation and migration to essential physiological processes. These insights refine our understanding of the mechanisms regulating tight junctions and offer potential therapeutic targets for enhancing epithelial barrier function and treating related diseases.

DOI: https://doi.org/10.1038/s41467-025-62107-z
FEBS Lett. 2025 Jul 22.

TRAF2 binds to TIFA via a novel motif and contributes to its autophagic degradation.

Tom Snelling, Kodi Hunter, Celest W S Tay, Nicola T Wood, Philip Cohen.

  A signal transduction pathway has been defined in which ADP-heptose activates the mammalian protein kinase ALPK1, which phosphorylates the adaptor protein TIFA, inducing its polymerisation and interaction with the E3 ubiquitin ligases TRAF2/c-IAP1 and TRAF6. These E3 ligases drive activation of the transcription factors NF-κB and AP-1, culminating in the production and secretion of inflammatory mediators to combat microbial infection. TRAF6 is essential in this process, but how TRAF2 interacts with TIFA and its role in the pathway is unclear. Here, we identify two conserved sequence motifs in TIFA essential for TRAF2 interaction, one of which (Pro159-Xaa-Xaa-Glu162) is novel. We additionally report that ADP-heptose induces TIFA degradation by autophagy and that both TRAF2 and TRAF6 contribute to this process. These findings advance understanding of how TRAF2 regulates the ALPK1-TIFA signalling pathway.

Keywords:   alphafold3 ; ALPK1; Autophagy; TIFA; TRAF2

DOI:  https://doi.org/10.1002/1873-3468.70110
iScience. 2025 Jul 18. 28(7): 112913

Engineering a cell-based orthogonal ubiquitin transfer cascade for profiling the substrates of RBR E3 Parkin.

Shuai Fang, Li Zhou, Geng Chen, Jing Zhang, Xiaoyu Wang, In Ho Jeong, Savannah E Jacobs, Bradley R Kossmann, Wei Wei, Shu Liu, Geon H Jeong, Yayun Xie, Duc Duong, Nicholas T Seyfried, Ivaylo Ivanov, Angela M Mabb, Hiroaki Kiyokawa, Bo Zhao, Jun Yin.

  The E3 ubiquitin (UB) ligase Parkin utilizes a Ring-Between-Ring (RBR) domain to mediate UB transfer to substrate proteins, and mutations affecting Parkin catalysis promote cancer and are associated with Parkinson's disease. An essential role of Parkin is to initiate mitophagy by ubiquitinating mitochondrial proteins. Still, it is unclear how Parkin carries out other cellular functions, such as the regulation of the cell cycle, metabolism, and the neuronal synapse. Here, we used phage display to engineer the RBR domain of Parkin and assembled an orthogonal ubiquitin transfer (OUT) cascade to profile Parkin substrates in living cells. Guided by the substrate profile from the OUT screen, we verified a panel of Rab GTPases and CDK5 as Parkin substrates. We also showed mitophagy stimulation enhanced Parkin-mediated ubiquitination of Rab proteins. Our work demonstrates that the OUT cascade can be an empowering tool for identifying Parkin substrates to elucidate its multifaceted cellular functions.

Keywords:  Biochemistry; Biomolecular engineering; Methodology in biological sciences; Protein; Structural biology

DOI:  https://doi.org/10.1016/j.isci.2025.112913
iScience. 2025 Aug 15. 28(8): 112990

The E3 ubiquitin ligase DTX3L and the deubiquitinase USP28 fine-tune DNA repair through mutual regulation of their protein levels.

Daniela Mennerich, Yashwanth Ashok, Carlos Vela-Rodríguez, Heli I Hentilä, Melanie Rall-Scharpf, Lisa Wiesmüller, Renata Prunskaite-Hyyryläinen, Lari Lehtiö, Thomas Kietzmann.

  The DNA damage response (DDR) relies on a complex protein network to maintain genomic integrity, yet the interplay between post-translational modifiers remains poorly understood. Here, we uncover a novel regulatory axis between the E3 ubiquitin ligase DTX3L and the deubiquitinase USP28 at DNA double-strand breaks (DSBs). Our results reveal a sophisticated feedback mechanism in which DTX3L ubiquitinates USP28, leading to its proteasomal degradation, while USP28 counteracts by deubiquitinating both itself and DTX3L. This cross-regulation fine-tunes DSB repair in multiple pathways, including non-homologous end-joining (NHEJ), homologous recombination (HR), single-strand annealing (SSA), and microhomology-mediated end joining (MMEJ). Strikingly, the detrimental effects of USP28 depletion on these repair pathways were rescued by concurrent DTX3L knockdown. Collectively, our work uncovers a novel layer of DDR regulation in which DTX3L and USP28's antagonistic activities calibrate cellular responses to genotoxic stress, thus identifying promising therapeutic targets to combat diseases associated with genomic instability.

Keywords:  Cell biology; Molecular biology

DOI:  https://doi.org/10.1016/j.isci.2025.112990
Cell Mol Gastroenterol Hepatol. 2025 Jul 19. pii: S2352-345X(25)00132-8. [Epub ahead of print] 101591

FMO5 plays a sex-specific role in goblet cell maturation and mucus barrier formation.

