bims-proteo 2025-03-02 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2025–03–02
forty-two papers selected by
Eric Chevet, INSERM

The pathway of unconventional protein secretion involves CUPS and a modified trans-Golgi network.
PROTAC-induced protein structural dynamics in targeted protein degradation.
Lysosomal degradation of ER client proteins by ER-phagy and related pathways.
RNA exosome-driven RNA processing instructs the duration of the unfolded protein response.
DET1 dynamics underlie cooperative ubiquitination by CRL4DET1-COP1 complexes.
Dynamic Modulation of IRE1α-XBP1 Signaling by Adenovirus.
Epsin1 enforces a condensation-dependent checkpoint for ubiquitylated cargo during clathrin-mediated endocytosis.
Control of Rhizobia Endosymbiosis by Coupling ER Expansion with Enhanced UPR.
Predictomes, a classifier-curated database of AlphaFold-modeled protein-protein interactions.
Structure-guided development of chemically tailored peptide binders of RNF43/ZNRF3 to enable versatile design of membrane protein-targeting PROTACs.
ER-mitochondria contact sites: a refuge for mitochondrial mRNAs under ER stress.
Integrating fragment-based screening with targeted protein degradation and genetic rescue to explore eIF4E function.
Molecular mechanisms of CAND2 in regulating SCF ubiquitin ligases.
RNF167 mediates atypical ubiquitylation and degradation of RLRs via two distinct proteolytic pathways.
Evolutionary conserved regulation of TFEB stability by the E3 ubiquitin ligase WWP2 modulates response to stress in vivo.
ER-to-Golgi trafficking through a dynamic intermediate cis-Golgi tubular network in Arabidopsis.
Protein degradation and growth dependent dilution substantially shape mammalian proteomes.
Chemoproteomics-Enabled De Novo Proteolysis Targeting Chimera Discovery Platform Identifies a Metallothionein Degrader to Probe Its Role in Cancer.
Spatiotemporal proteomics reveals the biosynthetic lysosomal membrane protein interactome in neurons.
The adaptor protein AP-3β disassembles heat-induced stress granules via 19S regulatory particle in Arabidopsis.
Exploiting the SunTag system to study the developmental regulation of mRNA translation.
Nellie: automated organelle segmentation, tracking and hierarchical feature extraction in 2D/3D live-cell microscopy.
Targeted Management: Unlocking the Crucial Role of PROTACs in Cancer Treatment.
Lineage plasticity of the integrated stress response is a hallmark of cancer evolution.
A bacterial RING ubiquitin ligase triggering stepwise degradation of BRISC via TOLLIP-mediated selective autophagy manipulates host inflammatory response.
Mapping Hsp104 interactions using cross-linking mass spectrometry.
Local structural flexibility drives oligomorphism in computationally designed protein assemblies.
Design of high-affinity binders to immune modulating receptors for cancer immunotherapy.
Endoplasmic reticulum stress as a driver and therapeutic target for kidney disease.
Maintenance of p-eIF2α levels by the eIF2B complex is vital for colorectal cancer.
Rapid and accurate prediction of protein homo-oligomer symmetry using Seq2Symm.
Insights from protein frustration analysis of BRD4-cereblon degrader ternary complexes show separation of strong from weak degraders.
Distinct outcomes from targeted perturbations of the multi-subunit SCFSkp2 E3 ubiquitin ligase in blocking Trp53/Rb1-null prostate tumorigenesis.
Ubiquitin is directly linked via an ester to protein-conjugated mono-ADP-ribose.
Cancer-associated mutations in autophagy-related proteins analyzed in yeast and human cells.
UFMylation of NLRP3 Prevents Its Autophagic Degradation and Facilitates Inflammasome Activation.
A Perspective on Therapeutic Targeting Against Ubiquitin Ligases to Stabilize Tumor Suppressor Proteins.
Reversible ubiquitination conferred by domain shuffling controls paired NLR immune receptor complex homeostasis in plant immunity.
Functional proteoform group deconvolution reveals a broader spectrum of ibrutinib off-targets.
Non-invasive in vivo monitoring of PROTAC-mediated protein degradation using an environment-sensitive reporter.
Hoi1 targets the yeast BLTP2 protein to ER-PM contact sites to regulate lipid homeostasis.
Systems-Level Interactome Mapping Reveals Actionable Protein Network Dysregulation Across the Alzheimer's Disease Spectrum.

J Cell Biol. 2025 May 05. pii: e202312120. [Epub ahead of print]224(5):

The pathway of unconventional protein secretion involves CUPS and a modified trans-Golgi network.

Amy J Curwin, Kazuo Kurokawa, Gonzalo Bigliani, Nathalie Brouwers, Akihiko Nakano, Vivek Malhotra.

Compartment for unconventional protein secretion (CUPS), a compartment for secretion of signal sequence-lacking proteins, forms through COPI-independent extraction of membranes from early Golgi cisternae, lacks Golgi-specific glycosyltransferases, and requires phosphatidylinositol 4-phosphate (PI4P) for biogenesis, as well as phosphatidylinositol 3-phosphate for stability. Our findings demonstrate that Drs2, a PI4P effector from the trans-Golgi network (TGN), is essential for CUPS formation, specifically through its interaction with Rcy1, and Rcy1 is crucial for the unconventional secretion. Using 4D super-resolution confocal live imaging microscopy, we observed that CUPS interact with a modified TGN that contains Drs2 in addition to proteins Tlg2 and Snc2, which are necessary for membrane fusion. Notably, while CUPS remain stable, the modified TGN undergoes remodeling during the later stages of unconventional secretion. In summary, we suggest that CUPS and the modified TGN, without the function of COPII and COPI, participate in collecting and sorting unconventionally secreted proteins, reflecting the role of Golgi membranes in receiving cargo from the ER during conventional secretion.

DOI: https://doi.org/10.1083/jcb.202312120
Elife. 2025 Feb 27. pii: RP101127. [Epub ahead of print]13

PROTAC-induced protein structural dynamics in targeted protein degradation.

Kingsley Y Wu, Ta I Hung, Chia-En A Chang.

  PROteolysis TArgeting Chimeras (PROTACs) are small molecules that induce target protein degradation via the ubiquitin-proteasome system. PROTACs recruit the target protein and E3 ligase; a critical first step is forming a ternary complex. However, while the formation of a ternary complex is crucial, it may not always guarantee successful protein degradation. The dynamics of the PROTAC-induced degradation complex play a key role in ubiquitination and subsequent degradation. In this study, we computationally modelled protein complex structures and dynamics associated with a series of PROTACs featuring different linkers to investigate why these PROTACs, all of which formed ternary complexes with Cereblon (CRBN) E3 ligase and the target protein bromodomain-containing protein 4 (BRD4BD1), exhibited varying degrees of degradation potency. We constructed the degradation machinery complexes with Culling-Ring Ligase 4A (CRL4A) E3 ligase scaffolds. Through atomistic molecular dynamics simulations, we illustrated how PROTAC-dependent protein dynamics facilitating the arrangement of surface lysine residues of BRD4BD1 into the catalytic pocket of E2/ubiquitin cascade for ubiquitination. Despite featuring identical warheads in this PROTAC series, the linkers were found to affect the residue-interaction networks, and thus governing the essential motions of the entire degradation machine for ubiquitination. These findings offer a structural dynamic perspective on ligand-induced protein degradation, providing insights to guide future PROTAC design endeavors.

Keywords:  conformational sampling; drug development; molecular biophysics; molecular mechanics; molecular modeling; none; structural biology; structural dynamic; targeted protein degradation

DOI:  https://doi.org/10.7554/eLife.101127
J Mol Biol. 2025 Feb 22. pii: S0022-2836(25)00101-9. [Epub ahead of print] 169035

Lysosomal degradation of ER client proteins by ER-phagy and related pathways.

Carla Salomo-Coll, Natalia Jimenez-Moreno, Simon Wilkinson.

The endoplasmic reticulum (ER) is a major site of cellular protein synthesis. Degradation of overabundant, misfolded, aggregating or unwanted proteins is required to maintain proteostasis and avoid the deleterious consequences of aberrant protein accumulation, at a cellular and organismal level. While extensive research has shown an important role for proteasomally-mediated, ER-associated degradation (ERAD) in maintaining proteostasis, it is becoming clear that there is a substantial role for lysosomal degradation of "client" proteins from the ER lumen or membrane (ER-to-lysosome degradation, ERLAD). Here we provide a brief overview of the broad categories of ERLAD - predominantly ER-phagy (ER autophagy) pathways and related processes. We collate the client proteins known to date, either individual species or categories of proteins. Where known, we summarise the molecular mechanisms by which they are selected for degradation, and the setting in which lysosomal degradation of the client(s) is important for correct cell or tissue function. Finally, we highlight the questions that remain open in this area.

DOI: https://doi.org/10.1016/j.jmb.2025.169035
Nucleic Acids Res. 2025 Feb 08. pii: gkaf088. [Epub ahead of print]53(4):

RNA exosome-driven RNA processing instructs the duration of the unfolded protein response.

Laura Matabishi-Bibi, Coralie Goncalves, Anna Babour.

