bims-proteo 2025-07-13 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2025–07–13
33 papers selected by
Eric Chevet, INSERM

ER-endosome contacts generate a local environment promoting phagophore formation.
Ub-POD: A Ubiquitin-Specific Proximity-Dependent Labeling Technique to Identify E3 Ubiquitin Ligase Substrates in Human Cells.
Rational Design of CDK12/13 and BRD4 Molecular Glue Degraders.
Sensing of extracellular l-proline availability by the integrated stress response determines the outcome of cell competition.
mRNA 3'UTRs chaperone intrinsically disordered regions to control protein activity.
Autophagic stress activates distinct compensatory secretory pathways in neurons.
Mechanism of cotranslational protein N-myristoylation in human cells.
CDR2 is a dynein adaptor recruited by kinectin to regulate ER sheet organization.
Identification of RNF114 as ADPr-Ub reader through non-hydrolysable ubiquitinated ADP-ribose.
Structural Insights into the GABARAP-ATG3 Backside Interaction and Apo ATG3 Conformation.
E3 ligase recruitment by UBQLN2 protects substrates from proteasomal degradation.
Serine ADPr on histones and PARP1 is a cellular target of ester-linked ubiquitylation.
EMP1 safeguards hematopoietic stem cells by suppressing sphingolipid metabolism and alleviating endoplasmic reticulum stress.
Inhibition of the UFD-1-NPL-4 complex triggers an aberrant immune response in Caenorhabditis elegans.
Proteins with cognition-associated structural changes in a rat model of aging exhibit reduced refolding capacity.
Stop codon context modulates NMD efficiency through translation termination kinetics.
Retention of DLK1 in the endoplasmic reticulum identifies roles for EGF-like-domain-specific O-glycans in the secretory pathway.
Benchmarking the Builders: A Comparative Analysis of PRosettaC and AlphaFold3 for Predicting PROTAC Ternary Complexes.
Endoplasmic Reticulum Stress-Driven Nucleotide Catabolism Fuels Prostate Cancer.
Nondisruptive inducible labeling of ER-membrane contact sites using the Lamin B receptor.
TRIM21-Driven Degradation of BRD4: Development of Heterobifunctional Degraders and Investigation of Recruitment and Selectivity Mechanisms.
Dynamic regulation of Sec24C by phosphorylation and O-GlcNAcylation during cell cycle progression.
UFMylation maintains tumor suppressor pVHL stability by activating the deubiquitinase BAP1.
Progressive Supranuclear Palsy PERK haplotype B selectively translates DLX1 promoting tau toxicity.
Advancing protein evolution with inverse folding models integrating structural and evolutionary constraints.
The role of metabolism in shaping enzyme structures over 400 million years.
Nuclear p62/SQSTM1 facilitates ubiquitin-independent proteasomal degradation of BMAL1.
Activation of endogenous PRKN by structural derepression is linked to increased turnover of the E3 ubiquitin ligase.
Cdk5rap3-mediated regulation of lysosomal and ER membrane proteins is pivotal for the survival and function of pancreatic acinar cells.
TBK1 phagosomal recruitment enhances antifungal immunity via positive feedback regulation with SRC.
Human protein interactome structure prediction at scale with Boltz-2.
mRNA interactions promote cotranslational association of heteromeric membrane proteins.
Endoplasmic Reticulum Stress and Unfolded Protein Response Sensor ERN1 Regulates Organic Dust Induction of Lung Inflammation.

Cell Rep. 2025 Jul 09. pii: S2211-1247(25)00764-8. [Epub ahead of print]44(7): 115993

ER-endosome contacts generate a local environment promoting phagophore formation.

Juliane Da Graça, Charlotte Thiola, Myckaëla Rouabah, Ida Chiara Guerrera, Naïma El Khallouki, Maryse Romao, Carla Alemany, Dobrawa Amiri, Sarah Dubois, Ashvini Srimoorthy, Francesca Giordano, Graça Raposo, Etienne Morel.

  Autophagy starts with the formation of a double-membrane vacuole called the autophagosome, initiated by a transient structure known as the phagophore. Previous studies reported that phagophore biogenesis primarily occurs at endoplasmic reticulum (ER) omegasome subdomains, but other evidence suggests that the phagophore derives from recycling endosomes. Our study demonstrates the importance of ER-endosome interactions, revealing the dynamic mobilization of endosome-ER contact sites (EERCSs) in response to starvation. We characterize a sequential tethering of Rab5 and Rab11 endosomes to omegasomes, facilitating phagophore biogenesis. Detailed analyses reveal that EERCS-associated molecular machinery creates a confined environment that promotes local Ca2+ accumulation and liquid-liquid phase separation at ER exit sites. This environment primes de novo phagophore formation through a Rab3a-RAB3GAP1/2-mediated nano-vesicle fusion. We propose that EERCS mobilization generates transient cytoplasmic confinement, fostering localized accumulation of components for phagophore biogenesis. Our study reveals a novel role for the ER-endosome interface in the nutrient deprivation response, emphasizing organelle coordination during autophagy initiation.

Keywords:  CP: Cell biology; ER; autophagy; calcium; endoplasmic reticulum; endosomes; membrane contact sites; phagophore; phase transition; starvation

DOI:  https://doi.org/10.1016/j.celrep.2025.115993
Bio Protoc. 2025 Jun 20. 15(12): e5351

Ub-POD: A Ubiquitin-Specific Proximity-Dependent Labeling Technique to Identify E3 Ubiquitin Ligase Substrates in Human Cells.

Urbi Mukhopadhyay, Sophie Levantovsky, Christian Behrends, Sagar Bhogaraju.

