bims-proteo 2025-04-13 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2025–04–13
43 papers selected by
Eric Chevet, INSERM

TMED9 coordinates the clearance of misfolded GPI-anchored proteins out of the ER and into the Golgi.
ADP ribosylation factor-like GTPase 6-interacting protein 5 (Arl6IP5) is an ER membrane-shaping protein that modulates ER-phagy.
Cytosolic and endoplasmic reticulum chaperones inhibit wt-p53 to increase cancer cells' survival by refluxing ER-proteins to the cytosol.
Structure of a Gcn2 dimer in complex with the large 60S ribosomal subunit.
ESCRT-I and PTPN23 mediate microautophagy of ubiquitylated tau aggregates.
Targeting the ER stress sensor IRE1 protects the liver from fibrosis through the downregulation of the proteostasis factor P4HB/PDIA1.
Shigella flexneri evades LPS ubiquitylation through IpaH1.4-mediated degradation of RNF213.
NUB1 traps unfolded FAT10 for ubiquitin-independent degradation by the 26S proteasome.
Rational Design of PROTAC Linkers Featuring Ferrocene as a Molecular Hinge to Enable Dynamic Conformational Changes.
TRIM21-NUP98 Interface Accommodates Structurally Diverse Molecular Glue Degraders.
Quiescent cell re-entry is limited by macroautophagy-induced lysosomal damage.
Neddylation modification stabilizes LC3B by antagonizing its ubiquitin-mediated degradation and promoting autophagy in skin.
Spatial mechanisms of quality control during chaperone-mediated assembly of the proteasome.
Selective Translation Under Heat Shock: Integrating HSP70 mRNA Regulation with Cellular Stress Responses in Yeast and Mammals.
Bro1 proteins determine tumor immune evasion and metastasis by controlling secretion or degradation of multivesicular bodies.
Protocol to determine the topology of integral endoplasmic reticulum membrane protein in Saccharomyces cerevisiae.
STING mediates lysosomal quality control and recovery through its proton channel function and TFEB activation in lysosomal storage disorders.
Self-assembled PROTACs enable glycoproteins degradation in the living cells.
Interferon-induced PARP14-mediated ADP-ribosylation in p62 bodies requires the ubiquitin-proteasome system.
Identification of RING E3 pseudoligases in the TRIM protein family.
HRI protein kinase in cytoplasmic heme sensing and mitochondrial stress response: relevance to hematological and mitochondrial diseases.
Harnessing the SPOP E3 Ubiquitin Ligase via a Bridged Proteolysis Targeting Chimera (PROTAC) Strategy for Targeted Protein Degradation.
Manipulation of targeted protein degradation in plant biology.
The autophagy protein ATG-9 regulates lysosome function and integrity.
The E3 ubiquitin ligase MGRN1 targets melanocortin receptors MC1R and MC4R via interactions with transmembrane adapters.
The bridge-like lipid transport protein VPS13C/PARK23 mediates ER-lysosome contacts following lysosome damage.
Interferon regulatory factor 4 mediates nonenzymatic IRE1 dependency in multiple myeloma cells.
Modulation of protein activity by small RNA base pairing internal to coding sequences.
Dissecting Branch-Specific Unfolded Protein Response Activation in Drug-Tolerant BRAF-Mutant Melanoma using Data-Independent Acquisition Mass Spectrometry.
A nonenzymatic dependency on inositol-requiring enzyme 1 controls cancer cell cycle progression and tumor growth.
MSP-tracker: A versatile vesicle tracking software tool used to reveal the spatial control of polarized secretion in Drosophila epithelial cells.
Advancing target validation with PROTAC technology.
Multiple cAMP/PKA complexes at the STIM1 ER/PM junction specified by E-Syt1 and E-Syt2 reciprocally gates ANO1 (TMEM16A) via Ca2.
A method to identify small molecule/protein pairs susceptible to protein ubiquitination by the CRBN E3 ligase.
Multimodal cell maps as a foundation for structural and functional genomics.
Suppression of TGF-β/SMAD signaling by an inner nuclear membrane phosphatase complex.
PITPβ promotes COPI vesicle fission through lipid transfer and membrane contact formation.
Large-scale discovery, analysis, and design of protein energy landscapes.
Activity-dependent synthesis of Emerin gates neuronal plasticity by regulating proteostasis.
RNA G-quadruplexes control mitochondria-localized mRNA translation and energy metabolism.
MLKL activity requires a splicing-regulated, druggable intramolecular interaction.
Comprehensive PTM profiling with SCASP-PTM uncovers mechanisms of p62 degradation and ALDOA-mediated tumor progression.
Structural basis of BAK sequestration by MCL-1 in apoptosis.

PLoS Biol. 2025 Apr 09. 23(4): e3003084

TMED9 coordinates the clearance of misfolded GPI-anchored proteins out of the ER and into the Golgi.

Elsa Ronzier, Prasanna Satpute-Krishnan.

The p24-family member, TMED9, has recently emerged as a player in secretory pathway protein quality control (PQC) that influences the trafficking and degradation of misfolded proteins. Here, we show that TMED9 plays a central role in the PQC of GPI-anchored proteins (GPI-APs). Typically, upon release from the endoplasmic reticulum (ER)-resident chaperone calnexin, misfolded GPI-APs traffic to the Golgi by an ER-export pathway called Rapid ER stress-induced Export (RESET). From the Golgi, they access the plasma membrane where they are rapidly internalized for lysosomal degradation. We used biochemical and imaging approaches in cultured cells to demonstrate that at steady-state, the majority of misfolded GPI-APs reside in the ER in association with calnexin and TMED9. During RESET, they dissociate from calnexin and increase their association with TMED9. Inhibition of TMED9's function through siRNA-induced depletion or chemical inhibitor, BRD4780, blocked ER-export of misfolded GPI-APs. In contrast, TMED9-inhibition did not prevent ER-export of wild-type GPI-APs, indicating a specific role for TMED9 in GPI-AP PQC. Intriguingly, we discovered that acute treatment with BRD4780 induced a shift in TMED9 localization away from the ER to the downstream Golgi cisternae and blocked the RESET pathway. Upon removal of BRD4780 following acute treatment, TMED9 regained access to the ER where TMED9 was able to associate with the RESET substrate and restore the RESET pathway. These results suggest that TMED9 plays a requisite role in RESET by capturing misfolded GPI-APs that are released by calnexin within the ER and conveying them to the Golgi.

DOI: https://doi.org/10.1371/journal.pbio.3003084
J Biol Chem. 2025 Apr 08. pii: S0021-9258(25)00342-4. [Epub ahead of print] 108493

ADP ribosylation factor-like GTPase 6-interacting protein 5 (Arl6IP5) is an ER membrane-shaping protein that modulates ER-phagy.

Yasunori Yamamoto, Toshiaki Sakisaka.

  The endoplasmic reticulum (ER) is the membrane-bound organelle characterized by the reticular network of tubules. It is well established that the ER tubules are shaped by ER membrane proteins containing the conserved reticulon-homology domain (RHD). Membrane shaping by the RHD-containing proteins is also involved in regulation of ER-phagy, selective autophagy of the ER. However, it remains unclear whether there exists ER membrane-shaping proteins other than the RHD-containing proteins. In this study, we characterize Arl6IP5, an ER membrane protein containing the conserved PRA1 domain, as an ER membrane-shaping protein. Upon overexpression, Arl6IP5 induces the extensive network of the ER tubules, and constricts the ER membrane as judged by exclusion of a luminal ER enzyme from the ER tubules. The membrane constriction by Arl6IP5 allows the cells to maintain the tubular ER network in the absence of microtubules. siRNA-mediated knockdown of Arl6IP5 impairs the ER morphology, and the phenotype of the Arl6IP5 knockdown cells is rescued by exogenous expression of Arl6IP1, an RHD-containing protein. Furthermore, exogenous expression of Arl6IP5 rescues the phenotype of Arl6IP1 knockdown cells, and the PRA1 domain is sufficient to rescue it. Upon disruption of the possible short hairpin structures of the PRA1 domain, Arl6IP5 abolishes membrane constriction. The siRNA-mediated knockdown of Arl6IP5 impairs flux of the ER-phagy mediated by FAM134B. These results indicate that Arl6IP5 acts as an ER membrane-shaping protein involved in regulation of ER-phagy, implying that the PRA1 domain may serve as a general membrane-shaping unit other than the RHD.

Keywords:  autophagy; endoplasmic reticulum (ER); membrane protein; membrane structure; protein domain

DOI:  https://doi.org/10.1016/j.jbc.2025.108493
Elife. 2025 Apr 09. pii: e102658. [Epub ahead of print]14

Cytosolic and endoplasmic reticulum chaperones inhibit wt-p53 to increase cancer cells' survival by refluxing ER-proteins to the cytosol.

Salam Dabsan, Gali Zur, Naim Abu-Freha, Shahar Sofer, Iris Grossman-Haham, Ayelet Gilad, Aeid Igbaria.

  The endoplasmic reticulum (ER) is an essential sensing organelle responsible for the folding and secretion of almost one-third of eukaryotic cells' total proteins. However, environmental, chemical, and genetic insults often lead to protein misfolding in the ER, accumulating misfolded proteins, and causing ER stress. To solve this, several mechanisms were reported to relieve ER stress by decreasing the ER protein load. Recently, we reported a novel ER surveillance mechanism by which proteins from the secretory pathway are refluxed to the cytosol to relieve the ER of its content. The refluxed proteins gain new prosurvival functions in cancer cells, thereby increasing cancer cell fitness. We termed this phenomenon ER to CYtosol Signaling (or 'ERCYS'). Here, we found that in mammalian cells, ERCYS is regulated by DNAJB12, DNAJB14, and the HSC70 cochaperone SGTA. Mechanistically, DNAJB12 and DNAJB14 bind HSC70 and SGTA - through their cytosolically localized J-domains to facilitate ER-protein reflux. DNAJB12 is necessary and sufficient to drive this phenomenon to increase AGR2 reflux and inhibit wt-p53 during ER stress. Mutations in DNAJB12/14 J-domain prevent the inhibitory interaction between AGR2-wt-p53. Thus, targeting the DNAJB12/14-HSC70/SGTA axis is a promising strategy to inhibit ERCYS and impair cancer cell fitness.