Upon stresses, cellular compartments initiate adaptive programs meant to restore homeostasis. Dedicated to the resolution of transient perturbations, these pathways are typically maintained at a basal level, activated upon stress, and critically downregulated upon reestablishment of cellular homeostasis. As such, prolonged activation of the unfolded protein response (UPR), a conserved adaptive transcriptional response to defective endoplasmic reticulum (ER) proteostasis, leads to cell death. Here, we elucidate an unanticipated role for the nuclear RNA exosome, an evolutionarily conserved ribonuclease complex that processes multiple classes of RNAs, in the control of UPR duration. Remarkably, the inactivation of Rrp6, an exclusively nuclear catalytic subunit of the RNA exosome, curtails UPR signaling, which is sufficient to promote the cell's resistance to ER stress. Mechanistically, accumulation of unprocessed RNA species diverts the processing machinery that maturates the messenger RNA encoding the master UPR regulator Hac1, thus restricting the UPR. Significantly, Rrp6 expression is naturally dampened upon ER stress, thereby participating in homeostatic UPR deactivation.

DOI: https://doi.org/10.1093/nar/gkaf088
Sci Adv. 2025 Feb 28. 11(9): eadq4187

DET1 dynamics underlie cooperative ubiquitination by CRL4DET1-COP1 complexes.

Abigail E Burgess, Tarren A Loughran, Liam S Turk, Hunter G Nyvall, Jessica L Dunlop, Sam A Jamieson, Jack R Curry, John E Burke, Pavel Filipcik, Simon H J Brown, Peter D Mace.

Transcription factor ubiquitination is a decisive regulator of growth and development. The DET1-DDB1-DDA1 (DDD) complex associates with the Cullin-4 ubiquitin ligase (CRL4) and a second ubiquitin ligase, COP1, to control ubiquitination of transcription factors involved in neurological, metabolic, and immune cell development. Here, we report the structure of the human DDD complex, revealing a specific segment of DET1 that can recruit ubiquitin-conjugating (E2) enzymes. Structural variability analysis, mass spectrometry, and mutagenesis based on AlphaFold predictions suggest that dynamic closure of DET1, stabilized by DDA1, underlies coordinated recruitment of E2 enzymes and COP1. Biochemical assays suggest that the E2 acts as a recruitment factor to bring COP1 to DET1 for more effective substrate ubiquitination, which parallels a catalytically inactive E2 enzyme (COP10) in plant DDD complexes. This work provides a clear architecture for regulation and cooperative CRL4DET1-COP1 complex assembly, which can affect degradation of diverse targets by COP1 complexes.

DOI: https://doi.org/10.1126/sciadv.adq4187
Pathogens. 2025 Feb 02. pii: 132. [Epub ahead of print]14(2):

Dynamic Modulation of IRE1α-XBP1 Signaling by Adenovirus.

Yumi Jang, Fred Bunz.

  The abundant production of foreign proteins and nucleic acids during viral infection elicits a variety of stress responses in host cells. Viral proteins that accumulate in the endoplasmic reticulum (ER) can trigger the unfolded protein response (UPR), a coordinated signaling program that culminates in the expression of downstream genes that collectively restore protein homeostasis. The model pathogen adenovirus serotype 5 (HAdV5) activates the UPR via the signaling axis formed by inositol-requiring enzyme type 1 (IRE1α) and the X-box binding protein 1 (XBP1), a transcription factor required for immune function. Recent studies have suggested that IRE1α-XBP1 activity supports adenovirus replication. Here, we show that HAdV5 exerted opposing effects on IRE1α and XBP1. IRE1α was activated in response to HAdV5, but the production of the XBP1 isoform, XBP1s, was post-transcriptionally blocked. The tumor suppressor p53, which is eliminated by HAdV5 after infection, inhibited IRE1α activation. The de-repression of IRE1α following the degradation of p53 conceivably reflects a novel antiviral mechanism, which HAdV5 ultimately evades by co-opting IRE1α and suppressing XBP1s. Our findings illustrate the opposing mechanisms used by adenoviruses and their host cells to exert control over the UPR, a critical determinant of cell fate.

Keywords:  XBP1 splicing; adenovirus; p53; unfolded protein response

DOI:  https://doi.org/10.3390/pathogens14020132
bioRxiv. 2025 Feb 16. pii: 2025.02.12.637885. [Epub ahead of print]

Epsin1 enforces a condensation-dependent checkpoint for ubiquitylated cargo during clathrin-mediated endocytosis.

Susovan Sarkar, Hao-Yang Liu, Feng Yuan, Brandon T Malady, Liping Wang, Jessica Perez, Eileen M Lafer, Jon M Huibregtse, Jeanne C Stachowiak.

Clathrin-mediated endocytosis internalizes proteins and lipids from the cell surface, supporting nutrient uptake, signaling, and membrane trafficking. Recent work has demonstrated that a flexible, liquid-like network of initiator proteins is responsible for catalyzing assembly of clathrin-coated vesicles in diverse organisms including yeast, mammals, and plants. How do cells regulate the assembly of this dynamic network to produce cargo-loaded vesicles? Here we reveal the ability of an endocytic adaptor protein, Epsin1, to conditionally stabilize the initiator protein network, creating a cargo-dependent checkpoint during clathrin-mediated endocytosis. Epsin1 is known to recruit ubiquitylated transmembrane proteins to endocytic sites. Using in vitro assays, we demonstrate that Epsin1 uses competitive binding and steric repulsion to destabilize condensation of initiator proteins in the absence of ubiquitin. However, when polyubiquitin is present, Epsin1 binds to both ubiquitin and initiator proteins, creating attractive interactions that stabilize condensation. Similarly, in mammalian cells, endocytic dynamics and ligand uptake are disrupted by removal of either ubiquitin or Epsin1. Surprisingly, when Epsin1 and ubiquitin are removed simultaneously, endocytic defects are rescued to near wildtype levels, although endocytic sites lose the ability to distinguish between ubiquitylated and non-ubiquitylated cargos. Taken together, these results suggest that Epsin1 tunes protein condensation to ensure the presence of ubiquitylated cargo during assembly of clathrin-coated vesicles. More broadly, these findings illustrate how a balance of attractive and repulsive molecular interactions controls the stability of liquid-like protein networks, providing dynamic control over key cellular events.

DOI: https://doi.org/10.1101/2025.02.12.637885
Adv Sci (Weinh). 2025 Feb 22. e2414519

Control of Rhizobia Endosymbiosis by Coupling ER Expansion with Enhanced UPR.

Jing Ren, Qi Wang, Xiaxia Zhang, Yongheng Cao, JingXia Wu, Juan Tian, Yanjun Yu, Qingqiu Gong, Zhaosheng Kong.

  Legumes establish symbiosis with rhizobia by forming a symbiotic interface that enables cross-kingdom exchanges of signaling molecules and nutrients. However, how host organelles interact with symbiosomes at the symbiotic interface remains elusive during rhizobia endosymbiosis. Here, symbiotic cells are reconstructed using 3D scanning electron microscopy (SEM) and uncover that the host endoplasmic reticulum (ER) undergoes dynamic expansion to gradually enwrap symbiosomes, facilitating their compartmentalization and endosymbiosis. Consistently, altering ER lamellar expansion by overexpressing MtRTNLBs, the reticulons responsible for ER tubulation, impairs rhizobia accommodation and symbiosome development. Intriguingly, unfolded protein response (UPR)-marker genes, bZIP60 and IRE1A/B, show continuously activated expression during nodule development, and the two UPR-deficient mutants, ire1b, and bzip60, exhibit compromised ER biogenesis and defective symbiosome development. Collectively, the findings underpin ER expansion and UPR activation as two key events in rhizobia accommodation and reveal an intrinsic coupling of ER morphology with proper UPR during root nodule symbiosis.

Keywords:  ER; UPR; compartmentalization; endosymbiosis; symbiosomes

DOI:  https://doi.org/10.1002/advs.202414519
Mol Cell. 2025 Feb 19. pii: S1097-2765(25)00105-4. [Epub ahead of print]

Predictomes, a classifier-curated database of AlphaFold-modeled protein-protein interactions.

Ernst W Schmid, Johannes C Walter.

  Protein-protein interactions (PPIs) are ubiquitous in biology, yet a comprehensive structural characterization of the PPIs underlying cellular processes is lacking. AlphaFold-Multimer (AF-M) has the potential to fill this knowledge gap, but standard AF-M confidence metrics do not reliably separate relevant PPIs from an abundance of false positive predictions. To address this limitation, we used machine learning on curated datasets to train a structure prediction and omics-informed classifier (SPOC) that effectively separates true and false AF-M predictions of PPIs, including in proteome-wide screens. We applied SPOC to an all-by-all matrix of nearly 300 human genome maintenance proteins, generating ∼40,000 predictions that can be viewed at predictomes.org, where users can also score their own predictions with SPOC. High-confidence PPIs discovered using our approach enable hypothesis generation in genome maintenance. Our results provide a framework for interpreting large-scale AF-M screens and help lay the foundation for a proteome-wide structural interactome.

Keywords:  AF-M database; AF-M score; AlphaFold-multimer; PPI database; PPI screen; SPOC; genome maintenance; in-silico interaction screen; predictomes; protein-protein interactions

DOI:  https://doi.org/10.1016/j.molcel.2025.01.034
Angew Chem Int Ed Engl. 2025 Feb 25. e202501488

Structure-guided development of chemically tailored peptide binders of RNF43/ZNRF3 to enable versatile design of membrane protein-targeting PROTACs.