Megan L Schaller, Madeline M Sykes, Joy Mecano, Sumeet Solanki, Wesley Huang, Ryan J Rebernick, Safa Beydoun, Emily Wang, Amara Bugarin-Lapuz, Yatrik M Shah, Scott F Leiser.

   BACKGROUND AND AIMS: The intestine plays a key role in metabolism, nutrient and water absorption, and provides both physical and immunological defense against dietary and luminal antigens. The protective mucosal lining in the intestine is a critical component of intestinal barrier that when compromised, can lead to increased permeability, a defining characteristic of inflammatory bowel disease (IBD), among other intestinal diseases. Here, we define a new role for the flavin-containing monooxygenase (FMO) family of enzymes in maintaining a healthy intestinal epithelium.
METHODS: Using Caenorhabditis elegans we measure intestinal barrier function, actin expression, and intestinal damage response. In mice, we utilize an intestine-specific, tamoxifen-inducible knockout model of the mammalian homolog of Cefmo-2, Fmo5, and assess histology, mucus barrier thickness, and goblet cell physiology. We also treat mice with the ER chaperone Tauroursodeoxycholic acid (TUDCA).
RESULTS: In nematodes, we find Cefmo-2 is necessary and sufficient for intestinal barrier function, intestinal actin expression, and is induced by intestinal damage. In mice, we find striking changes to the intestine within two weeks following FMO5 disruption. Alterations include sex-dependent changes in colon epithelial histology, goblet cell localization, and mucus barrier formation. These changes are significantly more severe in female mice, mirroring differences observed in IBD patients. Furthermore, we find increased protein folding stress in FMO5 knockout animals and successfully rescue the severe female phenotype with addition of a chemical ER chaperone.
CONCLUSIONS: Together, our results identify a highly conserved and novel role for FMO5 in the mammalian intestine and support a key role for FMO5 in maintenance of ER/protein homeostasis and proper mucus barrier formation.

Keywords:  ER stress; goblet cells; mucus barrier; sex-dependent

DOI:  https://doi.org/10.1016/j.jcmgh.2025.101591
PLoS Pathog. 2025 Jul;21(7): e1013275

A chaperonin complex regulates organelle proteostasis in malaria parasites.

Amanda Tissawak, Yarden Rosin, Shirly Katz Galay, Alia Qasem, Michal Shahar, Nirit Trabelsi, Ora Furman-Schueler, Steven M Johnson, Anat Florentin.

The apicoplast of Plasmodium parasites serves as a metabolic hub that synthesize essential biomolecules. Like other endosymbiotic organelles, 90% of the apicoplast proteome is encoded by the cell nucleus and transported to the organelle. Evidence suggests that the apicoplast has minimal control over the synthesis of its proteome and therefore it is unclear how organelle proteostasis is regulated. Here, we identified and investigated a large and conserved chaperonin (CPN) complex with a previously unknown function. Using genetic tools, we demonstrated that ablation of the apicoplast CPN60 subunit leads to parasite death due to organellar damage, immediately within its first replication cycle, deviating from the delayed death phenotype commonly observed for apicoplast translation inhibitors. Unlike its close orthologues in other prokaryotic and eukaryotic cells, CPN60 is not upregulated during heat shock (HS) and does not affect HS response in the parasite. Instead, we found that it is directly involved in proteostasis through interaction with the Clp (caseinolytic protease) proteolytic complex. We showed that CPN60 physically binds both the active and inactive forms of the Clp complex, and manipulates its stability. A computational structural model of a possible interaction between these two large complexes suggests a stable interface. Finally, we screened a panel of inhibitors for the bacterial CPN60 orthologue GroEL, to test the potential of chaperonin inhibition as antimalarial. These inhibitors demonstrated an anti-Plasmodium activity that was not restricted to apicoplast function, with additional targets outside of this organelle. Taken together, this work reveals how balanced activities of proteolysis and refolding safeguard the apicoplast proteome, and are essential for organelle biogenesis.

DOI: https://doi.org/10.1371/journal.ppat.1013275
Nat Commun. 2025 Jul 19. 16(1): 6653

Natural variation of an E3 ubiquitin ligase encoding gene Chalk9 regulates grain chalkiness in rice.

Zhi Hu, Hongchun Liu, Min Guo, Xiang Han, Youguang Li, Rujia Chen, Yifan Guo, Yihao Yang, Shengyuan Sun, Yong Zhou, Minghong Gu, Changjie Yan.

Grain chalkiness is an undesirable trait affecting rice quality, concerning both consumers and breeders. However, the genetic mechanisms underlying rice chalkiness remain largely elusive. Here, we identify Chalk9 as a major gene associated with grain chalkiness in a natural population. Chalk9 encodes an E3 ubiquitin ligase that targets OsEBP89 for its ubiquitination and degradation. Low expression of Chalk9 results in excessive accumulation of OsEBP89, disrupting the homeostasis of storage components and leading to the chalkiness phenotype. A 64-bp insertion/deletion in the Chalk9 promoter contributes to its differential transcriptional levels, thus causing chalkiness variation among rice varieties. Moreover, the elite allele Chalk9-L reduces grain chalkiness, without compromising yield. Chalk9-L is strongly selected in japonica but exhibits a complex evolutionary trajectory in indica. Our findings reveal the molecular and genetic mechanisms underlying chalkiness and provide a potential strategy for breeding rice varieties with improved quality.

DOI: https://doi.org/10.1038/s41467-025-61683-4