Keywords:  DNAJB12; ER stress; ERCYS; UPR; cancer biology; cell biology; chaperones; none; reflux

DOI:  https://doi.org/10.7554/eLife.102658
Proc Natl Acad Sci U S A. 2025 Apr 15. 122(15): e2415807122

Structure of a Gcn2 dimer in complex with the large 60S ribosomal subunit.

Helge Paternoga, Lu Xia, Lyudmila Dimitrova-Paternoga, Sihan Li, Liewei L Yan, Malte Oestereich, Sergo Kasvandik, Ankanahalli N Nanjaraj Urs, Bertrand Beckert, Tanel Tenson, Hani Zaher, Toshifumi Inada, Daniel N Wilson.

  The integrated stress response (ISR) is a central signaling network that enables eukaryotic cells to respond to a variety of different environmental stresses. Such stresses cause ribosome collisions that lead to activation of the kinase Gcn2, resulting in the phosphorylation and inactivation of eukaryotic initiation factor 2 and thereby promoting selective translation of mRNAs to restore homeostasis. Despite the importance of the ISR and intensive study over the past decades, structural insight into how Gcn2 interacts with ribosomal particles has been lacking. Using ex vivo affinity purification approaches, we have obtained a cryoelectron microscopy structure of a yeast Gcn2 dimer in complex with the ribosomal 60S subunit. The Gcn2 dimer is formed by dimerization of the histidine tRNA synthetase-like domains, which establish extensive interactions with the stalk-base and sarcin-ricin loop of the 60S subunit. The C-terminal domain of Gcn2 is also dimerized and occupies the A- and P-site tRNA binding sites at the peptidyl-transferase center of the 60S subunit. Complementary functional studies indicate that binding of Gcn2 to the 60S subunit does not require the coactivators Gcn1 or Gcn20, nor does it lead to phosphorylation of eIF2α. Instead, upon stress, we observe a shift of Gcn2 from the 60S subunit into the colliding ribosome fraction, suggesting that the Gcn2-60S complex represents an inactive stand-by state to enable a rapid redistribution to collided ribosomes, and thereby facilitating a quick and efficient response to stress.

Keywords:  Gcn2; eIF2; integrated stress response; ribosome; translation

DOI:  https://doi.org/10.1073/pnas.2415807122
J Cell Biol. 2025 Jun 02. pii: e202406120. [Epub ahead of print]224(6):

ESCRT-I and PTPN23 mediate microautophagy of ubiquitylated tau aggregates.

Yusen Men, Shoshiro Hirayama, Shinpei Ao, Yasuyuki Sakurai, Yuri Shibata, Megan Lo, Yusuke Sato, Shigeo Murata.

Protein aggregates are degraded by both the autophagy-lysosomal and the ubiquitin-proteasome pathways. Macroautophagy and microautophagy, two forms of the autophagy-lysosomal pathway, are widely conserved across eukaryotes. While macroautophagy has been extensively studied in the context of degradation of protein aggregates, microautophagy remains less explored. Here, we identify the UBAP1-containing ESCRT-I complex and PTPN23 as new regulators for degradation of aggregated proteins through an unbiased genome-wide CRISPR knockout screen, using a cell line expressing tau repeat domain (tauRD) aggregates. ESCRT-I recognizes ubiquitylated tauRD via the UEV domain of TSG101. The accessory protein PTPN23, instead of ESCRT-II, bridges ESCRT-I and ESCRT-III to complete the endosomal microautophagy of ubiquitylated tauRD aggregates. Our results uncover the molecular mechanism underlying the degradation of tau aggregates by endosomal microautophagy.

DOI: https://doi.org/10.1083/jcb.202406120
Hepatology. 2025 Apr 09.

Targeting the ER stress sensor IRE1 protects the liver from fibrosis through the downregulation of the proteostasis factor P4HB/PDIA1.

Younis Hazari, Lama Habbouche, Valeria A Garcia Lopez, Hery Urra, Javier Diaz, Giovanni Tamburini, Mateus Milani, Sylvere Durand, Fanny Aprahamian, Reese Baxter, Menghao Huang, X Charlie Dong, Luis Gonzalez-Rojas, Juan Francisco Silva, Ignacio Tapia-Dufey, Helena Vihinen, Vlad Ratziu, Fabienne Foufelle, Jan G Hengstler, Eija Jokitalo, Jessica L Maiers, Lars Plate, Guido Kroemer, Beatrice Bailly-Maitre, Claudio Hetz.

  Collagen is the main cargo of the secretory pathway, contributing to hepatic fibrogenesis due to extensive accumulation of extracellular matrix. An excess of collagen deposition is a characteristic feature of several chronic liver diseases. Collagen overproduction imposes pressure on the secretory pathway, altering endoplasmic reticulum (ER) proteostasis. Here we investigated the possible contribution of the unfolded protein response UPR, the main adaptive pathway that monitors and adjusts protein production capacity at the ER, to collagen biogenesis and liver disease. Genetic ablation of the ER stress sensor IRE1 in the liver using conditional knockout mice reduced liver damage and collagen deposition in models of fibrosis, steatosis, and acute hepatotoxicity. Proteomic profiling identified the prolyl 4-hydroxylase (P4HB, also known as PDIA1) as a major IRE1-regulated gene, a critical factor involved in collagen maturation. Cell culture studies demonstrated that IRE1 deficiency results in collagen retention at the ER, reducing its secretion, and this phenotype is rescued by P4HB/PDIA1 overexpression. Analyses of human MASH samples revealed a positive correlation between IRE1 signaling and P4HB/PDIA1 expression as well as the severity of the disease. Altogether, our results establish a role of the IRE1/P4HB axis in the regulation of collagen production and support its implication in the pathogenesis of liver fibrosis.

Keywords:  IRE1; PDIA1/ P4HB; UPR; collagen; endoplasmic reticulum; liver fibrosis

DOI:  https://doi.org/10.1097/HEP.0000000000001335
Nat Struct Mol Biol. 2025 Apr 09.

Shigella flexneri evades LPS ubiquitylation through IpaH1.4-mediated degradation of RNF213.

Katerina Naydenova, Keith B Boyle, Claudio Pathe, Prathyush Pothukuchi, Ana Crespillo-Casado, Felix Scharte, Pierre-Mehdi Hammoudi, Elsje G Otten, Neal M Alto, Felix Randow.

Pathogens have evolved diverse strategies to counteract host immunity. Ubiquitylation of lipopolysaccharide (LPS) on cytosol-invading bacteria by the E3 ligase RNF213 creates 'eat me' signals for antibacterial autophagy, but whether and how cytosol-adapted bacteria avoid LPS ubiquitylation remains poorly understood. Here, we show that the enterobacterium Shigella flexneri actively antagonizes LPS ubiquitylation through IpaH1.4, a secreted effector protein with ubiquitin E3 ligase activity. IpaH1.4 binds to RNF213, ubiquitylates it and targets it for proteasomal degradation, thus counteracting host-protective LPS ubiquitylation. To understand how IpaH1.4 recognizes RNF213, we determined the cryogenic electron microscopy structure of the IpaH1.4-RNF213 complex. The specificity of the interaction is achieved through the leucine-rich repeat of IpaH1.4, which binds the RING domain of RNF213 by hijacking the conserved RING interface required for binding to ubiquitin-charged E2 enzymes. IpaH1.4 also targets other E3 ligases involved in inflammation and immunity through binding to the E2-interacting face of their RING domains, including the E3 ligase LUBAC that is required for the synthesis of M1-linked ubiquitin chains on cytosol-invading bacteria downstream of RNF213. We conclude that IpaH1.4 has evolved to antagonize multiple antibacterial and proinflammatory host E3 ligases.

DOI: https://doi.org/10.1038/s41594-025-01530-8
Nat Struct Mol Biol. 2025 Apr 11.

NUB1 traps unfolded FAT10 for ubiquitin-independent degradation by the 26S proteasome.

Connor Arkinson, Ken C Dong, Christine L Gee, Shawn M Costello, Aimee Chi Soe, Greg L Hura, Susan Marqusee, Andreas Martin.

The ubiquitin-like modifier FAT10 targets hundreds of proteins in the mammalian immune system to the 26S proteasome for degradation. This degradation pathway requires the cofactor NUB1, yet the underlying mechanisms remain unknown. Here, we reconstituted a minimal in vitro system with human components and revealed that NUB1 uses the intrinsic instability of FAT10 to trap its N-terminal ubiquitin-like domain in an unfolded state and deliver it to the 26S proteasome for engagement, allowing the degradation of FAT10-ylated substrates in a ubiquitin-independent and p97-independent manner. Using hydrogen-deuterium exchange, structural modeling and site-directed mutagenesis, we identified the formation of an intricate complex with FAT10 that activates NUB1 for docking to the 26S proteasome, and our cryo-EM studies visualized the highly dynamic NUB1 complex bound to the proteasomal Rpn1 subunit during FAT10 delivery and the early stages of ATP-dependent degradation. These findings identified a previously unknown mode of cofactor-mediated, ubiquitin-independent substrate delivery to the 26S proteasome that relies on trapping partially unfolded states for engagement by the proteasomal ATPase motor.