Man Pan, Jibin Cui, Qingyun Zheng, Yicheng Weng, Xiaoguo Zhai, Zhen Su, YunXiang Du, Xiaoxiong Wei, Yuanyuan Yu, Qian Qu.

  Targeted membrane protein degradation using cell surface E3 ligases RNF43/ZNRF3 via proteolysis targeting chimeras (PROTACs) represents an effective strategy for treating membrane drug targets that cannot be fully inhibited using traditional inhibitors. Several ingenious chimeras have been developed to tether RNF43/ZNRF3 to target membrane proteins, resulting in the degradation of targets at sub-nanomolar concentrations both in vitro and in vivo. However, currently available RNF43/ZNRF3 binders are genetically encoded and have poor plasticity, which limits the design and promotion of such PROTACs. Here, we exploited the alphafold predicted complex structures of ligand-bound RNF43/ZNRF3 and developed a class of chemically tailored peptide binders for ZNRF3/RNF43. With these peptide binders that can be conveniently prepared by de novo peptide synthesis, we established a new membrane protein degradation platform that allows versatile modular design and targeted degradation of clinically relevant membrane proteins, i.e. PD-L1 and EGFR. This study presents a new subtype within the PROTAC field to develop therapeutic peptides targeting membrane proteins.

Keywords:  Targeted membrane protein degradation * Cell-surface E3 ligases * Peptide binders * Chemically tailored degraders * Synthesis and refolding of disulfide-rich peptide

DOI:  https://doi.org/10.1002/anie.202501488
Trends Cell Biol. 2025 Feb 25. pii: S0962-8924(25)00036-4. [Epub ahead of print]

ER-mitochondria contact sites: a refuge for mitochondrial mRNAs under ER stress.

Philippe Pihán, Lisa M Ellerby, Claudio Hetz.

  Tight mitochondria-endoplasmic reticulum (ER) contacts (MERCS) play essential roles in cellular homeostasis. Brar et al. reveal a novel mechanism where mitochondrial mRNAs escape global translational repression at novel context-specific MERCS during ER stress, uncovering spatially regulated translation as a critical adaptive strategy to cope with cellular stress.

Keywords:  ATAD3A; PERK; endoplasmic reticulum stress; mitochondria–ER contact sites (MERCS); spatial translation regulation

DOI:  https://doi.org/10.1016/j.tcb.2025.02.002
Nat Commun. 2024 Nov 20. 15(1): 10037

Integrating fragment-based screening with targeted protein degradation and genetic rescue to explore eIF4E function.

Swee Y Sharp, Marianna Martella, Sabrina D'Agostino, Christopher I Milton, George Ward, Andrew J Woodhead, Caroline J Richardson, Maria G Carr, Elisabetta Chiarparin, Benjamin D Cons, Joseph Coyle, Charlotte E East, Steven D Hiscock, Carlos Martinez-Fleites, Paul N Mortenson, Nick Palmer, Puja Pathuri, Marissa V Powers, Susanne M Saalau, Jeffrey D St Denis, Kate Swabey, Mladen Vinković, Hugh Walton, Glyn Williams, Paul A Clarke.

Eukaryotic initiation factor 4E (eIF4E) serves as a regulatory hub for oncogene-driven protein synthesis and is considered a promising anticancer target. Here we screen a fragment library against eIF4E and identify a ligand-binding site with previously unknown function. Follow-up structure-based design yields a low nM tool compound (4, Kd = 0.09 µM; LE 0.38), which disrupts the eIF4E:eIF4G interaction, inhibits translation in cell lysates, and demonstrates target engagement with eIF4E in intact cells (EC50 = 2 µM). By coupling targeted protein degradation with genetic rescue using eIF4E mutants, we show that disruption of both the canonical eIF4G and non-canonical binding sites is likely required to drive a strong cellular effect. This work highlights the power of fragment-based drug discovery to identify pockets in difficult-to-drug proteins and how this approach can be combined with genetic characterization and degrader technology to probe protein function in complex biological systems.

DOI: https://doi.org/10.1038/s41467-024-54356-1
Nat Commun. 2025 Feb 26. 16(1): 1998

Molecular mechanisms of CAND2 in regulating SCF ubiquitin ligases.

Kankan Wang, Lihong Li, Sebastian Kenny, Dailin Gan, Justin M Reitsma, Yun Zhou, Chittaranjan Das, Xing Liu.

Protein degradation orchestrated by SKP1·CUL1·F-box protein (SCF) ubiquitin ligases is a fundamental process essential for cellular and organismal function. The dynamic assembly of SCFs, facilitated by CAND1, ensures timely ubiquitination of diverse SCF target proteins. As a homolog of CAND1, CAND2 alone has been implicated in various human diseases, yet its functional mechanisms remain elusive. Here, we investigate the role of CAND2 in human cells and its distinct mode of action compared to CAND1. Using an array of quantitative assays, we demonstrate that CAND2 promotes SCF-mediated protein degradation as an F-box protein exchange factor. While CAND2 binds CUL1 with structure and affinity comparable to CAND1, it exhibits lower efficiency in exchanging F-box proteins. Kinetic measurements reveal a significantly higher KM for CAND2-catalyzed SCF disassembly than CAND1, which explains the lower exchange efficiency of CAND2 and is likely due to conformations of the CAND2·SCF exchange intermediate complex being less favorable for F-box protein dissociation. Our study provides mechanistic insights into the biochemical and structural properties of CAND2, as well as its role in regulating cellular dynamics of SCFs, laying a foundation for understanding contributions of CAND2 to healthy and diseased human cells.

DOI: https://doi.org/10.1038/s41467-025-57065-5
Nat Commun. 2025 Feb 24. 16(1): 1920

RNF167 mediates atypical ubiquitylation and degradation of RLRs via two distinct proteolytic pathways.

Miao He, Zixiao Yang, Luyang Xie, Junhai Chen, Shurui Liu, Liaoxun Lu, Zibo Li, Birong Zheng, Yu Ye, Yuxin Lin, Lang Bu, Jingshu Xiao, Yongheng Zhong, Penghui Jia, Qiang Li, Yinming Liang, Deyin Guo, Chun-Mei Li, Panpan Hou.

The precise regulation of the RIG-I-like receptors (RLRs)-mediated type I interferon (IFN-I) activation is crucial in antiviral immunity and maintaining host immune homeostasis in the meantime. Here, we identify an E3 ubiquitin ligase, namely RNF167, as a negative regulator of RLR-triggered IFN signaling. Mechanistically, RNF167 facilitates both atypical K6- and K11-linked polyubiquitination of RIG-I/MDA5 within CARD and CTD domains, respectively, which leads to degradation of the viral RNA sensors through dual proteolytic pathways. RIG-I/MDA5 conjugated with K6-linked ubiquitin chains in CARD domains is recognized by the autophagy cargo adaptor p62, that delivers the substrates to autolysosomes for selective autophagic degradation. In contrast, K11-linked polyubiquitination in CTD domains leads to proteasome-dependent degradation of RLRs. Thus, our study clarifies a function of atypical K6- and K11-linked polyubiquitination in the regulation of RLR signaling. We also unveil an elaborate synergistic effect of dual proteolysis systems to control amplitude and duration of IFN-I activation, hereby providing insights into physiological roles of the cross-talk between these two protein quality control pathways.

DOI: https://doi.org/10.1038/s41467-025-57245-3
iScience. 2025 Feb 21. 28(2): 111838

Evolutionary conserved regulation of TFEB stability by the E3 ubiquitin ligase WWP2 modulates response to stress in vivo.

Juan A Garcia-Sanchez, Estelle Bonnet, Céline Loubatier, Anne Doye, Guillaume Paillier, Fabien Segui, Frédéric Larbret, Paul Chaintreuil, Ludovic Batistic, Cédric Torre, Marcel Deckert, Jolanta Polanowska, Patrick Munro, Laurent Boyer, Orane Visvikis.

  Transcription factor EB (TFEB) is a key transcription factor that orchestrates the cellular response to stress. Dysregulation of TFEB is associated with a range of human diseases, and understanding the regulatory mechanisms of TFEB is crucial for identifying potential drug targets. In this study, we used Caenorhabditis elegans to screen for E3 ubiquitin ligases regulating the activity of TFEB's homolog, HLH-30, upon pathogenic infection. We identified WWP-1 as a regulator of HLH-30-dependent immune response controlling HLH-30 stability to mediate host defense in vivo. We found that HLH-30 interacts with WWP-1, supporting a model of WWP-1 directly regulating HLH-30. Furthermore, we found that WWP-1's human homolog WWP2 binds TFEB, directly induces TFEB ubiquitination and stabilizes TFEB. Finally, we found that WWP2 is required for TFEB-dependent host response in human monocytes-derived macrophages upon infection. Overall, our work has identified an evolutionarily conserved regulation of TFEB by WWP2 and highlighted its role in modulating stress response.

Keywords:  Cell biology; Functional aspects of cell biology

DOI:  https://doi.org/10.1016/j.isci.2025.111838
Nat Cell Biol. 2025 Feb 25.