  Ubiquitination is a post-translational protein modification that regulates a vast majority of processes during protein homeostasis. The covalent attachment of ubiquitin is a highly regulated process carried out by the sequential action of the three enzymes E1, E2, and E3. E3 ligases share a dual function of 1) transferring covalently attached ubiquitin from the catalytic cysteine of E2 (E2~Ub) to the substrate and 2) providing substrate specificity. Our current knowledge of their individual substrate pools is incomplete due to the difficult capture of these transient substrate-E3 ligase interactions. Here, we present an efficient protocol that enables the selective biotinylation of substrates of a given ubiquitin ligase. In brief, the candidate E3 ligase is fused to the biotin ligase BirA and ubiquitin to a biotin acceptor peptide, an Avi-tag variant (-2) AP. Cells are co-transfected with these fusion constructs and exposed to biotin, resulting in a BirA-E3 ligase-catalyzed biotinylation of (-2) AP-Ub when in complex with E2. As the next step, the biotinylated (-2) AP-Ub is transferred covalently to the substrate lysine, which enables an enrichment via denaturing streptavidin pulldown. Substrate candidates can then be identified via mass spectrometry (MS). Our ubiquitin-specific proximity-dependent labeling (Ub-POD) method allows robust biotinylation of the ubiquitylation substrates of a candidate E3 ligase thanks to the wild-type BirA and biotin acceptor peptide fused to the E3 and Ub, respectively. Because of the highly Ub-specific labeling, Ub-POD is more appropriate for identifying ubiquitination substrates compared to other conventional proximity labeling or immunoprecipitation (IP) approaches. Key features • A simple and cost-effective method using common chemicals makes Ub-POD easy to implement in any laboratory. • Can be an exploratory tool to identify new substrates via mass spectrometry (MS) or as a validation tool in combination with immunoblotting or immunofluorescence. • Knowledge of triggers and constraints of E3 ligase activity is beneficial.

Keywords:  Biotinylation; BirA; E3 ligase; Proximity labeling; RING; Substrate identification; U box; Ub-POD; Ubiquitin

DOI:  https://doi.org/10.21769/BioProtoc.5351
Angew Chem Int Ed Engl. 2025 Jul 08. e202508427

Rational Design of CDK12/13 and BRD4 Molecular Glue Degraders.

Nathanael Schiander Gray, Zhe Zhuang, Woong Sub Byun, Zuzanna Kozicka, Katherine Donovan, Brendan Dwyer, Abby Thornhill, Hannah Jones, Zixuan Jiang, Xijun Zhu, Eric Fischer, Nicolas Thomä.

  Targeted protein degradation (TPD) is an emerging therapeutic approach for the selective elimination of disease-related proteins. While molecular glue degraders exhibit drug-like properties, their discovery has traditionally been serendipitous and often requires post-hoc rationalization. In this study, we demonstrate the rational, mechanism-guided design of molecular glue degraders using gluing moieties. Building on established principles, by appending a chemical gluing moiety to several small molecule inhibitors, we successfully transformed them into degraders, obviating the need for a specific E3 ubiquitin ligase recruiter. Specifically, we found that incorporating a hydrophobic aromatic ring or a double bond into a cyclin-dependent kinase 12 and 13 (CDK12/13) dual inhibitor enabled the recruitment of DNA damage-binding protein 1 (DDB1), thereby transforming a high-molecular-weight bivalent CDK12 degrader into a potent monovalent CDK12/13 molecular glue degrader. We also showcase that attaching a cysteine-reactive warhead to a bromodomain-containing protein 4 (BRD4) inhibitor converts it into a degrader by recruiting the DDB1 and CUL4 associated factor 16 (DCAF16) E3 ligase.

Keywords:  Targeted protein degradation; bromodomain-containing protein 4; cyclin-dependent kinase 12/13; molecular glue

DOI:  https://doi.org/10.1002/anie.202508427
Sci Adv. 2025 Jul 11. 11(28): eadw1883

Sensing of extracellular l-proline availability by the integrated stress response determines the outcome of cell competition.

Shruthi Krishnan, Ana Lima, Sizhe Tan, Ying Thong Low, Salvador Perez Montero, Aida Di Gregorio, Adrian Perez Barreto, Sarah Bowling, Karen Vousden, Tristan A Rodriguez.

Cell competition is a conserved fitness quality control that eliminates cells that are less fit than their neighbors. How winner cells induce the elimination of losers is poorly understood. We tackle this question by studying the onset of embryonic differentiation in mice, where cell competition eliminates 35% of embryonic cells. These loser cells have mitochondrial dysfunction, which we show causes amino acid deprivation and activation of the integrated stress response (ISR), a pathway essential for their survival. We demonstrate that l-proline is a key amino acid sensed by the ISR and that proline represses the ISR and drives their elimination. These results indicate that cell competition acts as a previously unidentified tissue-sparing mechanism, regulated by the availability of extracellular amino acids, that allows for the elimination of dysfunctional cells when amino acids are plentiful but ensures their survival in nutrient-poor environments.

DOI: https://doi.org/10.1126/sciadv.adw1883
bioRxiv. 2025 Jul 03. pii: 2025.07.02.662873. [Epub ahead of print]

mRNA 3'UTRs chaperone intrinsically disordered regions to control protein activity.

Yang Luo, Yaofeng Zhong, Sudipto Basu, Christine Mayr.