DOI: https://doi.org/10.1038/s41594-025-01527-3
J Am Chem Soc. 2025 Apr 10.

Rational Design of PROTAC Linkers Featuring Ferrocene as a Molecular Hinge to Enable Dynamic Conformational Changes.

Alessandra Salerno, Lianne H E Wieske, Claudia J Diehl, Alessio Ciulli.

Proteolysis Targeting Chimeras (PROTACs) are bifunctional molecules that induce ubiquitination and degradation of a target protein via recruitment to an E3 ligase. The linker influences many steps of the PROTAC mode of action, from cellular permeability to ternary complex formation and target degradation. Much interest has therefore been devoted to linker design to fine-tune molecular and mechanistic properties of PROTACs. In this study, we present FerroTACs, a novel PROTAC design strategy incorporating ferrocene as the linker chemotype. We exemplify the approach across three different PROTAC systems: VHL-VHL (homo-PROTACs), VHL-CRBN, and VHL-BETs. We find that ferrocene's unique organometallic structure, featuring freely rotating cyclopentadienyl rings around a central Fe(II) ion, acts as a molecular hinge enabling structural adjustment to the environment that results in properties alteration, i.e., chameleonicity. Conformational analyses via NMR spectroscopy support ferrocene's role in fostering intramolecular interactions that result in a more folded state in an apolar environment. This property promotes compact conformations, improving cellular permeability and reducing efflux liabilities. Cellular assays demonstrate that FerroTACs exhibit robust target degradation and cell permeability profiles, en-par or enhanced compared to benchmark PROTACs CM11, 14a, and MZ1. These findings highlight ferrocene's potential as a new linker design strategy, offering a versatile platform to install and control molecular chameleonicity into next-generation PROTACs.

DOI: https://doi.org/10.1021/jacs.4c18354
ACS Chem Biol. 2025 Apr 09.

TRIM21-NUP98 Interface Accommodates Structurally Diverse Molecular Glue Degraders.

Yalong Cheng, Longzhi Cao, Panrui Lu, Lei Xue, Xiaomei Li, Qingyang Wang, Dengfeng Dou, Jin Li, Ting Han.

Molecular glue degraders enable targeted protein degradation by bridging interactions between target proteins and E3 ubiquitin ligases. Whereas some target-E3 interfaces exhibit the capacity to accommodate structurally diverse degraders, the extent of this adaptability across molecular glue targets remains unclear. We recently identified (S)-ACE-OH as a molecular glue degrader that recruits the E3 ubiquitin ligase TRIM21 to the nuclear pore complex by recognizing NUP98, thereby inducing the degradation of nuclear pore proteins. Here, we analyzed public compound toxicity data across a large collection of cell lines and identified two additional molecular glue degraders, PRLX 93936 and BMS-214662, which engage the TRIM21-NUP98 interface to induce selective degradation of nuclear pore proteins. Additionally, we confirmed that HGC652, another TRIM21-dependent molecular glue degrader, also binds at this interface. Together with our previously characterized degrader (S)-ACE-OH, these findings demonstrate that the TRIM21-NUP98 interface can accommodate structurally diverse molecular glue degraders.

DOI: https://doi.org/10.1021/acschembio.5c00036
Cell. 2025 Apr 04. pii: S0092-8674(25)00282-X. [Epub ahead of print]

Quiescent cell re-entry is limited by macroautophagy-induced lysosomal damage.

Andrew Murley, Ann Catherine Popovici, Xiwen Sophie Hu, Anina Lund, Kevin Wickham, Jenni Durieux, Larry Joe, Etai Koronyo, Hanlin Zhang, Naomi R Genuth, Andrew Dillin.

  To maintain tissue homeostasis, many cells reside in a quiescent state until prompted to divide. The reactivation of quiescent cells is perturbed with aging and may underlie declining tissue homeostasis and resiliency. The unfolded protein response regulators IRE-1 and XBP-1 are required for the reactivation of quiescent cells in developmentally L1-arrested C. elegans. Utilizing a forward genetic screen in C. elegans, we discovered that macroautophagy targets protein aggregates to lysosomes in quiescent cells, leading to lysosome damage. Genetic inhibition of macroautophagy and stimulation of lysosomes via the overexpression of HLH-30 (TFEB/TFE3) synergistically reduces lysosome damage. Damaged lysosomes require IRE-1/XBP-1 for their repair following prolonged L1 arrest. Protein aggregates are also targeted to lysosomes by macroautophagy in quiescent cultured mammalian cells and are associated with lysosome damage. Thus, lysosome damage is a hallmark of quiescent cells, and limiting lysosome damage by restraining macroautophagy can stimulate their reactivation.

Keywords:  aging; endoplasmic reticulum; lysosome; mTOR; macroautophagy; protein aggregates; quiescence

DOI:  https://doi.org/10.1016/j.cell.2025.03.009
Proc Natl Acad Sci U S A. 2025 Apr 15. 122(15): e2411429122

Neddylation modification stabilizes LC3B by antagonizing its ubiquitin-mediated degradation and promoting autophagy in skin.

Linlin Xu, Xinxing Lyu, Yibo Wang, Li Ni, Pin Li, Piao Zeng, Qixia Wang, Yunhao Chang, Chenglong Pan, Qingxia Hu, Shuhong Huang, Ningning Dang.

  The Atg8-family proteins, including LC3B (microtubule-associated protein 1 light chain 3 beta), are pivotal for key steps in the autophagy process. Proper regulation of LC3B homeostasis is essential for its function. Although LC3B is modulated by various posttranslational modifications (PTMs), the impact of these modifications on LC3B protein homeostasis remains unclear. Neddylation, a recently identified ubiquitin-like modification, plays diverse biological roles. Here, we identify LC3B as a specific target for neddylation. This modification weakens LC3B's interaction with the ubiquitin E3 ligases VHL and BIRC6, thereby reducing LC3B ubiquitination. Depletion of ubiquitin-conjugating enzyme E2M (UBE2M), the primary E2 enzyme in the neddylation pathway, destabilizes LC3B and suppresses autophagy activity. Heterozygous Ube2m knockout (Ube2m+/-) mice exhibit pronounced aging-like phenotypes, with reduced LC3B expression and impaired autophagy in skin tissues. Our findings demonstrate that LC3B neddylation is vital for maintaining its stability and regulating autophagy flux, offering a potential therapeutic avenue to mitigate aging-related processes.

Keywords:  LC3B; UBE2M; autophagy; neddylation; skin aging

DOI:  https://doi.org/10.1073/pnas.2411429122
Nat Commun. 2025 Apr 09. 16(1): 3358

Spatial mechanisms of quality control during chaperone-mediated assembly of the proteasome.

Eshita Das, Linh Le, Vladyslava Sokolova, James D Orth, Soyeon Park.

Cellular protein degradation requires a complex molecular machine, the proteasome. To mitigate the fundamental challenge of assembling the 66-subunit proteasome, cells utilize dedicated chaperones to order subunit addition. However, recent evidence suggests that proteasome assembly is not simply a series of subunit additions, but each step may be scrutinized so that only correct assembly events advance to proteasomes. Here, we find an unexpected mechanism of quality control (QC) during proteasome assembly-via the proteasomal nuclear localization signal (NLS). This mechanism specifically sequesters defective assembly intermediates to the nucleus, away from ongoing assembly in the cytoplasm, thereby antagonizing defective proteasome formation. This NLS, a bona fide proteasomal component, provides continuous surveillance throughout proteasome assembly. Even a single incorrect event activates spatial QC. Our findings illuminate a two-decade-old mystery in proteasome regulation; proteasomal NLSs, dispensable for proteasome localization, instead provide QC by compartmentalizing assembly defects to ensure that only correct proteasomes form.

DOI: https://doi.org/10.1038/s41467-025-58703-8
Mol Biol Cell. 2025 May 01. 36(5): re2

Selective Translation Under Heat Shock: Integrating HSP70 mRNA Regulation with Cellular Stress Responses in Yeast and Mammals.

Talar Ghadanian, Shruti Iyer, Luca Lazzari, Maria Vera.

Under stress, cells orchestrate a complex regulatory response to maintain protein homeostasis, leveraging differential translational regulation for constitutively expressed mRNAs and the transcriptionally induced heat shock protein HSP70 transcripts. Constitutive mRNAs typically experience partial translational suppression, consistent with their partitioning into stress-induced phase-separated condensates and the global reduction in protein synthesis. In contrast, inducible HSP70 mRNAs bypass this repression to remain in the cytosol where they recruit the available components of the translational machinery to ensure the rapid synthesis of HSP70. Although the components involved in the preferential translation of HSP70 mRNA during heat stress have not been fully elucidated, differences in the mRNA and translation factors between yeast and mammals suggest organism-specific mechanisms of HSP70 mRNA translation. In this review, we consider these differences to discuss the current knowledge on heat shock regulation of translation. We extend the discussion to go beyond the cytosolic needs of HSP70 to ponder the important interplay between the cytosol and mitochondria in activating HSP70 accumulation, which becomes vital for preserving intercompartmental proteostasis and cell survival.

DOI: https://doi.org/10.1091/mbc.E24-12-0564
Dev Cell. 2025 Mar 25. pii: S1534-5807(25)00155-8. [Epub ahead of print]

Bro1 proteins determine tumor immune evasion and metastasis by controlling secretion or degradation of multivesicular bodies.

Nai Yang Yeat, Li-Heng Liu, Yu-Hsuan Chang, Charles Pin-Kuang Lai, Ruey-Hwa Chen.