ER-to-Golgi trafficking through a dynamic intermediate cis-Golgi tubular network in Arabidopsis.

Louise Fougère, Magali Grison, Patricia Laquel, Matheus Montrazi, Fabrice Cordelières, Mónica Fernández-Monreal, Christel Poujol, Tomohiro Uemura, Akihiko Nakano, Yoko Ito, Yohann Boutté.

Endoplasmic reticulum (ER)-to-Golgi trafficking is a central process of the secretory system of eukaryotic cells that ensures proper spatiotemporal sorting of proteins and lipids. However, the nature of the ER-Golgi intermediate compartments (ERGICs) and the molecular mechanisms mediating the transition between ERGICs and the Golgi, as well as the universality of these processes among eukaryotes, remain undiscovered. Here we identify a reticulated tubulo-vesicular network, labelled by MEMBRIN proteins, that is mostly independent of the Golgi, highly dynamic at the ER-Golgi interface and crossed by ER-induced released luminal cargos. We find that plant ERGICs become stabilized by the interaction they establish with pre-existing Golgi and gradually mature into Golgi cisternae, this process being dependent on C24-ceramide sphingolipids. Our study is a major twist in the understanding of the Golgi, as it identifies that the ERGICs in plants comprise a Golgi-independent and highly dynamic tubular network from which arise more stable Golgi-associated pre-cisternae structures.

DOI: https://doi.org/10.1038/s41556-025-01624-x
bioRxiv. 2025 Feb 12. pii: 2025.02.10.637566. [Epub ahead of print]

Protein degradation and growth dependent dilution substantially shape mammalian proteomes.

Andrew Leduc, Nikolai Slavov.

Cellular protein concentrations are maintained through a balance of synthesis and clearance. Clearance occurs through both protein degradation and growth-dependent dilution. At slow growth, clearance is dominated by degradation, which leads to the accumulation of long lived proteins. At fast growth, however, it is dominated by dilution, preventing this accumulation. Thus, the concentration of long lived proteins will be reduced unless cells compensate by preferentially increasing synthesis rates. To determine the dominant regulatory mechanisms, we quantified the degree of compensation between activated and resting human B cells and across mouse tissues. The results indicate that growth-dependent dilution is insufficiently compensated for by changes in protein synthesis, and it accounts for over a third of the concentration changes between high and low growth conditions. Furthermore, we find that about 25 % of the differences in protein concentration across all tissues are controlled by protein clearance. When comparing only slowly growing tissues such as the brain and pancreas, clearance differences explain as much as 42 %. Within a tissue or cell type, clearance variation is sufficient to account for 50 % of the abundance variation for all measured proteins at slow growth, contrasted with 7 % at fast growth. Thus, our model unifies previous observations with our new results and highlights a context-dependent and larger than previously appreciated contribution of protein degradation in shaping protein variation both across the proteome and across cell states.

DOI: https://doi.org/10.1101/2025.02.10.637566
J Am Chem Soc. 2025 Feb 23.

Chemoproteomics-Enabled De Novo Proteolysis Targeting Chimera Discovery Platform Identifies a Metallothionein Degrader to Probe Its Role in Cancer.

Brittney Racioppo, Dany Pechalrieu, Daniel Abegg, Brendan Dwyer, Neal Thomas Ramseier, Ying S Hu, Alexander Adibekian.

Proteolysis targeting chimeras (PROTACs) represent powerful tools to modulate the activity of classically "undruggable" proteins, but their application has been limited to known ligands and a few select protein classes. Herein, we present our chemoproteomic strategy for simultaneous de novo discovery of novel degraders and ligands for challenging and previously "undruggable" targets. Using comparative PROTAC versus ligand global proteomics analyses, we rapidly identify proteins selectively downregulated by several "untargeted" PROTACs containing a VHL E3 ligase recruiter and various covalent and noncovalent ligands. We showcase our approach by identifying a first-in-class PROTAC for metallothionein 2A (MT2A), a small, cysteine-rich, metal-binding protein implicated in heavy metal detoxification, zinc homeostasis, and cellular invasion. Notably, isoform-specific MT overexpression has been shown to augment cellular migration and invasion across several cancer cell lines, although the precise mechanisms are unknown due to insufficient tools to study MTs. We show that optimized PROTAC AA-BR-157 covalently binds conserved C44, degrades overexpressed MT2A with nanomolar potency, and reduces the migration and invasion of MDA-MB-231 cells. We further demonstrate a time-dependent increase in intracellular zinc levels following MT2A degradation as well as downregulation of protein diaphanous homolog 3 (DIAPH3), a positive regulator of actin and cell motility. Super-resolution imaging of MDA-MB-231 cells shows that the downregulation of MT2A and DIAPH3 inhibits cell polarization and thereby migration, suggesting that MT2A may regulate motility via DIAPH3-dependent cytoskeletal remodeling. In summary, our strategy enables the de novo discovery of PROTACs and ligands for novel disease-related targets and lays the groundwork for expansion of the druggable proteome.

DOI: https://doi.org/10.1021/jacs.4c17827
Nat Commun. 2024 Dec 30. 15(1): 10829

Spatiotemporal proteomics reveals the biosynthetic lysosomal membrane protein interactome in neurons.

Chun Hei Li, Noortje Kersten, Nazmiye Özkan, Dan T M Nguyen, Max Koppers, Harm Post, Maarten Altelaar, Ginny G Farias.

Lysosomes are membrane-bound organelles critical for maintaining cellular homeostasis. Delivery of biosynthetic lysosomal proteins to lysosomes is crucial to orchestrate proper lysosomal function. However, it remains unknown how the delivery of biosynthetic lysosomal proteins to lysosomes is ensured in neurons, which are highly polarized cells. Here, we developed Protein Origin, Trafficking And Targeting to Organelle Mapping (POTATOMap), by combining trafficking synchronization and proximity-labelling based proteomics, to unravel the trafficking routes and interactome of the biosynthetic lysosomal membrane protein LAMP1 at specified time points. This approach, combined with advanced microscopy, enables us to identify the neuronal domain-specific trafficking machineries of biosynthetic LAMP1. We reveal a role in replenishing axonal lysosomes, in delivery of newly synthesized axonal synaptic proteins, and interactions with RNA granules to facilitate hitchhiking in the axon. POTATOMap offers a robust approach to map out dynamic biosynthetic protein trafficking and interactome from their origin to destination.

DOI: https://doi.org/10.1038/s41467-024-55052-w
Nat Commun. 2025 Feb 27. 16(1): 2039

The adaptor protein AP-3β disassembles heat-induced stress granules via 19S regulatory particle in Arabidopsis.

Lei Pang, Yuanzhi Huang, Yilin He, Dong Jiang, Ruixi Li.

To survive under adverse conditions, plants form stress granules (SGs) to temporally store mRNA and halt translation as a primary response. Dysregulation in SG disassembly can have detrimental effects on plant survival after stress release, yet the underlying mechanism remains poorly understood. Using Arabidopsis as a model system, we demonstrate that the β subunit of adaptor protein (AP) -3 complex (AP-3β) interacts with the SG core RNA-binding proteins Tudor staphylococcal nuclease 1/2 (TSN1/2) both in vitro and in vivo. We also show that AP-3β is rapidly recruited to SGs upon heat induction and plays a key role in disassembling SGs during stress recovery. Genetic evidences support that AP-3β serves as an adaptor to recruit the 19S regulatory particle (RP) of the proteasome to SGs. Notably, the 19S RP promotes SG disassembly through RP-associated deubiquitylation, independent of its proteolytic activity. This deubiquitylation process of SG components is crucial for translation reinitiation and growth recovery after heat release. Our findings uncover a previously unexplored role of the 19S RP in regulating SG disassembly and highlights the importance of endomembrane proteins in supporting RNA granule dynamics in plants.

DOI: https://doi.org/10.1038/s41467-025-57306-7
J Cell Sci. 2025 Feb 24. pii: jcs.263457. [Epub ahead of print]

Exploiting the SunTag system to study the developmental regulation of mRNA translation.

Alastair Pizzey, Catherine Sutcliffe, Jennifer C Love, Emmanuel Akabuogu, Magnus Rattray, Mark P Ashe, Hilary L Ashe.

  The ability to quantitatively study mRNA translation using SunTag imaging is transforming our understanding of the translation process. Here, we expand the SunTag method to study new aspects of translation regulation in Drosophila. Repression of the maternal hunchback (hb) mRNA in the posterior of the Drosophila embryo is a textbook example of translational control. Using SunTag imaging to quantitate translation of maternal SunTag-hb mRNAs, we show that repression in the posterior is leaky as ∼5% of SunTag-hb mRNAs are translated. In the anterior of the embryo, the maternal and zygotic SunTag-hb mRNAs show similar translation efficiency despite having different UTRs. We demonstrate that the SunTag-hb mRNA can be used as a reporter to study ribosome pausing at single-mRNA resolution, by exploiting the conserved xbp1 mRNA and A60 pausing sequences. Finally, we adapt the detector component of the SunTag system to visualise and quantitate translation of the short gastrulation (sog) mRNA, encoding an essential secreted extracellular BMP regulator, at the endoplasmic reticulum in fixed and live embryos. Together, these tools will facilitate the future dissection of translation regulatory mechanisms during development.