More than 2,700 human mRNA 3'UTRs have hundreds of highly conserved (HC) nucleotides, but their biological roles are unclear. Here, we show that mRNAs with HC 3'UTRs mostly encode proteins with long intrinsically disordered regions (IDRs), including MYC, UTX, and JMJD3. These proteins are only fully active when translated from mRNA templates that include their 3'UTRs, raising the possibility of functional interactions between 3'UTRs and IDRs. Rather than affecting protein abundance or localization, we find that HC 3'UTRs control transcriptional or histone demethylase activity through co-translationally determined protein oligomerization states that are kinetically stable. 3'UTR-dependent changes in protein folding require mRNA-IDR interactions, suggesting that mRNAs act as IDR chaperones. These mRNAs are multivalent, a biophysical RNA feature that enables their translation in network-like condensates, which provide favorable folding environments for proteins with long IDRs. These data indicate that the coding sequence is insufficient for the biogenesis of biologically active conformations of IDR-containing proteins and that RNA can catalyze protein folding.

DOI: https://doi.org/10.1101/2025.07.02.662873
Proc Natl Acad Sci U S A. 2025 Jul 15. 122(28): e2421886122

Autophagic stress activates distinct compensatory secretory pathways in neurons.

Sierra D Palumbos, Jacob Popolow, Juliet Goldsmith, Erika L F Holzbaur.

  Autophagic dysfunction is a hallmark of neurodegenerative disease, leaving neurons vulnerable to the accumulation of damaged organelles and aggregated proteins. However, the late onset of diseases suggests that compensatory quality control mechanisms may be engaged to delay these deleterious effects. Neurons expressing common familial Parkinson's disease-associated mutations in the leucine-rich repeat kinase 2 (LRRK2) exhibit defective autophagy. Here, we demonstrate that both primary murine neurons and human induced Pluripotent Stem Cells (iPSC)-derived neurons harboring pathogenic LRRK2 upregulate the secretion of extracellular vesicles. We used unbiased proteomics to characterize the secretome of LRRK2G2019S neurons and found that autophagic cargos including mitochondrial proteins were enriched. Based on these observations, we hypothesize that autophagosomes are rerouted toward secretion when cell-autonomous degradation is compromised to mediate clearance of undegraded cellular waste. Immunoblotting confirmed the release of autophagic cargos and live-cell imaging demonstrated that secretory autophagy is upregulated in LRRK2G2019S neurons. We also found that LRRK2G2019S neurons upregulate the release of exosomes containing microRNAs. Live-cell imaging confirmed that this upregulation of exosomal release is dependent on hyperactive LRRK2 activity, while pharmacological experiments indicate that this release staves off apoptosis. Finally, we show that markers of both vesicle populations are upregulated in plasma from mice expressing pathogenic LRRK2. In sum, we find that neurons expressing pathogenic LRRK2 upregulate secretory autophagy and the compensatory release of exosomes to mediate waste disposal and transcellular communication, respectively. We propose that this increased secretion contributes to the maintenance of cellular homeostasis, delaying neurodegenerative disease progression over the short term while potentially contributing to neuroinflammation over the longer term.

Keywords:  Parkinson’s disease; autophagy; neurodegeneration; secretion

DOI:  https://doi.org/10.1073/pnas.2421886122
Mol Cell. 2025 Jul 04. pii: S1097-2765(25)00544-1. [Epub ahead of print]

Mechanism of cotranslational protein N-myristoylation in human cells.

Martin Gamerdinger, Blanca Echeverria, Alfred M Lentzsch, Nicolas Burg, Ziyi Fan, Mateusz Jaskolowski, Alain Scaiola, Selina Piening, Shu-Ou Shan, Nenad Ban, Elke Deuerling.

  N-myristoyltransferases (NMTs) cotranslationally transfer the fatty acid myristic acid to the N terminus of newly synthesized proteins, regulating their function and cellular localization. These enzymes are important drug targets for the treatment of cancer and viral infections. N-myristoylation of nascent proteins occurs specifically on N-terminal glycine residues after the excision of the initiator methionine by methionine aminopeptidases (METAPs). How NMTs interact with ribosomes and gain timely and specific access to their substrates remains unknown. Here, we show that human NMT1 exchanges with METAP1 at the ribosomal tunnel exit to form an active cotranslational complex together with the nascent polypeptide-associated complex (NAC). NMT1 binding is sequence selective and specifically triggered by methionine excision, which exposes the N-myristoylation motif in the nascent chain. The revealed mode of interaction of NMT1 with NAC and the methionine-cleaved nascent protein elucidates how a specific subset of proteins can be efficiently N-myristoylated in human cells.

Keywords:  Coenzyme A; N-acetyltransferase; N-terminal modification; NAC; NMT; methionine aminopeptidase; myristic acid; nascent polypeptide-associated complex; ribosome

DOI:  https://doi.org/10.1016/j.molcel.2025.06.015
J Cell Biol. 2025 Sep 01. pii: e202411034. [Epub ahead of print]224(9):

CDR2 is a dynein adaptor recruited by kinectin to regulate ER sheet organization.

Vanessa Teixeira, Kashish Singh, José B Gama, Matilde Moreira, Ricardo Celestino, Ana Xavier Carvalho, Paulo S Pereira, Carla M C Abreu, Tiago J Dantas, Andrew P Carter, Reto Gassmann.

The ER relies on the microtubule cytoskeleton for the organization of its extended membrane network, but how microtubule-based motors contribute remains unclear. Using biochemical and cell-based assays, we identify cerebellar degeneration-related protein 2 (CDR2) and its paralog CDR2-like (CDR2L), onconeural antigens with poorly understood functions, as ER adaptors for cytoplasmic dynein-1 (dynein). We demonstrate in human cancer cells that CDR2 is recruited by the integral ER membrane protein kinectin (KTN1) and that double knockout of CDR2 and CDR2L enhances KTN1-dependent ER sheet stacking, reversal of which by exogenous CDR2 requires its dynein-binding CC1 box motif. Exogenous CDR2 expression additionally promotes CC1 box-dependent clustering of ER sheets near centrosomes. CDR2 competes with the eEF1Bβ subunit of translation elongation factor 1 for binding to KTN1, and eEF1Bβ knockdown increases endogenous CDR2 levels on ER sheets, inducing their centrosome-proximal clustering. Our study describes a novel molecular pathway that implicates dynein in ER sheet organization and may be involved in the pathogenesis of paraneoplastic cerebellar degeneration.