  Exosomes play pleiotropic tumor-promoting functions and are secreted by fusion of multivesicular bodies (MVBs) with the plasma membrane. However, MVBs are also directed to lysosomes for degradation, and the mechanism controlling different fates of MVBs remains elusive. Here, we show that the pro-tumor protein WDR4 enhances exosome secretion from mouse and human cancer cells through degrading the endosomal sorting complex required for transport (ESCRT)-associated Bro1-family protein PTPN23. Mechanistically, PTPN23 and ALIX compete for binding to syntenin, thereby directing MVBs toward degradation and secretion, respectively. ALIX, but not PTPN23, recruits actin-capping proteins CAPZA1/CAPZB to prevent branched filamentous actin (F-actin) accumulation around MVBs, thus enabling MVBs trafficking to the cell periphery for secretion. Functionally, WDR4/ALIX-dependent exosomes load a set of pro-tumor proteins through LAMP2A, thereby potentiating metastasis and immune evasion in mice. Our study highlights a previously unappreciated coupling between the biogenesis mechanism and the fate decision of MVBs and its importance in determining exosomal cargos, which have a profound impact on tumor progression.

Keywords:  ESCRT complex; actin cytoskeletons; endocytic trafficking; exosome biogenesis; multivesicular body; tumor microenvironment

DOI:  https://doi.org/10.1016/j.devcel.2025.03.008
STAR Protoc. 2025 Apr 03. pii: S2666-1667(25)00133-9. [Epub ahead of print]6(2): 103727

Protocol to determine the topology of integral endoplasmic reticulum membrane protein in Saccharomyces cerevisiae.

Ritika Chaudhary, Vineet Choudhary.

  Here, we present a protocol to determine the topology of Fld1 (few lipid droplets), an integral endoplasmic reticulum (ER) membrane protein in Saccharomyces cerevisiae. We describe steps to generate functional N-terminal GFP and C-terminal mCherry fusion with Fld1. We detail the strategy to perform subcellular fractionation to isolate ER-derived microsomes that were subjected to salt detergent extraction analysis. We then provide procedures to determine the topology of Fld1 using proteinase K treatment.

Keywords:  cell biology; microscopy; molecular biology

DOI:  https://doi.org/10.1016/j.xpro.2025.103727
Mol Cell. 2025 Mar 27. pii: S1097-2765(25)00201-1. [Epub ahead of print]

STING mediates lysosomal quality control and recovery through its proton channel function and TFEB activation in lysosomal storage disorders.

Zhen Tang, Cong Xing, Antonina Araszkiewicz, Kun Yang, Wanwan Huai, Devon Jeltema, Nicole Dobbs, Yihe Zhang, Lu O Sun, Nan Yan.

  Lysosomes are essential organelles for cellular homeostasis. Defective lysosomes are associated with diseases like lysosomal storage disorders (LSDs). How lysosomal defects are detected and lysosomal function restored remain incompletely understood. Here, we show that STING mediates a neuroinflammatory gene signature in three distinct LSD mouse models, Galctwi/twi, Ppt1-/-, and Cln7-/-. Transcriptomic analysis of Galctwi/twi mouse brain tissue revealed that STING also mediates the expression of lysosomal genes that are regulated by transcriptional factor EB (TFEB). Immunohistochemical and single-nucleus RNA-sequencing (snRNA-seq) analysis show that STING regulates lysosomal gene expression in microglia. Mechanistically, we show that STING activation leads to TFEB dephosphorylation, nuclear translocation, and expression of lysosomal genes. This process requires STING's proton channel function, the V-ATPase-ATG5-ATG8 cascade, and is independent of immune signaling. Furthermore, we show that the STING-TFEB axis facilitates lysosomal repair. Together, our data identify STING-TFEB as a lysosomal quality control mechanism that responds to lysosomal dysfunction.

Keywords:  Krabbe disease; Niemann-Pick disease; STING; TFEB; innate immunity; lysosomal storage disorder; lysosome repair; neuroinflammation; non-canonical autophagy

DOI:  https://doi.org/10.1016/j.molcel.2025.03.008
Chem Sci. 2025 Apr 02.

Self-assembled PROTACs enable glycoproteins degradation in the living cells.

Haoyu Chen, Liu Zang, Pavel Kielkowski.

We report here a two-component proteolysis targeting chimeras (PROTACs) strategy selectively targeting O-GalNAcylated and O-GlcNAcylated proteins for proteasomal degradation, which leads to severe toxicity in human cancer cell lines through perturbation of critical metabolic and signaling pathways governed by glycoproteins. Our approach termed as GlyTAC leverages from metabolic incorporation of easily accessible and cell-permeable peracetylated N-acetylglucosamine (GlcNAc) or N-acetylgalactosamine (GalNAc) analogues bearing an azido group into glycoproteins. In the living cells, the azido-modified glycoproteins serve as covalent anchors for the introduction of thalidomide moiety by strain-promoted azide-alkyne cycloaddition (SPAAC) to recruit E3 ligase cereblon (CRBN), resulting in stepwise ubiquitination of 'sensitized' proteins and their degradation by proteasome. We show the efficiency of the system in a series of human cancer cell lines and verify the mechanistic pathway by performing control experiments at each stage of the process. Given the characteristic features of cancer cells including fast nutrient turnover, and overall increase of protein glycosylation, as well as the low cytotoxicity of the individual components, our approach may open a feasible strategy in cancer therapy.

DOI: https://doi.org/10.1039/d5sc00400d
EMBO J. 2025 Apr 07.

Interferon-induced PARP14-mediated ADP-ribosylation in p62 bodies requires the ubiquitin-proteasome system.

Rameez Raja, Banhi Biswas, Rachy Abraham, Yiran Wang, Che-Yuan Chang, Ivo A Hendriks, Sara C Buch-Larsen, Hongrui Liu, Xingyi Yang, Chenyao Wang, Hien Vu, Anne Hamacher-Brady, Danfeng Cai, Anthony K L Leung.

  Biomolecular condensates are cellular compartments without enveloping membranes, enabling them to dynamically adjust their composition in response to environmental changes through post-translational modifications. Recent work has revealed that interferon-induced ADP-ribosylation (ADPr), which can be reversed by a SARS-CoV-2-encoded hydrolase, is enriched within a condensate. However, the identity of the condensate and the responsible host ADP-ribosyltransferase remain elusive. Here, we demonstrate that interferon induces ADPr through transcriptional activation of PARP14, requiring both the physical presence and catalytic activity of PARP14 for condensate formation. Interferon-induced ADPr colocalizes with PARP14 and its associated E3 ligase, DTX3L. These PARP14/ADPr condensates contain key components of p62 bodies-including the selective autophagy receptor p62, its binding partner NBR1 and the associated protein TAX1BP1, along with K48-linked and K63-linked polyubiquitin chains-but lack the autophagosome marker LC3B. Knockdown of p62 disrupts the formation of these ADPr condensates. Importantly, these structures are unaffected by autophagy inhibition, but depend on ubiquitination and proteasome activity. Taken together, these findings demonstrate that interferon triggers PARP14-mediated ADP-ribosylation in p62 bodies, which requires an active ubiquitin-proteasome system.

Keywords:  ADP-Ribosylation; Condensates; Interferon; Ubiquitin-Proteasome System; p62

DOI:  https://doi.org/10.1038/s44318-025-00421-4
Nat Commun. 2025 Apr 11. 16(1): 3456

Identification of RING E3 pseudoligases in the TRIM protein family.

Jane Dudley-Fraser, Diego Esposito, Katherine A McPhie, Coltrane Morley-Williams, Tania Auchynnikava, Katrin Rittinger.

TRIpartite Motif (TRIM) family proteins have diverse roles across a broad variety of cellular functions, which are largely presumed to depend on their ubiquitin E3 ligase activity, conferred by a RING domain. However, recent reports have shown that some TRIMs lack detectable ubiquitination activity in isolation, despite containing a RING domain. Here, we present parallel in cellulo, in vitro, and in silico structure-function analyses of the ubiquitin E3 ligase activity and RING domain structural characteristics of the whole TRIM protein family. In-depth follow-up studies of this comprehensive dataset reveals a number of 'pseudoligases', whose RING domains have structurally diverged at either the homodimerisation or E2~ubiquitin interfaces, thereby disrupting their ability to catalyse ubiquitin transfer. Together, these data raise intriguing open questions regarding the unknown TRIM functions in physiology and disease.

DOI: https://doi.org/10.1038/s41467-025-58807-1
J Biol Chem. 2025 Apr 08. pii: S0021-9258(25)00343-6. [Epub ahead of print] 108494

HRI protein kinase in cytoplasmic heme sensing and mitochondrial stress response: relevance to hematological and mitochondrial diseases.

Jane-Jane Chen.

  Most iron in humans is bound in heme used as a prosthetic group for hemoglobin. Heme-regulated inhibitor (HRI) is responsible for coordinating heme availability and protein synthesis. Originally characterized in rabbit reticulocyte lysates, HRI was shown in 1976 to phosphorylate the α-subunit of eIF2, revealing a new molecular mechanism for regulating protein synthesis. Since then, HRI research has mostly been focused on the biochemistry of heme inhibition through direct binding, and heme sensing in balancing heme and globin synthesis to prevent proteotoxicity in erythroid cells. Beyond inhibiting translation of highly translated mRNAs, eIF2α phosphorylation also selectively increases translation of certain poorly translated mRNAs, notably ATF4 mRNA, for reprogramming of gene expression to mitigate stress, known as the integrated stress response (ISR). In recent years, there have been novel mechanistic insights of HRI-ISR in oxidative stress, mitochondrial function and erythroid differentiation during heme deficiency. Furthermore, HRI-ISR is activated upon mitochondrial stress in several cell types, establishing the bifunctional nature of HRI protein. The role of HRI and ISR in cancer development and vulnerability is also emerging. Excitingly, the UBR4 ubiquitin ligase complex has been demonstrated to silence the HRI-ISR by degradation of activated HRI proteins, suggesting additional regulatory processes. Together, these recent advancements indicate that the HRI-ISR mechanistic axis is a target for new therapies for hematological and mitochondrial diseases, as well as oncology. This review covers the historical overview of HRI biology, the biochemical mechanisms of regulating HRI, and the biological impacts of the HRI-ISR pathway in human diseases.