Keywords:   Drosophila embryo; Hunchback; Ribosome pausing; Sog; SunTag; Translation

DOI:  https://doi.org/10.1242/jcs.263457
Nat Methods. 2025 Feb 27.

Nellie: automated organelle segmentation, tracking and hierarchical feature extraction in 2D/3D live-cell microscopy.

Austin E Y T Lefebvre, Gabriel Sturm, Ting-Yu Lin, Emily Stoops, Magdalena Preciado López, Benjamin Kaufmann-Malaga, Kayley Hake.

Cellular organelles undergo constant morphological changes and dynamic interactions that are fundamental to cell homeostasis, stress responses and disease progression. Despite their importance, quantifying organelle morphology and motility remains challenging due to their complex architectures, rapid movements and the technical limitations of existing analysis tools. Here we introduce Nellie, an automated and unbiased pipeline for segmentation, tracking and feature extraction of diverse intracellular structures. Nellie adapts to image metadata and employs hierarchical segmentation to resolve sub-organellar regions, while its radius-adaptive pattern matching enables precise motion tracking. Through a user-friendly Napari-based interface, Nellie enables comprehensive organelle analysis without coding expertise. We demonstrate Nellie's versatility by unmixing multiple organelles from single-channel data, quantifying mitochondrial responses to ionomycin via graph autoencoders and characterizing endoplasmic reticulum networks across cell types and time points. This tool addresses a critical need in cell biology by providing accessible, automated analysis of organelle dynamics.

DOI: https://doi.org/10.1038/s41592-025-02612-7
Curr Drug Discov Technol. 2025 Feb 26.

Targeted Management: Unlocking the Crucial Role of PROTACs in Cancer Treatment.

Priyanka Gupta, Sumit Dutta, Prashant Kumar, Monika Kaushik, Sumel Ashique, Mithun Bhowmick.

  Targeted Protein Degradation (TPD) offers a solution, eliminating disease-related proteins and overcoming challenges associated with unintended toxicity and lack of precision. PROTACs (Proteolysis Targeting Chimeras) represent an innovative strategy for the specific degradation of tar-get proteins through the UPS (Ubiquitin-Proteasome System). In comparison to conventional protein inhibitor medications, PROTAC offers advantages in terms of efficacy, selectivity, and the ability to overcome drug resistance in cancer treatment, contributing novel perspectives to the field of anti-cancer drug discovery. Proteins play vital roles in an organism's health, and misfolded contributes to diseases like neurodegenerative disorders and cancer. Cells maintain protein balance through quality control systems, primarily the UPS and autophagy. Protac, a Targeted Protein Degradation (TPD) strategy, utilizes UPS, employing small molecules to induce targeted protein degradation. PROTAC exhibits promise in preclinical studies and clinical trials for diverse cancers. Notable examples in-clude breast cancer, where PROTAC targets CDK4/6 (cyclin-dependent kinase) and Estrogen Recep-tors (ER), prostate cancer, addressing Androgen Receptor (AR) degradation, hematologic malignan-cies, focusing on AURORA-A and CDKs, and NSCLC (Non-Small-Cell Lung Cancer), targeting Estimated Glomerular Filtration Rate (EGFR), and KRAS. Despite their potential, PROTAC faces challenges, including compensatory protein expression in response to targeted therapies. This com-prehensive review explores recent advancements in PROTAC and related technologies, emphasizing the mechanisms and structures of PROTAC and their applications in proteins targeting cancer.

Keywords:  AbTAC; PROTACs; RNA-PROTAC; cancer therapy; targeted protein degradation; ubiquitin-proteasome system.

DOI:  https://doi.org/10.2174/0115701638324854250218053353
bioRxiv. 2025 Feb 13. pii: 2025.02.10.637516. [Epub ahead of print]

Lineage plasticity of the integrated stress response is a hallmark of cancer evolution.

Shiqi Diao, Jia Yi Zou, Shuo Wang, Nour Ghaddar, Jason E Chan, Hyungdong Kim, Nicolas Poulain, Constantinos Koumenis, Maria Hatzoglou, Peter Walter, Nahum Sonenberg, John Le Quesne, Tuomas Tammela, Antonis E Koromilas.

The link between the "stress phenotype"-a well-established hallmark of cancer-and its role in tumor progression and intratumor heterogeneity remains poorly defined. The integrated stress response (ISR) is a key adaptive pathway that enables tumor survival under oncogenic stress. While ISR has been implicated in promoting tumor growth, its precise role in driving tumor evolution and heterogeneity has not been elucidated. In this study, using a genetically engineered mouse models, we demonstrate that ISR activation-indicated by elevated levels of phosphorylated eIF2 (p-eIF2) and ATF4-is essential for the emergence of dedifferentiated, therapy-resistant cell states. ISR, through the coordinated actions of ATF4 and MYC, facilitates the development of tumor cell populations characterized by high plasticity, stemness, and an epithelial-mesenchymal transition (EMT)-prone phenotype. This process is driven by ISR-mediated expression of genes that maintain mitochondrial integrity and function, critical for sustaining tumor progression. Importantly, genetic, or pharmacological inhibition of the p-eIF2-ATF4 signaling axis leads to mitochondrial dysfunction and significantly impairs tumor growth in mouse models of lung adenocarcinoma (LUAD). Moreover, ISR-driven dedifferentiation is associated with poor prognosis and therapy resistance in advanced human LUAD, underscoring ISR inhibition as a promising therapeutic strategy to disrupt tumor evolution and counteract disease progression.

DOI: https://doi.org/10.1101/2025.02.10.637516
Autophagy. 2025 Feb 27. 1-20

A bacterial RING ubiquitin ligase triggering stepwise degradation of BRISC via TOLLIP-mediated selective autophagy manipulates host inflammatory response.

Xinming Pan, Yangyang Sun, Jianan Liu, Rong Chen, Zhen Zhang, Caiying Li, Huochun Yao, Jiale Ma.

  Numerous bacterial pathogens have evolved tactics to interfere with the host ubiquitination network to evade clearance by the innate immune system. Nevertheless, the subtle antagonism between a bacterial ubiquitinase and a host deubiquitinase, through which they modify their respective targets within a multifaceted network, has yet to be characterized. BRCC3 isopeptidase complex (BRISC) is a newly identified K63-specific deubiquitinase complex that plays a crucial role in cellular signaling pathways such as inflammation. NleG, a type III secretion system (T3SS) effector, contains a conserved RING E3 ubiquitin ligase domain that interacts with host ubiquitination machinery, along with a distinct substrate-recognition domain that targets host proteins. Here, one particular variant, NleG6, was identified as mediating K27- and K29-linked polyubiquitination at residues K89 and K114 of ABRAXAS2/FAM175B, a scaffolding protein within the BRISC complex, leading to its degradation through TOLLIP (toll interacting protein)-mediated selective autophagy. Further investigations elucidated that ABRAXAS2 degradation triggered the subsequent degradation of adjacent BRCC3, which in turn, hindered TNIP1/ABIN1 degradation, ultimately inhibiting NFKB/NF-κB (nuclear factor kappa B)-mediated inflammatory responses. This chain of events offers valuable insights into the NFKB activation by the K63-specific deubiquitinating role of BRISC, unveiling how bacteria manipulate ubiquitin regulation and selective autophagy within the BRISC network to inhibit the host's inflammatory response and thus dominate a pathogen-host tug-of-war.Abbreviations: 3-MA: 3-methyladenine; A/E: attaching and effacing; ATG7: autophagy related 7; BafA1: bafilomycin A1; BNIP3L/Nix: BCL2 interacting protein 3 like; BRISC: BRCC3 isopeptidase complex; Cas9: CRISPR-associated system 9; co-IP: co-immunoprecipitation; CQ: chloroquine; CRISPR: clustered regulatory interspaced short palindromic repeat; DAPI: 4',6-diamidino2-phenylindole; DMSO: dimethyl sulfoxide; DUB: deubiquitinating enzyme; E. coli: Escherichia coli; EHEC: enterohemorrhagic Escherichia coli; EPEC: enteropathogenic Escherichia coli; GFP: green fluorescent protein; LEE: locus of enterocyte effacement; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MG132: cbz-leu-leu-leucinal; MOI: multiplicity of infection; NBR1: NBR1 autophagy cargo receptor; NC: negative control; NFKB/NF-κB: nuclear factor kappa B; NH4Cl: ammonium chloride; OPTN: optineurin; SQSTM1/p62: sequestosome 1; sgRNAs: small guide RNAs; T3SS: type III secretion system; TNF: tumor necrosis factor; TOLLIP: toll interacting protein; TRAF: TNF receptor associated factor; TUBB: tubulin beta class I; WCL: whole cell lysate; WT: wide type.

Keywords:  BRISC complex; E3 ubiquitin ligase; NFKB; NleG; selective autophagy; type III secretion system

DOI:  https://doi.org/10.1080/15548627.2025.2468140
FEBS Open Bio. 2025 Feb 24.

Mapping Hsp104 interactions using cross-linking mass spectrometry.

Kinga Westphal, Karolina Michalska, Andrzej Joachimiak, Lukasz A Joachimiak.