DOI: https://doi.org/10.1083/jcb.202411034
Nat Commun. 2025 Jul 09. 16(1): 6319

Identification of RNF114 as ADPr-Ub reader through non-hydrolysable ubiquitinated ADP-ribose.

Max S Kloet, Chatrin Chatrin, Rishov Mukhopadhyay, Bianca D M van Tol, Rebecca Smith, Sarah A Rotman, Rayman T N Tjokrodirijo, Kang Zhu, Andrii Gorelik, Lucy Maginn, Paul R Elliott, Peter A van Veelen, Dragana Ahel, Ivan Ahel, Gerbrand J van der Heden van Noort.

Crosstalk between the post-translational modification processes of ubiquitination and ADP-ribosylation occurs in DNA-damage- and immune-responses, in addition the physical linkage of ADP-ribose and ubiquitin is found during bacterial infection. Here, we study the ubiquitination of ADP-ribose mediated by human Deltex E3 ligases and the subsequent fate of the formed hybrid post-translational modification. We prepare a non-hydrolysable ADPr-Ub probe that we employ in a proteomics approach and identify RNF114 as an interacting protein. Using biophysical and biochemical experiments, we validate that RNF114 preferentially interacts with ubiquitinated ADP-ribose over non-modified ubiquitin. Subsequently, RNF114 can elongate the ubiquitinated ADP-ribose with a K11-linked ubiquitin chain. Using domain deletion analysis, we pinpoint the tandem zinc fingers and ubiquitin interacting motif (ZnF2 + ZnF3+UIM) domains of RNF114 to be crucial for recognising ubiquitinated ADP-ribose. Moreover, these domains are essential for the recruitment of RNF114 to the sites of laser-induced DNA damage.

DOI: https://doi.org/10.1038/s41467-025-61111-7
Biochemistry. 2025 Jul 08.

Structural Insights into the GABARAP-ATG3 Backside Interaction and Apo ATG3 Conformation.

Kazuto Ohashi, Gerard J Kroon, Takanori Otomo.

Members of the ATG8 family of ubiquitin-like proteins (Ubls) are covalently attached to phosphatidylethanolamine (PE) on nascent autophagosomal membranes, where they recruit cargo receptors and promote membrane expansion. Although the overall lipidation pathway is well established, the molecular details-particularly those involving the E2 enzyme ATG3-remain incompletely defined. Here, we uncover a previously unrecognized, noncovalent binding mode between the mammalian ATG8 protein GABARAP and the backside of ATG3's catalytic E2 domain. In crystals, an isopeptide-linked GABARAP∼ATG3 conjugate self-assembles into a helical filament via this backside interface, mirroring architectures observed for canonical Ub/Ubl∼E2 conjugates. The E2 backside-binding surface on GABARAP is topologically distinct from those of other Ub/Ubl proteins and overlaps the LC3-interacting region (LIR) motif-binding site. Solution NMR confirms this interaction, and targeted mutagenesis shows that disrupting the interface impairs PE conjugation. Complementary NMR and AlphaFold modeling of apo ATG3 reveal an intramolecular contact between a segment of its flexible region (FR) and the catalytic core that suppresses conjugation. Together, these findings establish backside engagement as a critical feature of ATG8 lipidation and illuminate the dynamic architecture and regulation of ATG3.

DOI: https://doi.org/10.1021/acs.biochem.4c00485
bioRxiv. 2025 Jul 01. pii: 2024.07.04.602059. [Epub ahead of print]

E3 ligase recruitment by UBQLN2 protects substrates from proteasomal degradation.

Ashley N Scheutzow, Sachini Thanthirige, Gracie Siffer, Alexander D Sorkin, Matthew L Wohlever.

Ubiquilins are a family of proteins critical to cellular proteostasis that are also linked to several neurodegenerative diseases, with specific mutations in UBQLN2 causing dominant, X-linked ALS. Despite an initial characterization as proteasomal shuttle factors, Ubiquilins have paradoxically been reported to stabilize numerous substrates. The basis of this triage decision remains enigmatic. Many other fundamental aspects of Ubiquilin function are unclear at the mechanistic level, such as the physiological significance of Ubiquilin phase separation, the unique role of each Ubiquilin paralog, and the mechanistic defects of ALS mutants. To address these questions, we utilized a library of triple knockout (TKO) rescue cell lines with physiological expression of single Ubiquilin paralogs or disease mutants in an isogenic background. Our findings reveal that UBQLN2 has a unique ability to protect substrates from degradation and that substrate stabilization correlates with the recruitment of multiple E3 ligases, including SCF bxo7 . We propose that E3 ligase recruitment promotes UBQLN2 phase separation, which protects substrates from proteasomal degradation. Consistent with this model, we demonstrate that ALS mutants, which were previously shown to have altered phase separation properties, also show a defect in substrate stabilization. Finally, we show that substrate stabilization appears to be a general feature of proteins that interact with the UBQLN2 Sti1 domains as amyloid precursor protein (APP) is also protected from proteasomal degradation by the formation of biomolecular condensates. This proposal unifies many existing observations in the field and presents a new paradigm for understanding Ubiquilin function in neurodegenerative disease.

DOI: https://doi.org/10.1101/2024.07.04.602059
Nat Chem Biol. 2025 Jul 09.