Keywords:  ATF4; E3 ubiquitin ligase; Erythropoiesis; Heme; Mitochondrial stress; Protein kinase; Protein synthesis; Proteostasis; Stress response; eIF2

DOI:  https://doi.org/10.1016/j.jbc.2025.108494
J Med Chem. 2025 Apr 09.

Harnessing the SPOP E3 Ubiquitin Ligase via a Bridged Proteolysis Targeting Chimera (PROTAC) Strategy for Targeted Protein Degradation.

Zhijie Deng, Jerrel Catlett, Youngeun Lee, Qiong Wu, Zhongli Xu, Ling Xie, Xian Chen, Yan Xiong, H Ümit Kaniskan, Jian Jin.

Proteolysis Targeting Chimeras (PROTACs) represent promising therapeutic modalities for degrading disease-causing proteins. However, the development of effective PROTACs has been limited by the availability of suitable E3 ligase ligands. In this study, we demonstrate for the first time that SPOP, an unexplored E3 ligase, can be recruited to degrade target proteins of interest. We developed a bridged PROTAC strategy and successfully discovered a proof-of-concept PROTAC degrader 9 (MS479), which recruits the E3 ligase SPOP by directly binding its substrate GLP as a bridge protein. This approach facilitates the polyubiquitination and subsequent degradation of BRD4/3/2 by the 26S proteasome. 9 effectively reduced the protein level of BRD4 short isoform in a time-, concentration-, GLP-, SPOP-, and ubiquitin-proteasome system (UPS)-dependent manner. Additionally, 9 effectively inhibited the proliferation of colorectal cancer (CRC) cells. Overall, our study expands the limited repertoire of the E3 ligases that can be harnessed for targeted protein degradation.

DOI: https://doi.org/10.1021/acs.jmedchem.5c00295
Biochem Soc Trans. 2025 Apr 09. pii: BST20230939. [Epub ahead of print]53(2):

Manipulation of targeted protein degradation in plant biology.

Marcela Rojas-Pierce, Sebastian Y Bednarek.

  Inducible protein degradation systems are an important but untapped resource for the study of protein function in plant cells. Unlike mutagenesis or transcriptional control, regulated degradation of proteins of interest allows the study of the biological mechanisms of highly dynamic cellular processes involving essential proteins. While systems for targeted protein degradation are available for research and therapeutics in animals, there are currently limited options in plant biology. Targeted protein degradation systems rely on target ubiquitination by E3 ubiquitin ligases. Systems that are available or being developed in plants can be distinguished primarily by the type of E3 ubiquitin ligase involved, including those that utilize Cullin-RING ligases, bacterial novel E3 ligases, and N-end rule pathway E3 ligases, or they can be controlled by proteolysis targeting chimeras. Target protein ubiquitination leads to degradation by the proteasome or targeting to the vacuole, with both pathways being ubiquitous and important for the endogenous control of protein abundance in plants. Targeted proteolysis approaches for plants will likely be an important tool for basic research and to yield novel traits for crop biotechnology.

Keywords:  degron; plant biology; proteolysis; targeted protein degradation; ubiquitin ligases; ubiquitination

DOI:  https://doi.org/10.1042/BST20230939
J Cell Biol. 2025 Jun 02. pii: e202411092. [Epub ahead of print]224(6):

The autophagy protein ATG-9 regulates lysosome function and integrity.

Kangfu Peng, Guoxiu Zhao, Hongyu Zhao, Nobuo N Noda, Hong Zhang.

The transmembrane autophagy protein ATG9 has multiple functions essential for autophagosome formation. Here, we uncovered a novel function of ATG-9 in regulating lysosome biogenesis and integrity in Caenorhabditis elegans. Through a genetic screen, we identified that mutations attenuating the lipid scrambling activity of ATG-9 suppress the autophagy defect in epg-5 mutants, in which non-degradative autolysosomes accumulate. The scramblase-attenuated ATG-9 mutants promote lysosome biogenesis and delivery of lysosome-localized hydrolases and also facilitate the maintenance of lysosome integrity. Through manipulation of phospholipid levels, we found that a reduction in phosphatidylethanolamine (PE) also suppresses the autophagy defects and lysosome damage associated with impaired lysosomal degradation. Our results reveal that modulation of phospholipid composition and distribution, e.g., by attenuating the scramblase activity of ATG-9 or reducing the PE level, regulates lysosome function and integrity.

DOI: https://doi.org/10.1083/jcb.202411092
bioRxiv. 2025 Mar 26. pii: 2025.03.25.645338. [Epub ahead of print]

The E3 ubiquitin ligase MGRN1 targets melanocortin receptors MC1R and MC4R via interactions with transmembrane adapters.

Pragya Parashara, Lei Gao, Alyssa Riglos, Sonia B Sidhu, Dorothy Lartey, Tessa Marks, Carys Williams, Grace Siauw, Anna I L Ostrem, Christian Siebold, Maia Kinnebrew, Michael Riffle, Teresa M Gunn, Jennifer H Kong.

E3 ubiquitin ligases play a crucial role in modulating receptor stability and signaling at the cell surface, yet the mechanisms governing their substrate specificity remain incompletely understood. Mahogunin Ring Finger 1 (MGRN1) is a membrane-tethered E3 ligase that fine-tunes signaling sensitivity by targeting surface receptors for ubiquitination and degradation. Unlike cytosolic E3 ligases, membrane-tethered E3s require transmembrane adapters to selectively recognize and regulate surface receptors, yet few such ligases have been studied in detail. While MGRN1 is known to regulate the receptor Smoothened (SMO) within the Hedgehog pathway through its interaction with the transmembrane adapter Multiple Epidermal Growth Factor-like 8 (MEGF8), the broader scope of its regulatory network has been speculative. Here, we identify Attractin (ATRN) and Attractin-like 1 (ATRNL1) as additional transmembrane adapters that recruit MGRN1 and regulate cell surface receptor turnover. Through co-immunoprecipitation, we show that ATRN and ATRNL1 likely interact with the RING domain of MGRN1. Functional assays reveal that MGRN1 requires these transmembrane adapters to ubiquitinate and degrade the melanocortin receptors MC1R and MC4R, in a process analogous to its regulation of SMO. Loss of MGRN1 leads to increased surface and ciliary localization of MC4R in fibroblasts and elevated MC1R levels in melanocytes, with the latter resulting in enhanced eumelanin production. These findings expand the repertoire of MGRN1-regulated receptors and provide new insight into a shared mechanism by which membrane-tethered E3 ligases utilize transmembrane adapters to dictate substrate receptor specificity. By elucidating how MGRN1 selectively engages with surface receptors, this work establishes a broader framework for understanding how this unique class of E3 ligases fine-tunes receptor homeostasis and signaling output.

DOI: https://doi.org/10.1101/2025.03.25.645338
Nat Cell Biol. 2025 Apr 10.

The bridge-like lipid transport protein VPS13C/PARK23 mediates ER-lysosome contacts following lysosome damage.

Xinbo Wang, Peng Xu, Amanda Bentley-DeSousa, William Hancock-Cerutti, Shujun Cai, Benjamin T Johnson, Francesca Tonelli, Lin Shao, Gabriel Talaia, Dario R Alessi, Shawn M Ferguson, Pietro De Camilli.

Based on genetic studies, lysosome dysfunction is thought to play a pathogenetic role in Parkinson's disease. Here we show that VPS13C, a bridge-like lipid-transport protein and a Parkinson's disease gene, is a sensor of lysosome stress or damage. Following lysosome membrane perturbation, VPS13C rapidly relocates from the cytosol to the surface of lysosomes where it tethers their membranes to the ER. This recruitment depends on Rab7 and requires a signal at the damaged lysosome surface that releases an inhibited state of VPS13C, which hinders access of its VAB domain to lysosome-bound Rab7. Although another Parkinson's disease protein, LRRK2, is also recruited to stressed or damaged lysosomes, its recruitment occurs at much later stages and by different mechanisms. Given the role of VPS13 proteins in bulk lipid transport, these findings suggest that lipid delivery to lysosomes by VPS13C is part of an early protective response to lysosome damage.

DOI: https://doi.org/10.1038/s41556-025-01653-6
PLoS Biol. 2025 Apr 11. 23(4): e3003096

Interferon regulatory factor 4 mediates nonenzymatic IRE1 dependency in multiple myeloma cells.

Ioanna Oikonomidi, Vasumathi Kameswaran, Victoria C Pham, Iratxe Zuazo-Gaztelu, Lauren M Gutgesell, Scot Marsters, Bence Daniel, Jennie R Lill, Zora Modrusan, Avi Ashkenazi.