  Molecular machines from the AAA+ (ATPases Associated with diverse cellular Activity) superfamily of protein disaggregases play important roles in protein folding, disaggregation and DNA processing. Recent cryo-EM structures of AAA+ molecular machines have uncovered nuanced changes in their conformation that underlie their specialized functions. Structural knowledge of these molecular machines in complex with substrates begins to explain their mechanism of activity. Here, we explore how cross-linking mass spectrometry (XL-MS) can be used to interpret changes in conformation induced by ATP in Hsp104 and how a substrate may interact with Hsp104. We applied a panel of cross-linking reagents to produce cross-linking maps of Hsp104 and interpret our data on previously determined X-ray and cryo-EM structures of Hsp104 from a thermophilic yeast, Calcarisporiella thermophila. We developed an analysis pipeline to differentiate between intra-subunit and inter-subunit contacts within the hexameric homo-oligomer. We identify cross-links that break the asymmetry that is present in Hsp104 in an ATP-hydrolysis competent conformation but is absent in an ATP-hydrolysis-defective mutant. Finally, we identify contacts between Hsp104 and a selected protein (proprotein convertase subtilisin/kexin type 9 PCSK9) to reveal contacts on the central channel of Hsp104 across the length of this protein indicating that we might have trapped interactions consistent with its translocation. Our simple and robust XL-MS-based experiments and methods help interpret how these molecular machines change conformation and bind to other proteins even in the context of homo-oligomeric assemblies enabling coupling state-of-the-art modeling approaches with XL-MS.

Keywords:  Hsp104; PCSK9; XL‐MS; protein–protein interactions

DOI:  https://doi.org/10.1002/2211-5463.70007
Nat Struct Mol Biol. 2025 Feb 26.

Local structural flexibility drives oligomorphism in computationally designed protein assemblies.

Alena Khmelinskaia, Neville P Bethel, Farzad Fatehi, Bhoomika Basu Mallik, Aleksandar Antanasijevic, Andrew J Borst, Szu-Hsueh Lai, Ho Yeung Chim, Jing Yang 'John' Wang, Marcos C Miranda, Andrew M Watkins, Cassandra Ogohara, Shane Caldwell, Mengyu Wu, Albert J R Heck, David Veesler, Andrew B Ward, David Baker, Reidun Twarock, Neil P King.

Many naturally occurring protein assemblies have dynamic structures that allow them to perform specialized functions. Although computational methods for designing novel self-assembling proteins have advanced substantially over the past decade, they primarily focus on designing static structures. Here we characterize three distinct computationally designed protein assemblies that exhibit unanticipated structural diversity arising from flexibility in their subunits. Cryo-EM single-particle reconstructions and native mass spectrometry reveal two distinct architectures for two assemblies, while six cryo-EM reconstructions for the third likely represent a subset of its solution-phase structures. Structural modeling and molecular dynamics simulations indicate that constrained flexibility within the subunits of each assembly promotes a defined range of architectures rather than nonspecific aggregation. Redesigning the flexible region in one building block rescues the intended monomorphic assembly. These findings highlight structural flexibility as a powerful design principle, enabling exploration of new structural and functional spaces in protein assembly design.

DOI: https://doi.org/10.1038/s41594-025-01490-z
Nat Commun. 2025 Feb 26. 16(1): 2001

Design of high-affinity binders to immune modulating receptors for cancer immunotherapy.

Wei Yang, Derrick R Hicks, Agnidipta Ghosh, Tristin A Schwartze, Brian Conventry, Inna Goreshnik, Aza Allen, Samer F Halabiya, Chan Johng Kim, Cynthia S Hinck, David S Lee, Asim K Bera, Zhe Li, Yujia Wang, Thomas Schlichthaerle, Longxing Cao, Buwei Huang, Sarah Garrett, Stacey R Gerben, Stephen Rettie, Piper Heine, Analisa Murray, Natasha Edman, Lauren Carter, Lance Stewart, Steven C Almo, Andrew P Hinck, David Baker.

Immune receptors have emerged as critical therapeutic targets for cancer immunotherapy. Designed protein binders can have high affinity, modularity, and stability and hence could be attractive components of protein therapeutics directed against these receptors, but traditional Rosetta based protein binder methods using small globular scaffolds have difficulty achieving high affinity on convex targets. Here we describe the development of helical concave scaffolds tailored to the convex target sites typically involved in immune receptor interactions. We employed these scaffolds to design proteins that bind to TGFβRII, CTLA-4, and PD-L1, achieving low nanomolar to picomolar affinities and potent biological activity following experimental optimization. Co-crystal structures of the TGFβRII and CTLA-4 binders in complex with their respective receptors closely match the design models. These designs should have considerable utility for downstream therapeutic applications.

DOI: https://doi.org/10.1038/s41467-025-57192-z
Nat Rev Nephrol. 2025 Feb 24.

Endoplasmic reticulum stress as a driver and therapeutic target for kidney disease.

Jae Hyun Byun, Paul F Lebeau, Jackie Trink, Nikhil Uppal, Matthew B Lanktree, Joan C Krepinsky, Richard C Austin.

The endoplasmic reticulum (ER) has crucial roles in metabolically active cells, including protein translation, protein folding and quality control, lipid biosynthesis, and calcium homeostasis. Adverse metabolic conditions or pathogenic genetic variants that cause misfolding and accumulation of proteins within the ER of kidney cells initiate an injurious process known as ER stress that contributes to kidney disease and its cardiovascular complications. Initiation of ER stress activates the unfolded protein response (UPR), a cellular defence mechanism that functions to restore ER homeostasis. However, severe or chronic ER stress rewires the UPR to activate deleterious pathways that exacerbate inflammation, apoptosis and fibrosis, resulting in kidney injury. This insidious crosstalk between ER stress, UPR activation, oxidative stress and inflammation forms a vicious cycle that drives kidney disease and vascular damage. Furthermore, genetic variants that disrupt protein-folding mechanisms trigger ER stress, as evidenced in autosomal-dominant tubulointerstitial kidney disease and Fabry disease. Emerging therapeutic strategies that enhance protein-folding capacity and reduce the burden of ER stress have shown promising results in kidney diseases. Thus, integrating knowledge of how genetic variants cause protein misfolding and ER stress into clinical practice will enhance treatment strategies and potentially improve outcomes for various kidney diseases and their vascular complications.

DOI: https://doi.org/10.1038/s41581-025-00938-1
EMBO J. 2025 Feb 27.

Maintenance of p-eIF2α levels by the eIF2B complex is vital for colorectal cancer.

Ivana Paskov Škapik, Chiara Giacomelli, Sarah Hahn, Hanna Deinlein, Peter Gallant, Mathias Diebold, Josep Biayna, Anne Hendricks, Leon Olimski, Christoph Otto, Carolin Kastner, Elmar Wolf, Christina Schülein-Völk, Katja Maurus, Andreas Rosenwald, Nikolai Schleussner, Rene-Filip Jackstadt, Nicolas Schlegel, Christoph-Thomas Germer, Martin Bushell, Martin Eilers, Stefanie Schmidt, Armin Wiegering.

  Protein synthesis is an essential process, deregulated in multiple tumor types showing differential dependence on translation factors compared to untransformed tissue. We show that colorectal cancer (CRC) with loss-of-function mutation in the APC tumor suppressor depends on an oncogenic translation program regulated by the ability to sense phosphorylated eIF2α (p-eIF2α). Despite increased protein synthesis rates following APC loss, eIF2α phosphorylation, typically associated with translation inhibition, is enhanced in CRC. Elevated p-eIF2α, and its proper sensing by the decameric eIF2B complex, are essential to balance translation. Knockdown or mutation of eIF2Bα and eIF2Bδ, two eIF2B subunits responsible for sensing p-eIF2α, impairs CRC viability, demonstrating that the eIF2B/p-eIF2α nexus is vital for CRC. Specifically, the decameric eIF2B linked by two eIF2Bα subunits is critical for translating growth-promoting mRNAs which are induced upon APC loss. Depletion of eIF2Bα in APC-deficient murine and patient-derived organoids establishes a therapeutic window, validating eIF2Bα as a target for clinical intervention. In conclusion, we demonstrate how the expression of the oncogenic signature in CRC is crucially controlled at the translational level.

Keywords:  APC; Colorectal Cancer; Translation; eIF2B; eIF2α

DOI:  https://doi.org/10.1038/s44318-025-00381-9
Nat Commun. 2025 Feb 27. 16(1): 2017

Rapid and accurate prediction of protein homo-oligomer symmetry using Seq2Symm.

Meghana Kshirsagar, Artur Meller, Ian R Humphreys, Samuel Sledzieski, Yixi Xu, Rahul Dodhia, Eric Horvitz, Bonnie Berger, Gregory R Bowman, Juan Lavista Ferres, David Baker, Minkyung Baek.

The majority of proteins must form higher-order assemblies to perform their biological functions, yet few machine learning models can accurately and rapidly predict the symmetry of assemblies involving multiple copies of the same protein chain. Here, we address this gap by finetuning several classes of protein foundation models, to predict homo-oligomer symmetry. Our best model named Seq2Symm, which utilizes ESM2, outperforms existing template-based and deep learning methods achieving an average AUC-PR of 0.47, 0.44 and 0.49 across homo-oligomer symmetries on three held-out test sets compared to 0.24, 0.24 and 0.25 with template-based search. Seq2Symm uses a single sequence as input and can predict at the rate of ~80,000 proteins/hour. We apply this method to 5 proteomes and ~3.5 million unlabeled protein sequences, showing its promise to be used in conjunction with downstream computationally intensive all-atom structure generation methods such as RoseTTAFold2 and AlphaFold2-multimer. Code, datasets, model are available at: https://github.com/microsoft/seq2symm .