Serine ADPr on histones and PARP1 is a cellular target of ester-linked ubiquitylation.

Andreas Kolvenbach, Maria Dilia Palumbieri, Thomas Colby, Diyaraj Nadarajan, Remo Bode, Ivan Matić.

ADP-ribosylation and ubiquitylation regulate various cellular processes, with the complexity of their interplay becoming increasingly clear, as illustrated by ADP-ribosylation-dependent ubiquitylation mediated by Legionella. Biochemical studies have reported ester-linked ubiquitylation of ADP-ribose by DELTEX ubiquitin ligases, yet the modification sites on cellular targets remain unknown. Here, our search for interactors of RNF114 revealed DNA-damage-induced serine mono-ADP-ribosylation as a cellular target for ester-linked ubiquitylation. By developing proteomics strategies tailored to the chemical features of this composite modification, combined with an enrichment method using the zfDi19 and ubiquitin interaction motif domain (ZUD) of RNF114 and specific chemical elution, we identified ADP-ribosyl-linked serine ubiquitylation sites in cells, including on histones and poly(ADP-ribose) polymerase 1. Engineering ZUD into a modular reagent enabled the detection of this dual modification by immunoblotting. We establish ADP-ribosyl-ubiquitylation as an endogenous serine post-translational modification and propose that our multifaceted, tailored methodology will uncover its widespread occurrence, along with other conjugation chemistries, across diverse signaling pathways.

DOI: https://doi.org/10.1038/s41589-025-01974-5
Nat Commun. 2025 Jul 07. 16(1): 6247

EMP1 safeguards hematopoietic stem cells by suppressing sphingolipid metabolism and alleviating endoplasmic reticulum stress.

Lei Li, Yufei Lei, Yan Li, Yuxin Xie, Pusheng Hui, Xiaoyan Zang, Weiru Wu, Feng Wu, Jiankun Fan, Jianming Wang, Jieping Chen, Zhe Chen, Yu Hou.

The long-term maintenance of hematopoietic stem cells (HSCs) relies on the regulation of endoplasmic reticulum (ER) stress at a low level, but the underlying mechanism remains poorly understood. Here, we demonstrate that suppression of ER stress improves the functions of HSCs and protects HSCs against ionizing radiation (IR)-induced injury. We identify epithelial membrane protein 1 (EMP1) as a key regulator that mitigates ER stress in HSCs. Emp1 deficiency leads to the accumulation of protein aggregates and elevated ER stress, ultimately resulting in impaired HSC maintenance and self-renewal. Mechanistically, EMP1 is located within the ER and interacts with ceramide synthase 2 (CERS2) to limit the production of a class of sphingolipids, dihydroceramides (dhCers). DhCers accumulate in Emp1-deficient HSCs and induce protein aggregation. Furthermore, Emp1 deficiency renders HSCs more susceptible to IR, while overexpression of Emp1 or inhibition of CERS2 protects HSCs against IR-induced injury. These findings highlight the critical role played by the EMP1-CERS2-dhCers axis in constraining ER stress and preserving HSC potential.

DOI: https://doi.org/10.1038/s41467-025-61552-0
bioRxiv. 2025 Jul 06. pii: 2023.12.12.571255. [Epub ahead of print]

Inhibition of the UFD-1-NPL-4 complex triggers an aberrant immune response in Caenorhabditis elegans.

Rajneesh Rao, Alejandro Aballay, Jogender Singh.

The UFD-1 (ubiquitin fusion degradation 1)-NPL-4 (nuclear protein localization homolog 4) heterodimer is involved in extracting ubiquitinated proteins from several plasma membrane locations, including the endoplasmic reticulum. This heterodimer complex helps in the degradation of ubiquitinated proteins via the proteasome with the help of the AAA+ ATPase CDC-48. While the ubiquitin-proteasome system is known to have important roles in maintaining innate immune responses, the role of the UFD-1-NPL-4 complex in regulating immunity remains elusive. In this study, we investigate the role of the UFD-1-NPL-4 complex in maintaining Caenorhabditis elegans innate immune responses. Inhibition of the UFD-1-NPL-4 complex activates an aberrant immune response that reduces the survival of the wild-type worms on the pathogenic bacterium Pseudomonas aeruginosa despite diminishing colonization of the gut with the bacterium. This aberrant immune response improves the survival of severely immunocompromised worms on pathogenic bacteria but is detrimental on nonpathogenic bacteria. Transcriptomics studies reveal that the GATA transcription factor ELT-2 mediates the aberrant immune response upon inhibition of the UFD-1-NPL-4 complex. Collectively, our findings show that inhibition of the UFD-1-NPL-4 complex triggers an aberrant immune response that is detrimental to immunocompetent worms under infection conditions but can be advantageous for immunocompromised worms.

DOI: https://doi.org/10.1101/2023.12.12.571255
Sci Adv. 2025 Jul 11. 11(28): eadt3778

Proteins with cognition-associated structural changes in a rat model of aging exhibit reduced refolding capacity.

Haley E Tarbox, Audrey Branch, Stephen D Fried.

Cognitive decline during aging represents a major societal burden, causing both personal and economic hardship in an increasingly aging population. Many studies have found that the proteostasis network, which functions to keep proteins properly folded, is impaired with age, suggesting that there may be many proteins that incur structural alterations with age. Here, we used limited proteolysis mass spectrometry, a structural proteomic method, to globally interrogate protein conformational changes in a rat model of cognitive aging. Specifically, we compared soluble hippocampal proteins from aged rats with preserved cognition to those from aged rats with impaired cognition. We identified a couple hundred proteins as having undergone cognition-associated structural changes (CASCs). We report that CASC proteins are substantially more likely to be nonrefoldable than non-CASC proteins, meaning that they typically cannot spontaneously refold to their native conformations after being chemically denatured. These findings suggest that noncovalent, conformational alterations may be general features in cognitive decline.