Multiple myeloma (MM) arises through oncogenic transformation of immunoglobulin-secreting plasma cells. MM often co-opts the central endoplasmic reticulum (ER)-stress mitigator, inositol-requiring enzyme 1 (IRE1), to sustain malignant growth. While certain MMs require enzymatic IRE1-dependent activation of the transcription factor XBP1s, others display a nonenzymatic IRE1 dependency that is not yet mechanistically understood. Here we identify interferon regulatory factor 4 (IRF4), which stimulates genes that promote immune-cell proliferation, as a key conduit for IRE1's nonenzymatic control of cell-cycle progression in MM. IRE1 silencing increased inhibitory S114/S270 phosphorylation on IRF4, disrupting IRF4's chromatin-binding and transcriptional activity. IRF4 knockdown recapitulated, whereas IRF4 repletion reversed the anti-proliferative phenotype of IRE1 silencing. Furthermore, phospho-deficient, but not phospho-mimetic, IRF4 mutants rescued proliferation under IRE1 silencing. Functional studies revealed that IRF4 engages the E2F1 and CDC25A genes and promotes CDK2 activation to drive cell-cycle progression. Our results advance mechanistic understanding of IRE1 and IRF4 in MM.

DOI: https://doi.org/10.1371/journal.pbio.3003096
Mol Cell. 2025 Mar 30. pii: S1097-2765(25)00258-8. [Epub ahead of print]

Modulation of protein activity by small RNA base pairing internal to coding sequences.

Narumon Thongdee, Miranda M Alaniz, Ekaterina Samatova, Aoshu Zhong, Caroline Esnault, Hongen Zhang, Ryan K Dale, Marina V Rodnina, Gisela Storz.

  Most characterized interactions between bacterial small RNAs (sRNAs) and their target mRNAs occur near ribosome binding sites, resulting in changes in translation initiation or target mRNA decay. To understand the consequences of sRNA pairing internal to coding sequences detected by global RNA-RNA interactome approaches, we examined the impact of sRNA overexpression on seven target proteins. Overexpression of the sRNA led to decreased target protein levels for two pairs, but there were no differences for the others. By further examining ArcZ-ligA and ArcZ-hemK, we discovered that ArcZ pairing with the mRNAs leads to translation pausing and increased protein activity. A ligA point mutation that eliminates sRNA pairing resulted in increased sensitivity to DNA damage, revealing the physiological consequences of the regulation. Thus, regulatory RNA pairing in coding sequences can locally slow translation elongation, likely impacting co-translational protein folding and allowing improved incorporation of co-factors or more optimal folding under specific conditions.

Keywords:  ArcZ; DNA damage; DNA ligase; HemK; Hfq; co-factor incorperation; protein folding; regulatory RNAs; translation elongation

DOI:  https://doi.org/10.1016/j.molcel.2025.03.014
bioRxiv. 2025 Mar 24. pii: 2025.03.20.644425. [Epub ahead of print]

Dissecting Branch-Specific Unfolded Protein Response Activation in Drug-Tolerant BRAF-Mutant Melanoma using Data-Independent Acquisition Mass Spectrometry.

Lea A Barny, Jake N Hermanson, Sarah K Garcia, Philip E Stauffer, Lars Plate.

Cells rely on the Unfolded Protein Response (UPR) to maintain ER protein homeostasis (proteostasis) when faced with elevated levels of misfolded and aggregated proteins. The UPR is comprised of three main branches-ATF6, IRE1, and PERK-that coordinate the synthesis of proteins involved in folding, trafficking, and degradation of nascent proteins to restore ER function. Dysregulation of the UPR is linked to numerous diseases, including neurodegenerative disorders, cancer, and diabetes. Despite its importance, identifying UPR targets has been challenging due to their heterogeneous induction, which varies by cell type and tissue. Additionally, defining the magnitude and range of UPR-regulated genes is difficult because of intricate temporal regulation, feedback between UPR branches, and extensive cross-talk with other stress-signaling pathways. To comprehensively identify UPR-regulated proteins and determine their branch specificity, we developed a data-independent acquisition (DIA) liquid-chromatography mass spectrometry (LC-MS) pipeline. Our optimized workflow improved identifications of low-abundant UPR proteins and leveraged an automated SP3-based protocol on the Biomek i5 liquid handler for label-free peptide preparation. Using engineered stable cell lines that enable selective pharmacological activation of each UPR branch without triggering global UPR activation, we identified branch-specific UPR proteomic targets. These targets were subsequently applied to investigate proteomic changes in multiple patient-derived BRAF-mutant melanoma cell lines treated with a BRAF inhibitor (PLX4720, i.e., vemurafenib). Our findings revealed differential regulation of the XBP1s branch of the UPR in the BRAF-mutant melanoma cell lines after PLX4720 treatment, likely due to calcium activation, suggesting that the UPR plays a role as a non-genetic mechanism of drug tolerance in melanoma. In conclusion, the validated branch-specific UPR proteomic targets identified in this study provide a robust framework for investigating this pathway across different cell types, drug treatments, and disease conditions in a high-throughput manner.

DOI: https://doi.org/10.1101/2025.03.20.644425
PLoS Biol. 2025 Apr 10. 23(4): e3003086

A nonenzymatic dependency on inositol-requiring enzyme 1 controls cancer cell cycle progression and tumor growth.

Iratxe Zuazo-Gaztelu, David Lawrence, Ioanna Oikonomidi, Scot Marsters, Ximo Pechuan-Jorge, Catarina J Gaspar, David Kan, Ehud Segal, Kevin Clark, Maureen Beresini, Marie-Gabrielle Braun, Joachim Rudolph, Zora Modrusan, Meena Choi, Wendy Sandoval, Mike Reichelt, David C DeWitt, Pekka Kujala, Suzanne van Dijk, Judith Klumperman, Avi Ashkenazi.

Endoplasmic-reticulum resident inositol-requiring enzyme 1α (IRE1) supports protein homeostasis via its cytoplasmic kinase-RNase module. Known cancer dependency on IRE1 entails its enzymatic activation of the transcription factor XBP1s and regulated RNA decay. We discovered that some cancer cells surprisingly require IRE1 but not its enzymatic activity. IRE1 knockdown but not enzymatic IRE1 inhibition or XBP1 disruption attenuated cell cycle progression and tumor growth. IRE1 silencing led to activation of TP53 and CDKN1A/p21 in conjunction with increased DNA damage and chromosome instability, while decreasing heterochromatin as well as DNA and histone H3K9me3 methylation. Immunoelectron microscopy detected endogenous IRE1 at the nuclear envelope. Thus, cancer cells co-opt IRE1 either enzymatically or nonenzymatically, which has significant implications for IRE1's biological role and therapeutic targeting.

DOI: https://doi.org/10.1371/journal.pbio.3003086
PLoS Biol. 2025 Apr 10. 23(4): e3003099

MSP-tracker: A versatile vesicle tracking software tool used to reveal the spatial control of polarized secretion in Drosophila epithelial cells.

Jennifer H Richens, Mariia Dmitrieva, Helen L Zenner, Nadine Muschalik, Richard Butler, Jade Glashauser, Carolina Camelo, Stefan Luschnig, Sean Munro, Jens Rittscher, Daniel St Johnston.

Understanding how specific secretory cargoes are targeted to distinct domains of the plasma membrane in epithelial cells requires analyzing the trafficking of post-Golgi vesicles to their sites of secretion. We used the RUSH (retention using selective hooks) system to synchronously release an apical cargo, Cadherin 99C (Cad99C), and a basolateral cargo, the ECM protein Nidogen, from the endoplasmic reticulum and follow their movements to the plasma membrane. We also developed an interactive vesicle tracking framework, MSP-tracker and viewer, that exploits developments in computer vision and deep learning to determine vesicle trajectories in a noisy environment without the need for extensive training data. MSP-tracker outperformed other tracking software in detecting and tracking post-Golgi vesicles, revealing that Cad99c vesicles predominantly move apically with a mean speed of 1.1µm/sec. This is reduced to 0.85 µm/sec by a dominant slow dynein mutant, demonstrating that dynein transports Cad99C vesicles to the apical cortex. Furthermore, both the dynein mutant and microtubule depolymerization cause lateral Cad99C secretion. Thus, microtubule organization plays a central role in targeting apical secretion, suggesting that Drosophila does not have distinct apical versus basolateral vesicle fusion machinery. Nidogen vesicles undergo planar-polarized transport to the leading edge of follicle cells as they migrate over the ECM, whereas most Collagen is secreted at trailing edges. The follicle cells therefore bias secretion of different ECM components to opposite sides of the cell, revealing that the secretory pathway is more spatially organized than previously thought.

DOI: https://doi.org/10.1371/journal.pbio.3003099
Expert Opin Drug Discov. 2025 Apr 10. 1-13

Advancing target validation with PROTAC technology.

M Leora Spitz, Aseel Kashkush, Raphael I Benhamou.

   INTRODUCTION: Targeted protein degradation (TPD) is a cutting-edge technology that provides new avenues for drug discovery and development. PROteolysis TArgeting Chimeras (PROTACs) are the most established and advanced TPD strategy, enabling the selective degradation of disease-associated and 'undruggable' proteins of interest (POIs) by leveraging the cell's natural protein degradation machinery. To confirm that PROTAC-induced proximity drives protein degradation, target validation and ternary complex formation must be thoroughly assessed.
AREAS COVERED: In this perspective, the authors detail some of the most widely used in silico, structural, in vitro, and in cellulo methods to validate PROTAC target engagement and ternary complex formation. Additionally, they discuss the growing use of PROTACs as chemical probes for novel target identification and validation.
EXPERT OPINION: Target validation is essential in the PROTAC approach, and ongoing studies should prioritize confirming ternary complex formation using assays conducted under physiologically relevant cellular conditions. Proteomics analyses are among the most valuable tools for elucidating PROTAC mechanisms, selectivity, and outcomes. The authors are optimistic about the future of PROTACs in drug development and their use as probes to confirm target engagement. PROTAC technology holds vast opportunities for future exploration, offering significant potential to further both chemical and biological research.