DOI: https://doi.org/10.1038/s41467-025-57148-3
RSC Med Chem. 2025 Feb 10.

Insights from protein frustration analysis of BRD4-cereblon degrader ternary complexes show separation of strong from weak degraders.

Tianyi Yang, Elizaveta Mukhaleva, Wenyuan Wei, Dahlia Weiss, Ning Ma, Veerabahu Shanmugasundaram, Nagarajan Vaidehi.

PROteolysis TArgeting Chimeras (PROTACs), also known as ligand-directed degraders (LDDs), are an innovative class of small molecules that leverage the ubiquitin-proteasome system to induce the degradation of target proteins. Structure based design methods are not readily applicable for designing LDDs due to the dynamic nature of the ternary complexes. This study investigates the dynamic properties of five LDD-mediated BRD4-cereblon complexes, focusing on the challenges of evaluating linker efficiency due to the difficulty in identifying suitable computational metrics that correlate well with the cooperativity or degradation propensity of LDDs. We uncovered that protein frustration, a concept originally developed to understand protein folding, calculated for the residues in the protein-protein interface of the LDD-mediated ternary complexes recapitulate the strength of degradation of the LDDs. Our findings indicated that hydrophobic residues in the interface are among the highly frustrated residues pairs, and they are crucial in distinguishing strong degraders from weak ones. By analyzing frustration patterns, we identified key residues and interactions critical to the effectiveness of the ternary complex. These insights provide practical guidelines for designing and prioritizing more efficient degraders, paving the way for the development of next-generation LDDs with improved therapeutic potential.

DOI: https://doi.org/10.1039/d4md00962b
Commun Biol. 2025 Feb 22. 8(1): 278

Distinct outcomes from targeted perturbations of the multi-subunit SCFSkp2 E3 ubiquitin ligase in blocking Trp53/Rb1-null prostate tumorigenesis.

Yingjiao Xue, Liang Zhu, Saumen Karan, Joseph D Locker, Craig Branch, Jinghang Zhang, Bang Hoang, Juan Pablo Maianti, Hongling Zhao, Edward L Schwartz.

Identifying effective therapies targeting multi-protein complexes that lack catalytic sites or cofactor pockets remains a long-standing challenge. The proto-oncogene, ubiquitin E3 ligase SCFSkp2, is one such target. SCFSkp2 promotes the proteasomal degradation of the cyclin-dependent kinase inhibitor p27, which controls cell cycle progression. Targeted knockout of Rb1/Trp53 causes metastatic prostate cancer in mice; additional knockout of Skp2 completely blocks tumorigenesis. We compared gene-edited mice that carried two different single amino acid changes in the SCFSkp2 complex, structurally predicted to inhibit the degradation of p27. Mutation of the SCFSkp2 accessory protein Cks1 (Cks1N45R) completely blocked Rb1/Trp53-driven prostate tumorigenesis, phenocopying Skp2 knockout, whereas a mutation directly stabilizing p27 (p27T187A) did not. This was consistent with structural models that predicted the binding of both p27 and p27T187A to the SCFSkp2/Cks1/Cdk2/CyclinA/p27 complex, and their subsequent ubiquitination and degradation, albeit at different rates. Two binding modes, which differ in their dependence on phosphorylated T187, are predicted by the model. Studies confirmed the role of p27 in mediating tumorigenesis in Rb1/Trp53 mutant tumors and revealed a mutually destabilizing Skp2 and p27 feedback loop. The integration of gene editing, drug-surrogate mutations, and mouse tumor models offers a blueprint for studying SCFSkp2 and other multi-subunit biomedical targets.

DOI: https://doi.org/10.1038/s42003-025-07662-3
EMBO J. 2025 Feb 25.

Ubiquitin is directly linked via an ester to protein-conjugated mono-ADP-ribose.

Daniel S Bejan, Rachel E Lacoursiere, Jonathan N Pruneda, Michael S Cohen.

  The prevailing view on post-translational modifications (PTMs) is that a single amino acid is modified with a single PTM at any given time. However, recent work has demonstrated crosstalk between different PTMs, some occurring on the same residue. Such interplay is seen with ADP-ribosylation and ubiquitylation. For example, DELTEX E3 ligases were reported to ubiquitylate a hydroxyl group on free NAD+ and ADP-ribose in vitro, generating a noncanonical ubiquitin ester-linked species. In this report, we show, for the first time, that this dual PTM occurs in cells on mono-ADP-ribosylated (MARylated) PARP10 on Glu/Asp sites to form a MAR ubiquitin ester. We call this process mono-ADP-ribosyl ubiquitylation or MARUbylation. Using chemical and enzymatic treatments, including a newly characterized bacterial deubiquitinase with esterase-specific activity, we discovered that multiple PARPs are MARUbylated and extended with K11-linked polyubiquitin chains when exogenously expressed. Finally, we show that in response to type I interferon stimulation, MARUbylation can occur endogenously on PARP targets. Thus, MARUbylation represents a new dual PTM that broadens our understanding of the function of PARP-mediated ADP-ribosylation in cells.

Keywords:  ADP-ribosylation; Interferon Response; PARPs; Post-translational Modifications; Ubiquitylation

DOI:  https://doi.org/10.1038/s44318-025-00391-7
Autophagy. 2025 Feb 28.

Cancer-associated mutations in autophagy-related proteins analyzed in yeast and human cells.

Yuchen Lei, Louise Uoselis, Dimitra Dialynaki, Ying Yang, Michael Lazarou, Daniel J Klionsky.

  Macroautophagy/autophagy is a conserved process among eukaryotes and is essential to maintain cell homeostasis; the dysregulation of autophagy has been linked with multiple human diseases, including cancer. However, not many studies have focused on the cancer-related mutations in ATG (autophagy related) proteins, which are likely to affect the protein function, influence autophagy activity and further contribute to the progression of the disease. In this study, we focused on the four ATG4 isoforms, which have a higher mutation frequency compared with the other core ATG proteins (i.e. those involved in autophagosome formation). We first studied the mutations in conserved residues and characterized one cancer-associated mutation that significantly impairs protein function and autophagy activity. Extending the study, we determined a region around the mutant residue to be essential for protein function, which had yet to be examined in previous studies. In addition, we created a yeast system expressing the human ATG4B protein to study mutations in the residues that are not conserved from human to yeast. Using this yeast model, we identified six cancer-associated mutations affecting autophagy. The effects of these mutations were further tested in mammalian cells using a quadruple ATG4 gene knockout cell line. Our study proves the principle of using human disease-associated mutations to study Atg proteins in yeast and generates a yeast tool that is helpful for a rapid screen of mutations to determine the autophagy phenotype, providing a new perspective in studying autophagy and its relation with cancer.

Keywords:  ATG4; autophagy; cancer; mutation

DOI:  https://doi.org/10.1080/15548627.2025.2471142
Adv Sci (Weinh). 2025 Feb 22. e2406786

UFMylation of NLRP3 Prevents Its Autophagic Degradation and Facilitates Inflammasome Activation.

Jiongjie Jing, Fan Yang, Ke Wang, Mintian Cui, Ni Kong, Shixi Wang, Xiaoyue Qiao, Fanyu Kong, Dongyang Zhao, Jinlu Ji, Lunxian Tang, Jiaxin Gao, Yu-Sheng Cong, Deqiang Ding, Kun Chen.

  NLRP3 (NOD, LRR and pyrin domain-containing protein 3) inflammasome is important for host defense against infections and maintaining homeostasis. Aberrant activation of NLRP3 inflammasome is closely related to various inflammatory diseases. Post-translational modifications are critical for NLRP3 inflammasome regulation. However, the mechanism of NLRP3 inflammasome activation remains incompletely understood. Here, it is demonstrated that the Ufm1 E3 ligase Ufl1 mediated UFMylation is essential for NLRP3 inflammasome activation. Mechanistically, Ufl1 binds and UFMylates NLRP3 in the priming stage of NLRP3 activation, thereby sustaining the stability of NLRP3 by preventing NLRP3 K63-linked ubiquitination and the subsequent autophagic degradation. It is further demonstrated that myeloid cell-specific Ufl1 or Ufm1 deficiency in mice significantly alleviated inflammatory responses and tissue damage following lipopolysaccharide (LPS)-induced endotoxemia and alum-induced peritonitis. Thus, the findings offer new insights into potential therapeutic targets for NLRP3 inflammasome-related diseases by targeting the UFMylation system.

Keywords:  NLRP3 inflammasome; UFMylation; autophagic degradation; therapeutic target

DOI:  https://doi.org/10.1002/advs.202406786
Cancers (Basel). 2025 Feb 13. pii: 626. [Epub ahead of print]17(4):

A Perspective on Therapeutic Targeting Against Ubiquitin Ligases to Stabilize Tumor Suppressor Proteins.

Ishaar P Ganesan, Hiroaki Kiyokawa.