DOI: https://doi.org/10.1126/sciadv.adt3778
Cell Genom. 2025 Jul 09. pii: S2666-979X(25)00204-6. [Epub ahead of print]5(7): 100948

Stop codon context modulates NMD efficiency through translation termination kinetics.

Dasa Longman, Laura Monaghan, Javier F Cáceres.

Nonsense-mediated mRNA decay efficiency varies unpredictably across transcripts containing premature termination codons. A new study by Kolakada et al.1 demonstrates that glycine residues preceding stop codons create an extended translation termination window that enhances NMD activity, offering new mechanistic explanations for NMD variability and improved clinical variant interpretation.

DOI: https://doi.org/10.1016/j.xgen.2025.100948
FEBS J. 2025 Jul 10.

Retention of DLK1 in the endoplasmic reticulum identifies roles for EGF-like-domain-specific O-glycans in the secretory pathway.

Yuko Tashima, Yohei Tsukamoto, Natsumi Tsukamoto, Yuji Kondo, Ehsan Uddin, Wakako Furukawa, Shiori Go, Hiromu Arakawa, Hiroyuki Kaji, Hideyuki Takeuchi, Tetsuya Okajima.

  In the endoplasmic reticulum (ER), O-glycosylation by O-fucose, O-glucose, and O-N-acetylglucosamine (O-GlcNAc) occur in the epidermal growth factor-like (EGF) domains of secreted or transmembrane glycoproteins. Previous studies on Notch receptors have revealed the pivotal role of these O-glycans in the cell surface expression of Notch and secretion of truncated Notch fragments. Although it has been demonstrated that O-fucose, O-glucose and O-GlcNAc stabilize individual EGF domains, their role in the secretory pathway following the completion of the folding process remains unexplored. In this study, we used delta-like homolog 1 (DLK1) containing six consecutive EGF domains as a model glycoprotein to investigate the role of EGF-domain-specific O-glycans in the secretory pathway. Semi-quantitative site-specific glycoproteomics of recombinantly expressed DLK1 revealed multiple O-fucose and O-glucose modifications, along with an unusual EGF-domain-specific O-linked N-acetylglucosamine transferase (EOGT)-dependent O-hexose modification. Consistent with the results of the secretion assay, inactivation of the glycosyltransferases modifying O-fucose and O-glucose, but not the newly identified O-hexose, perturbed the transport of DLK1 from the ER during retention, as assessed using the selective hooks (RUSH) system. Importantly, the absence of O-fucose did not affect O-glucose modification within the same EGF domain, and vice versa. Since protein O-fucosyltransferase 1 and protein O-glucosyltransferase 1 activities depend on the folded state of the EGF domains, O-glycans affected DLK1 transport independently of the folding process required for O-glycosylation in the ER. These findings highlight the distinct roles of O-glycans in facilitating the transport of DLK1 from the ER to the cell surface.

Keywords:  DLK1; EGF; O‐glycan; glycoproteomics; transport

DOI:  https://doi.org/10.1111/febs.70168
Res Sq. 2025 Jul 02. pii: rs.3.rs-6866610. [Epub ahead of print]

Benchmarking the Builders: A Comparative Analysis of PRosettaC and AlphaFold3 for Predicting PROTAC Ternary Complexes.

Joseph M Schulz, Sarah I Schürer, Robert C Reynolds, Stephan C Schürer.

Targeted protein degradation via PROTACs offers a promising therapeutic strategy, yet accurate modeling of ternary complexes remains a critical challenge in degrader design. In this study, we systematically benchmark two leading structure prediction tools, AlphaFold3 and PRosettaC, against a curated dataset of 36 crystallographically resolved ternary complexes. Using DockQ as a quantitative interface scoring metric, we assess the structural fidelity of predicted complexes under both scaffold-inclusive and stripped configurations. Our results demonstrate that AlphaFold3's performance is often inflated by accessory proteins such as Elongin B/C or DDB1, which contribute to overall interface area but not degrader-specific binding. PRosettaC, on the other hand, leverages chemically defined anchor points to yield more geometrically accurate models in select systems, though it frequently fails when linker sampling is insufficient or misaligned. To overcome the limitations of static benchmarking, we introduce a dynamic evaluation strategy using molecular dynamics simulations of the crystal structures. This frame-resolved analysis reveals that several PRosettaC models, while poorly aligned to the static crystal conformation, transiently achieve high DockQ alignment with specific frames along the MD trajectory. These findings underscore the importance of incorporating protein flexibility into benchmarking workflows and suggest that transient conformational compatibility may be overlooked in conventional evaluations. By combining constraint-based modeling with dynamic frame matching, this study provides a more nuanced framework for assessing ternary complex predictions and informs the selection of in silico tools for rational PROTAC development.

DOI: https://doi.org/10.21203/rs.3.rs-6866610/v1
Cancer Lett. 2025 Jul 03. pii: S0304-3835(25)00456-2. [Epub ahead of print] 217888

Endoplasmic Reticulum Stress-Driven Nucleotide Catabolism Fuels Prostate Cancer.

Ke Deng, Nora Pällmann, Marte Livgård, Wanja Kildal, Manohar Pradhan, Ladan Fazli, Paul S Rennie, Yang Jin, Fahri Saatcioglu, Omer Kuzu.