Keywords:  Drug design; PROTAC; small molecules; target validation; targeted degradation; ternary complex; ubiquitination

DOI:  https://doi.org/10.1080/17460441.2025.2490248
Nat Commun. 2025 Apr 09. 16(1): 3378

Multiple cAMP/PKA complexes at the STIM1 ER/PM junction specified by E-Syt1 and E-Syt2 reciprocally gates ANO1 (TMEM16A) via Ca2.

Wei-Yin Lin, Woo Young Chung, Seonghee Park, Ava Movahed Abtahi, Benjamin Leblanc, Malini Ahuja, Shmuel Muallem.

ANO1 plays a crucial role in determining numerous physiological functions, including epithelial secretion, yet its regulatory mechanisms remain incompletely understood. Here, we describe a fundamental dynamic regulation of ANO1 surface expression and Ca2+-dependent gating via the cAMP/PKA pathway at the STIM1 ER/PM junctions. At these junctions, STIM1 assembles AC-AKAP-PKA complexes, while E-Syt1 mediates formation of ANO1-VAPA-IRBIT-E-Syt1-AC8-AKAP5-PKA complex, that phosphorylates ANO1 S673, increasing ANO1 Ca2+ affinity. Within these complexes, the Ca2+ and cAMP pathways act synergistically to enhance ANO1 function. By contrast, E-Syt2 dissociates the ANO1-VAPA interaction, forming ANO1-IRBIT-E-Syt2-AC6-AKAP11-PKA complex that phosphorylates ANO1 S221, which markedly reduces ANO1 Ca2+ affinity. The effects of the E-Syts are primarily mediated by their reciprocal regulation of junctional PI(4)P, PI(4,5)P2 and PtdSer. Accordingly, IRBIT deletion in mice impairs receptor-stimulated activation of ANO1 and fluid secretion. These findings should have broad implications for ANO1 roles and functions across various tissues.

DOI: https://doi.org/10.1038/s41467-025-58682-w
Chem Sci. 2025 Mar 31.

A method to identify small molecule/protein pairs susceptible to protein ubiquitination by the CRBN E3 ligase.

Pinwen Cai, Chiara Disraeli, Basilius Sauter, Saule Zhanybekova, Dennis Gillingham.

Although using DNA-encoded libraries (DELs) to find small molecule binders of target proteins is well-established, identifying molecules with functions beyond binding remains challenging in pooled screens. Here, we develop an approach for multiplexing functional screens that simultaneously evaluates encoded small molecules and encoded collections of protein targets in functional selections. We focus on ubiquitin (Ub) transfer with the cereblon-bound CRL4 E3 ligase because of its proven versatility in drug discovery. The functional selections recover small molecule/G-hairpin loop pairs based on their ability to promote Ub-transfer onto the G-hairpin loop. As Ub-transfer is the first step in tagging proteins for proteasomal destruction, finding small molecules capable of selectively reprogramming it is a significant challenge in contemporary drug development. Our work lays the foundation for functional DEL selections that match small molecule Ub-transfer catalysts with their optimal protein substrates.

DOI: https://doi.org/10.1039/d5sc01251a
Nature. 2025 Apr 09.

Multimodal cell maps as a foundation for structural and functional genomics.

Leah V Schaffer, Mengzhou Hu, Gege Qian, Kyung-Mee Moon, Abantika Pal, Neelesh Soni, Andrew P Latham, Laura Pontano Vaites, Dorothy Tsai, Nicole M Mattson, Katherine Licon, Robin Bachelder, Anthony Cesnik, Ishan Gaur, Trang Le, William Leineweber, Aji Palar, Ernst Pulido, Yue Qin, Xiaoyu Zhao, Christopher Churas, Joanna Lenkiewicz, Jing Chen, Keiichiro Ono, Dexter Pratt, Peter Zage, Ignacia Echeverria, Andrej Sali, J Wade Harper, Steven P Gygi, Leonard J Foster, Edward L Huttlin, Emma Lundberg, Trey Ideker.

Human cells consist of a complex hierarchy of components, many of which remain unexplored1,2. Here we construct a global map of human subcellular architecture through joint measurement of biophysical interactions and immunofluorescence images for over 5,100 proteins in U2OS osteosarcoma cells. Self-supervised multimodal data integration resolves 275 molecular assemblies spanning the range of 10-8 to 10-5 m, which we validate systematically using whole-cell size-exclusion chromatography and annotate using large language models3. We explore key applications in structural biology, yielding structures for 111 heterodimeric complexes and an expanded Rag-Ragulator assembly. The map assigns unexpected functions to 975 proteins, including roles for C18orf21 in RNA processing and DPP9 in interferon signalling, and identifies assemblies with multiple localizations or cell type specificity. It decodes paediatric cancer genomes4, identifying 21 recurrently mutated assemblies and implicating 102 validated new cancer proteins. The associated Cell Visualization Portal and Mapping Toolkit provide a reference platform for structural and functional cell biology.

DOI: https://doi.org/10.1038/s41586-025-08878-3
Nat Commun. 2025 Apr 11. 16(1): 3474

Suppression of TGF-β/SMAD signaling by an inner nuclear membrane phosphatase complex.

Zhe Ji, Wing-Yan Skyla Siu, Maria Emilia Dueñas, Leonie Müller, Matthias Trost, Pedro Carvalho.

Cytokines of the TGF-β superfamily control essential cell fate decisions via receptor regulated SMAD (R-SMAD) transcription factors. Ligand-induced R-SMAD phosphorylation in the cytosol triggers their activation and nuclear accumulation. We determine how R-SMADs are inactivated by dephosphorylation in the cell nucleus to counteract signaling by TGF-β superfamily ligands. We show that R-SMAD dephosphorylation is mediated by an inner nuclear membrane associated complex containing the scaffold protein MAN1 and the CTDNEP1-NEP1R1 phosphatase. Structural prediction, domain mapping and mutagenesis reveals that MAN1 binds independently to the CTDNEP1-NEP1R1 phosphatase and R-SMADs to promote their inactivation by dephosphorylation. Disruption of this complex causes nuclear accumulation of R-SMADs and aberrant signaling, even in the absence of TGF-β ligands. These findings establish CTDNEP1-NEP1R1 as the R-SMAD phosphatase, reveal the mechanistic basis for TGF-β signaling inactivation and highlight how this process is disrupted by disease-associated MAN1 mutations.

DOI: https://doi.org/10.1038/s41467-025-58681-x
J Cell Biol. 2025 May 05. pii: e202407166. [Epub ahead of print]224(5):

PITPβ promotes COPI vesicle fission through lipid transfer and membrane contact formation.

Kunyou Park, Sungeun Ju, Hyewon Choi, Peng Gao, Geul Bang, Jung Hoon Choi, Jiwon Jang, Andrew J Morris, Byung-Ho Kang, Victor W Hsu, Seung-Yeol Park.

Intracellular transport among organellar compartments occurs in two general ways: by membrane-bound carriers and membrane contacts. Specific circumstances that involve the coordination of these two modes of transport remain to be defined. By studying coat protein I (COPI) transport, we find that phosphatidylcholine with short acyl chains (sPC) is delivered through membrane contact from the endoplasmic reticulum (ER) to sites of COPI vesicle formation at the Golgi to support the fission stage. Phosphatidylinositol transfer protein beta (PITPβ) plays a key role in this process, with the elucidation of this role shedding new insights into how PITPβ acts, providing a mechanistic understanding of a specific circumstance when vesicular transport requires membrane contact and contributing to the general understanding of how intracellular transport carriers are formed.

DOI: https://doi.org/10.1083/jcb.202407166
bioRxiv. 2025 Mar 25. pii: 2025.03.20.644235. [Epub ahead of print]

Large-scale discovery, analysis, and design of protein energy landscapes.

Állan J R Ferrari, Sugyan M Dixit, Jane Thibeault, Mario Garcia, Scott Houliston, Robert W Ludwig, Pascal Notin, Claire M Phoumyvong, Cydney M Martell, Michelle D Jung, Kotaro Tsuboyama, Lauren Carter, Cheryl H Arrowsmith, Miklos Guttman, Gabriel J Rocklin.

All folded proteins continuously fluctuate between their low-energy native structures and higher energy conformations that can be partially or fully unfolded. These rare states influence protein function, interactions, aggregation, and immunogenicity, yet they remain far less understood than protein native states. Although native protein structures are now often predictable with impressive accuracy, conformational fluctuations and their energies remain largely invisible and unpredictable, and experimental challenges have prevented large-scale measurements that could improve machine learning and physics-based modeling. Here, we introduce a multiplexed experimental approach to analyze the energies of conformational fluctuations for hundreds of protein domains in parallel using intact protein hydrogen-deuterium exchange mass spectrometry. We analyzed 5,778 domains 28-64 amino acids in length, revealing hidden variation in conformational fluctuations even between sequences sharing the same fold and global folding stability. Site-resolved hydrogen exchange NMR analysis of 13 domains showed that these fluctuations often involve entire secondary structural elements with lower stability than the overall fold. Computational modeling of our domains identified structural features that correlated with the experimentally observed fluctuations, enabling us to design mutations that stabilized low-stability structural segments. Our dataset enables new machine learning-based analysis of protein energy landscapes, and our experimental approach promises to reveal these landscapes at unprecedented scale.

DOI: https://doi.org/10.1101/2025.03.20.644235
Cell Rep. 2025 Apr 09. pii: S2211-1247(25)00210-4. [Epub ahead of print]44(4): 115439

Activity-dependent synthesis of Emerin gates neuronal plasticity by regulating proteostasis.