  The loss of functions of tumor suppressor (TS) genes plays a key role in not only tumor initiation but also tumor progression leading to poor prognosis. While therapeutic inhibition of oncogene-encoded kinases has shown clinical success, restoring TS functions remains challenging due to conceptual and technical limitations. E3 ubiquitin ligases that ubiquitinate TS proteins for accelerated degradation in cancers emerge as promising therapeutic targets. Unlike proteasomal inhibitors with a broad spectrum, inhibitors of an E3 ligase would offer superior selectivity and efficacy in enhancing expression of its substrate TS proteins as far as the TS proteins retain wild-type structures. Recent advances in developing E3 inhibitors, including MDM2 inhibitors, highlight their potential and ultimately guide the framework to establish E3 inhibition as effective strategies to treat specific types of cancers. This review explores E3 ligases that negatively regulate bona fide TS proteins, the developmental status of E3 inhibitors, and their promise and pitfalls as therapeutic agents for anti-cancer precision medicine.

Keywords:  CDK inhibitor; FBXW7; MDM2; PML; PTEN; UBE3A; drug development; p53; tumor suppressor; ubiquitin

DOI:  https://doi.org/10.3390/cancers17040626
Nat Commun. 2025 Feb 26. 16(1): 1984

Reversible ubiquitination conferred by domain shuffling controls paired NLR immune receptor complex homeostasis in plant immunity.

Zhiyi Chen, Jianhua Huang, Jianyu Li, Frank L H Menke, Jonathan D G Jones, Hailong Guo.

Plant intracellular NLR immune receptors can function individually or in pairs to detect pathogen effectors and activate immune responses. NLR homeostasis has to be tightly regulated to ensure proper defense without triggering autoimmunity. However, in contrast to singleton NLRs, the mechanisms controlling the paired NLRs complex homeostasis are less understood. The paired Arabidopsis RRS1/RPS4 immune receptor complex confers disease resistance through effector recognition mediated by the integrated WRKY domain of RRS1. Here, through proximity labeling, we reveal a ubiquitination-deubiquitination cycle that controls the homeostasis of the RRS1/RPS4 complex. E3 ligase RARE directly binds and ubiquitinates RRS1's WRKY domain to promote its proteasomal degradation, thereby destabilizing RPS4 indirectly and compromising the stability and function of the RRS1/RPS4 complex. Conversely, the deubiquitinating enzymes UBP12/UBP13 deubiquitinate RRS1's WRKY domain, counteracting RARE's effects. Interestingly, the abundance of WRKY transcription factors WRKY70 and WRKY41 is also regulated by RARE and UBP12/UBP13. Phylogenetic analysis suggests this regulation likely transferred from WRKY70/WRKY41 to RRS1 upon WRKY domain integration. Our findings improve our understanding of homeostatic regulation of paired NLR complex and uncover a paradigm whereby domain integration can co-opt preexisting post-translational modification to regulate novel protein functions.

DOI: https://doi.org/10.1038/s41467-025-57231-9
Nat Commun. 2025 Feb 25. 16(1): 1948

Functional proteoform group deconvolution reveals a broader spectrum of ibrutinib off-targets.

Isabelle Rose Leo, Elena Kunold, Anastasia Audrey, Marianna Tampere, Jürgen Eirich, Janne Lehtiö, Rozbeh Jafari.

Proteome-wide profiling has revealed that targeted drugs can have complex protein interaction landscapes. However, it's a challenge to profile drug targets while systematically accounting for the dynamic protein variations that produce populations of multiple proteoforms. We address this problem by combining thermal proteome profiling (TPP) with functional proteoform group detection to refine the target landscape of ibrutinib. In addition to known targets, we implicate additional specific functional proteoform groups linking ibrutinib to mechanisms in immunomodulation and cellular processes like Golgi trafficking, endosomal trafficking, and glycosylation. Further, we identify variability in functional proteoform group profiles in a CLL cohort, linked to treatment status and ex vivo response and resistance. This offers deeper insights into the impacts of functional proteoform groups in a clinical treatment setting and suggests complex biological effects linked to off-target engagement. These results provide a framework for interpreting clinically observed off-target processes and adverse events, highlighting the importance of functional proteoform group-level deconvolution in understanding drug interactions and their functional impacts with potential applications in precision medicine.

DOI: https://doi.org/10.1038/s41467-024-54654-8
Nat Commun. 2025 Feb 22. 16(1): 1892

Non-invasive in vivo monitoring of PROTAC-mediated protein degradation using an environment-sensitive reporter.

Tao Li, Qingyu Zong, He Dong, Ihsan Ullah, Zhenhai Pan, Youyong Yuan.

Proteolysis targeting chimeras (PROTACs) represent a groundbreaking therapeutic technology for selectively degrading proteins of interest (POIs). The structural variations in PROTACs unpredictably influence their protein degradation efficiency, which is predominantly assessed by quantifying POIs abundance through western blotting. This approach, however, falls short of enabling non-invasive monitoring of protein degradation within living cells let alone assessing directly the degradation effects in vivo. Herein, we develop an environment-sensitive reporter (ESR) for the quantification of protein degradation events triggered by PROTACs in vivo. By simultaneously integrating POIs targeting ligand and an environment-sensitive fluorophore, the ESR signals exhibit a strong fluorescence correlation with the levels of POIs. This non-invasive monitoring reporter offers a high-throughput and convenient way to screen POIs targeting degraders and predict PROTACs-mediated therapeutic outcomes in mouse models. These properties suggest the potential of ESR strategy as a general modular scheme for non-invasive quantification of protein degradation of cancer-related therapeutic targets.

DOI: https://doi.org/10.1038/s41467-025-57191-0
bioRxiv. 2025 Feb 19. pii: 2025.02.11.637747. [Epub ahead of print]

Hoi1 targets the yeast BLTP2 protein to ER-PM contact sites to regulate lipid homeostasis.

Samantha K Dziurdzik, Vaishnavi Sridhar, Hailey Eng, Sarah D Neuman, Junran Yan, Michael Davey, Stefan Taubert, Arash Bashirullah, Elizabeth Conibear.

Membrane contact sites between organelles are important for maintaining cellular lipid homeostasis. Members of the recently identified family of bridge-like lipid transfer proteins (BLTPs) span opposing membranes at these contact sites to enable the rapid transfer of bulk lipids between organelles. While the VPS13 and ATG2 family members use organelle-specific adaptors for membrane targeting, the mechanisms that regulate other bridge-like transporters remain unknown. Here, we identify the conserved protein Ybl086c, which we name Hoi1 (Hob interactor 1), as an adaptor that targets the yeast BLTP2-like proteins Fmp27/Hob1 and Hob2 to ER-PM contact sites. Two separate Hoi1 domains interface with alpha-helical projections that decorate the central hydrophobic channel on Fmp27, and loss of these interactions disrupts cellular sterol homeostasis. The mutant phenotypes of BLTP2 and HOI1 orthologs indicate these proteins act in a shared pathway in worms and flies. Together, this suggests that Hoi1-mediated recruitment of BLTP2-like proteins represents an evolutionarily conserved mechanism for regulating lipid transport at membrane contact sites.

DOI: https://doi.org/10.1101/2025.02.11.637747
Res Sq. 2025 Feb 12. pii: rs.3.rs-5930673. [Epub ahead of print]

Systems-Level Interactome Mapping Reveals Actionable Protein Network Dysregulation Across the Alzheimer's Disease Spectrum.

Gabriela Chiosis, Sadik Bay, Anna Rodina, Florence Haut, Tanaya Roychowdhury, Eleni K Argyrousi, Agnieszka Staniszevski, Kyung Han, Sahil Sharma, Souparna Chakrabarty, Chander Digwal, Aleksandra Stanisavljevic, Amanda Labuza, Melissa J Alldred, Palak Panchal, Ramaian Anand, Laura Tuffery, Zhuoning Li, Arsalan Hashmi, Eric Rosiek, Eric Chan, Mara Monetti, Hiiroki Sasaguri, Takaomi Saido, Julie Schneider, David Bennett, Paul Fraser, Hediye Erdjument-Bromage, Thomas Neubert, Stephen D Ginsberg, Ottavio Arancio.

Alzheimer's disease (AD) progresses as a continuum, from preclinical stages to late-stage cognitive decline, yet the molecular mechanisms driving this progression remain poorly understood. Here, we provide a systems-level map of protein-protein interaction (PPI) network dysfunction across the AD spectrum and uncover epichaperomes-stable scaffolding platforms formed by chaperones and co-factors-as central drivers of this process. Using over 100 human brain specimens, mouse models, and human neurons, we show that epichaperomes emerge early, even in preclinical AD, and progressively disrupt multiple PPI networks critical for synaptic function and neuroplasticity. Glutamatergic neurons, essential for learning and memory, exhibit heightened vulnerability, with their dysfunction driven by protein sequestration into epichaperome scaffolds, independent of changes in protein expression. Notably, pharmacological disruption of epichaperomes with PU-AD restores PPI network integrity and reverses synaptic and cognitive deficits, directly linking epichaperome-driven network dysfunction to AD pathology. These findings establish epichaperomes as key mediators of molecular collapse in AD and identify network-centric intervention strategies as a promising avenue for disease-modifying therapies.

DOI: https://doi.org/10.21203/rs.3.rs-5930673/v1