Endoplasmic reticulum (ER) stress is a critical regulator of cancer cell metabolism and survival. In this study, we elucidate the coordinated roles of two key ER stress mediators, Activating Transcription Factor 4 (ATF4) and X-box Binding Protein 1 spliced (XBP1s), in regulating purine homeostasis in prostate cancer (PCa) cells. We demonstrate that ATF4 directly upregulates Molybdenum Cofactor Sulfurase (MOCOS), a key enzyme in purine catabolism, while XBP1s induces the expression of xanthine dehydrogenase (XDH), the principal MOCOS target in this pathway. Knockdown of MOCOS significantly impairs PCa cell proliferation as well as prostatosphere and colony formation in vitro and inhibits tumor growth in preclinical mouse models of PCa. Mechanistically, MOCOS suppression leads to purine accumulation, disrupts pyrimidine synthesis, and causes nucleotide imbalance, resulting in replication fork stalling. This imbalance is also accompanied by a compromised glutathione-mediated antioxidant response, rendering the cells more susceptible to DNA damage. Importantly, targeting XDH, either genetically or biochemically, also significantly hinders PCa cell growth. Collectively, our data highlight the pivotal role of ER stress-mediated purine homeostasis in sustaining PCa cell growth.

DOI: https://doi.org/10.1016/j.canlet.2025.217888
PLoS Biol. 2025 Jul;23(7): e3003249

Nondisruptive inducible labeling of ER-membrane contact sites using the Lamin B receptor.

Laura Downie, Nuria Ferrandiz, Elizabeth Courthold, Megan Jones, Stephen J Royle.

Membrane contact sites (MCSs) are areas of close proximity between organelles that allow the exchange of material, among other roles. The endoplasmic reticulum (ER) has MCSs with a variety of organelles in the cell. MCSs are dynamic, responding to changes in cell state, and are, therefore, best visualized through inducible labeling methods. However, existing methods typically distort ER-MCSs, by expanding contacts or creating artificial ones. Here, we describe a new method for inducible labeling of ER-MCSs using the Lamin B receptor (LBR) and a generic anchor protein on the partner organelle. Termed LaBeRling, this versatile, one-to-many approach allows labeling of different types of ER-MCSs (mitochondria, plasma membrane, lysosomes, early endosomes, lipid droplets, and Golgi), on-demand, in interphase or mitotic human cells. LaBeRling is nondisruptive and does not change ER-MCSs in terms of the contact number, extent or distance measured; as determined by light microscopy or a deep-learning volume electron microscopy approach. We applied this method to study the changes in ER-MCSs during mitosis and to label novel ER-Golgi contact sites at different mitotic stages in live cells.

DOI: https://doi.org/10.1371/journal.pbio.3003249
J Chem Inf Model. 2025 Jul 07.

TRIM21-Driven Degradation of BRD4: Development of Heterobifunctional Degraders and Investigation of Recruitment and Selectivity Mechanisms.

Liyun Zhang, Qingyang Wang, Xiaomei Li, Xuemin Cheng, Longying Cai, Anping Guo, Lanjun Zhang, Hong Hu, Mei Wu, Xianyang Li, Huilin Li, Xiaoyun Meng, Yaping Qiu, Guiping Tu, Lifang Zhang, Xiaofei Dong, Yu Liang, Yushuang Wang, Jin Li, Dengfeng Dou, Xuemei Pu.

TRIM21 is a highly efficient and versatile E3 ubiquitin ligase that plays a crucial role in targeted protein degradation through its specific binding capabilities. To investigate its role in chemically mediated degradation, we utilized two high-affinity binders identified from a DNA-encoded compound library (DEL) to design and synthesize several TrimTACs (TRIM21-based degraders) targeting BRD4. Degradation assays revealed that TrimTACs incorporating distinct TRIM21-binding moieties produced markedly different levels of BRD4 degradation. Further, to elucidate mechanisms underlying ternary complex formation and selective degradation, we employed precise ternary complex modeling combined with Gaussian accelerated molecular dynamics (GaMD) simulations. These computational analyses demonstrated that the diversity of TRIM21-binding moieties significantly affects the stability and conformation of the complex. Structurally stable complexes efficiently induce BRD4 degradation, and the binding orientation of BRD4 directly affects ubiquitin transfer and degradation efficiency. Moreover, key residues critical for complex formation were identified, shedding light on the cooperative interactions driving TRIM21-mediated degradation. Building on these findings, we conducted degradation experiments to validate novel TrimTAC designs derived from our ternary complex-based free energy perturbation (FEP) calculations. The results confirmed the reliability and accuracy of the ternary complex model, providing key insights for the rational design and optimization of TrimTACs, thereby advancing the development of targeted protein degraders.

DOI: https://doi.org/10.1021/acs.jcim.5c00473
J Biol Chem. 2025 Jul 03. pii: S0021-9258(25)02306-3. [Epub ahead of print] 110456

Dynamic regulation of Sec24C by phosphorylation and O-GlcNAcylation during cell cycle progression.

George R Georgiou, Tetsuya Hirata, Erik Soderblom, Rose Homoelle, Jakob Maiwald, Michael Boyce.

  During mitosis, eukaryotic cells cease anterograde trafficking from the endoplasmic reticulum (ER) towards the Golgi. This cessation corresponds with the dispersal of the COPII transport protein, Sec24C, from juxtanuclear ER exit sites (ERES) into a diffusely cytosolic pool. Redistribution of Sec24 paralogs and other core COPII proteins may underlie the mitotic pause in secretion and may be required for the equal inheritance of endomembrane organelles and machinery by both daughter cells. Therefore, it is important to understand the mechanisms governing the mitotic relocalization of COPII components. Here, we explore the role of post-translational modifications (PTMs) of the model COPII protein Sec24C in this phenotypic switch during mitosis. In interphase, Sec24C is modified by O-linked β-N-acetylglucosamine (O-GlcNAc), and we show that this glycan is rapidly removed upon mitotic entry, influencing the timing of Sec24C dispersal. Additionally, we identify novel, cell cycle phase-enriched phosphorylation events on Sec24C, including phosphosites that regulate the stability and localization of the protein, providing the first systematic characterization of dynamic PTMs on any Sec24 protein. Together, our data support the hypothesis that phosphorylation and glycosylation of Sec24C act in concert to induce rapid dispersal upon mitotic entry and may promote equal partitioning of the endomembrane system to daughter cells after division.