Yi Xie, Ruoxi Wang, Daniel B McClatchy, Yuanhui Ma, Jolene Diedrich, Manuel Sanchez-Alavez, Michael Petrascheck, John R Yates, Hollis T Cline.

  Neurons dynamically regulate their proteome in response to sensory input, a key process underlying experience-dependent plasticity. We characterized the visual experience-dependent nascent proteome in mice within a brief, defined time window after stimulation using an optimized metabolic labeling approach. Visual experience induced cell-type-specific and age-dependent alterations in the nascent proteome, including proteostasis-related proteins. Emerin is the top activity-induced candidate plasticity protein. Activity-induced neuronal Emerin synthesis is rapid and transcription independent. Emerin broadly inhibits protein synthesis, decreasing translation regulators and synaptic proteins. Decreasing Emerin shifted the dendritic spine population from a predominantly mushroom morphology to filopodia and decreased network connectivity. Blocking visual experience-induced Emerin reduced visually evoked electrophysiological responses and impaired behaviorally assessed visual information processing. Our findings support a proteostatic model in which visual experience-induced Emerin provides a feedforward block on further protein synthesis, refining temporal control of activity-induced plasticity proteins and optimizing visual system function.

Keywords:  CP: Molecular biology; CP: Neuroscience; Emerin; cell-type-specific proteomics; metabolic protein labeling; protein synthesis; proteostasis; sensory experience-dependent plasticity; synaptic plasticity; visual depth perception; visual response

DOI:  https://doi.org/10.1016/j.celrep.2025.115439
Nat Commun. 2025 Apr 07. 16(1): 3292

RNA G-quadruplexes control mitochondria-localized mRNA translation and energy metabolism.

Leïla Dumas, Sauyeun Shin, Quentin Rigaud, Marie Cargnello, Beatriz Hernández-Suárez, Pauline Herviou, Nathalie Saint-Laurent, Marjorie Leduc, Morgane Le Gall, David Monchaud, Erik Dassi, Anne Cammas, Stefania Millevoi.

Cancer cells rely on mitochondria for their bioenergetic supply and macromolecule synthesis. Central to mitochondrial function is the regulation of mitochondrial protein synthesis, which primarily depends on the cytoplasmic translation of nuclear-encoded mitochondrial mRNAs whose protein products are imported into mitochondria. Despite the growing evidence that mitochondrial protein synthesis contributes to the onset and progression of cancer, and can thus offer new opportunities for cancer therapy, knowledge of the underlying molecular mechanisms remains limited. Here, we show that RNA G-quadruplexes (RG4s) regulate mitochondrial function by modulating cytoplasmic mRNA translation of nuclear-encoded mitochondrial proteins. Our data support a model whereby the RG4 folding dynamics, under the control of oncogenic signaling and modulated by small molecule ligands or RG4-binding proteins, modifies mitochondria-localized cytoplasmic protein synthesis. Ultimately, this impairs mitochondrial functions, affecting energy metabolism and consequently cancer cell proliferation.

DOI: https://doi.org/10.1038/s41467-025-58118-5
Mol Cell. 2025 Apr 03. pii: S1097-2765(25)00259-X. [Epub ahead of print]

MLKL activity requires a splicing-regulated, druggable intramolecular interaction.

Uris Ros, Veronica Martinez-Osorio, Pedro A Valiente, Yasmin Abdelwahab, Milos Gojkovic, Raed Shalaby, Silvia Zanna, Julia Saggau, Laurens Wachsmuth, Harshal N Nemade, Jonathan Zoeller, Hannah Lottermoser, Yu-Guang Chen, Mohamed Ibrahim, Konstantinos Kelepouras, Lazaros Vasilikos, Paula Bedoya, Rafael A Espiritu, Stefan Müller, Veronika Altmannova, D Peter Tieleman, John Weir, Julian Langer, Matti Adam, Henning Walczak, W Wei-Lynn Wong, Gianmaria Liccardi, Martin Mollenhauer, Manolis Pasparakis, Nieves Peltzer, Ana J García-Sáez.

  Necroptosis is an inflammatory form of regulated cell death implicated in a range of human pathologies, whose execution depends on the poorly understood pseudokinase mixed lineage kinase domain-like (MLKL). Here, we report that splicing-dependent insertion of a short amino acid sequence in the C-terminal α-helix (Hc) of MLKL abolishes cell killing activity and creates an anti-necroptotic isoform that counteracts cell death induced by the necroptosis-proficient protein in mice and humans. We show that interaction of Hc with a previously unrecognized hydrophobic groove is essential for necroptosis, which we exploited in a strategy to identify small molecules that inhibit MLKL and substantially ameliorate disease in murine models of necroptosis-driven dermatitis and abdominal aortic aneurysm. Thus, alternative splicing of microexons controls the ability of MLKL to undergo an intramolecular rearrangement essential for necroptosis with potential to guide the development of allosteric MLKL inhibitors for the treatment of human disease.

Keywords:  MLKL; abdominal aortic aneurysm; drug discovery; inflammatory diseases; membrane permeabilization; microexons; necroptosis; skin inflammation; small molecule inhibitors; splicing variants

DOI:  https://doi.org/10.1016/j.molcel.2025.03.015
Cell Rep. 2025 Apr 03. pii: S2211-1247(25)00271-2. [Epub ahead of print]44(4): 115500

Comprehensive PTM profiling with SCASP-PTM uncovers mechanisms of p62 degradation and ALDOA-mediated tumor progression.

Zhan-Peng Lin, Guohong Gan, Xiao Xu, Chengwen Wen, Xin Ding, Xiang-Yu Chen, Kaijie Zhang, Wen-Yu Guo, Mingxin Lin, Yu-Yang Wang, Xi Chen, Changchuan Xie, Jinling Wang, Minjie Li, Chuan-Qi Zhong.

  Multiple post-translational modification (PTM) proteomics typically combines PTM enrichment with multiplex isobaric labeling and peptide fractionation. However, effective methods for sequentially enriching multiple PTMs from a single sample for data-independent acquisition mass spectrometry (DIA-MS) remain lacking. We present SDS-cyclodextrin-assisted sample preparation (SCASP)-PTM, an approach that enables desalting-free enrichment of diverse PTMs, including phosphopeptides, ubiquitinated peptides, acetylated peptides, glycopeptides, and biotinylated peptides. SCASP-PTM uses SDS for protein denaturation, which is sequestered by cyclodextrins before trypsin digestion, facilitating sequential PTM enrichment without additional purification steps. Combined with DIA-MS, SCASP-PTM quantifies the proteome, ubiquitinome, phosphoproteome, and glycoproteome in HeLa-S3 cell samples, identifying serine 28 phosphorylation as a key driver of poly(I:C)-induced p62 degradation. This method also quantifies PTMs in clinical tissue samples, revealing the critical role of ALDOA K330 ubiquitination/acetylation in tumor progression. SCASP-PTM offers a streamlined workflow for comprehensive PTM analysis in both basic research and clinical applications.

Keywords:  ALDOA; CP: Cancer; CP: Molecular biology; data-independent acquisition; diaPASEF; post-translational modifications

DOI:  https://doi.org/10.1016/j.celrep.2025.115500
Mol Cell. 2025 Mar 28. pii: S1097-2765(25)00251-5. [Epub ahead of print]

Structural basis of BAK sequestration by MCL-1 in apoptosis.

Shagun Srivastava, Giridhar Sekar, Adedolapo Ojoawo, Anup Aggarwal, Elisabeth Ferreira, Emiko Uchikawa, Meek Yang, Christy R Grace, Raja Dey, Yi-Lun Lin, Cristina D Guibao, Seetharaman Jayaraman, Somnath Mukherjee, Anthony A Kossiakoff, Bin Dong, Alexander Myasnikov, Tudor Moldoveanu.

  Apoptosis controls cell fate, ensuring tissue homeostasis and promoting disease when dysregulated. The rate-limiting step in apoptosis is mitochondrial poration by the effector B cell lymphoma 2 (BCL-2) family proteins BAK and BAX, which are activated by initiator BCL-2 homology 3 (BH3)-only proteins (e.g., BIM) and inhibited by guardian BCL-2 family proteins (e.g., MCL-1). We integrated structural, biochemical, and pharmacological approaches to characterize the human prosurvival MCL-1:BAK complex assembled from their BCL-2 globular core domains. We reveal a canonical interaction with BAK BH3 bound to the hydrophobic groove of MCL-1 and disordered and highly dynamic BAK regions outside the complex interface. We predict similar conformations of activated effectors in complex with other guardians or effectors. The MCL-1:BAK complex is a major cancer drug target. We show that MCL-1 inhibitors are inefficient in neutralizing the MCL-1:BAK complex, requiring high doses to initiate apoptosis. Our study underscores the need to design superior clinical candidate MCL-1 inhibitors.

Keywords:  BCL-2 antagonist killer BAK; BCL-2 family proteins; BCL-2-like protein 11 BIM; BH3 mimetics; BH3-interacting domain death agonist BID; BH3-only initiator; MCL-1 inhibitors; NMR spectroscopy; Phorbol-12-myristate-13-acetate-induced protein 1 NOXA; X-ray crystallography; apoptosis; apoptosis resistance; cryo-electron microscopy; direct BAK activation; induced myeloid leukemia cell differentiation protein MCL-1; mitochondrial outer membrane permeabilization MOMP; mode II MCL-1:BAK sequestration; mode II neutralization; prodeath effector; prosurvival guardian

DOI:  https://doi.org/10.1016/j.molcel.2025.03.013