Keywords:  COPII; O-GlcNAcylation; cell cycle; intracellular trafficking; mitosis; phosphorylation

DOI:  https://doi.org/10.1016/j.jbc.2025.110456
Sci Adv. 2025 Jul 11. 11(28): eadt8800

UFMylation maintains tumor suppressor pVHL stability by activating the deubiquitinase BAP1.

Xiao Yang, Yalei Wen, Shengying Qin, Yang Zhou, Caishi Zhang, Lei Huang, Mei Li, Xiuqing Ma, Rui Wan, Jiaqi Chen, Rong-Rong He, Hao Gao, Colin R Goding, Oscar Junhong Luo, Xiangchun Shen, Rutao Cui, Tongzheng Liu.

BRCA1-associated protein 1 (BAP1) can function as a tumor suppressor or oncogene depending on context, but its role in colorectal cancer (CRC) is not well understood. Here, we demonstrate that BAP1 suppresses CRC progression primarily by deubiquitinating and stabilizing von Hippel-Lindau tumor suppressor protein (pVHL). BAP1 undergoes covalent modification by ubiquitin-fold modifier 1 (UFM1) at Lys51, Lys61, Lys187, and Lys205, enhancing its interaction with pVHL and promoting pVHL stabilization. Loss of this modification through UFL1 depletion or reconstitution with a UFMylation-defective BAP1 mutant (4KR) impairs pVHL stabilization and promotes tumor progression in CRC cell line-based and patient-derived xenograft models. Clinically, down-regulation of UFL1 and BAP1 correlates with reduced pVHL level and poor prognosis in patients with CRC. These findings identify a previously unrecognized posttranslational mechanism regulating BAP1 activity and highlight UFMylation as essential for maintaining pVHL tumor-suppressive function. Targeting BAP1 UFMylation may represent a potential therapeutic strategy in CRC and other cancers with wild-type BAP1 and VHL.

DOI: https://doi.org/10.1126/sciadv.adt8800
bioRxiv. 2025 Jul 04. pii: 2025.06.30.662315. [Epub ahead of print]

Progressive Supranuclear Palsy PERK haplotype B selectively translates DLX1 promoting tau toxicity.

Christian B Lessard, Diego Rubio-Rubio, Samantha Tolton, Marangelie Criado-Marrero, Sakthivel Ravi, Tristyn N Garza, John Koren, Jennifer Phillips, Pritha Bagchi, Karen N McFarland, Deepak Chhangani, Todd Golde, Benoit I Giasson, Jada Lewis, Paramita Chakrabarty, Matthew J LaVoie, David R Borchelt, Nicholas T Seyfried, Stefan Prokop, Diego E Rincon-Limas, Jose Abisambra.

The unfolded protein response sensor PERK exists in haplotypes A and B. PERK-B confers increased risk for tauopathies like progressive supranuclear palsy (PSP), but the mechanisms distinguishing its function from PERK-A and contributing to its association with tauopathy remain unknown. Here, we developed a controlled cellular model for a pair-wise comparison of the two PERK haplotypes, finding their UPR functions nearly indistinguishable. However, a careful examination employing puromycin-based proteomics revealed that a subset of mRNA translation events were permissible under PERK-B, but not PERK-A, dependent UPR. One of the targets that escaped PERK-B suppression was the transcription factor DLX1, which is genetically linked to PSP risk. We found that DLX1 solubility shifted in human PSP brain tissue. Furthermore, silencing the fly homolog of DLX1 was sufficient to decrease tau-induced toxicity, in vivo. Our results detail the haplotype-specific PERK-B/DLX-1 pathway as a novel driver of tau pathology in cells, flies, and likely human brain, revealing new insights into PSP pathogenesis and potential therapeutic targets.

DOI: https://doi.org/10.1101/2025.06.30.662315
Cell. 2025 Jul 03. pii: S0092-8674(25)00680-4. [Epub ahead of print]

Advancing protein evolution with inverse folding models integrating structural and evolutionary constraints.

Hongyuan Fei, Yunjia Li, Yijing Liu, Jingjing Wei, Aojie Chen, Caixia Gao.

  Protein engineering enables artificial protein evolution through iterative sequence changes, but current methods often suffer from low success rates and limited cost effectiveness. Here, we present AI-informed constraints for protein engineering (AiCE), an approach that facilitates efficient protein evolution using generic protein inverse folding models, reducing dependence on human heuristics and task-specific models. By sampling sequences from inverse folding models and integrating structural and evolutionary constraints, AiCE identifies high-fitness single and multi-mutations. We applied AiCE to eight protein engineering tasks, including deaminases, a nuclear localization sequence, nucleases, and a reverse transcriptase, spanning proteins from tens to thousands of residues, with success rates of 11%-88%. We also developed base editors for precision medicine and agriculture, including enABE8e (5-bp window), enSdd6-CBE (1.3-fold improved fidelity), and enDdd1-DdCBE (up to 14.3-fold enhanced mitochondrial activity). These results demonstrate that AiCE is a versatile, user-friendly mutation-design method that outperforms conventional approaches in efficiency, scalability, and generalizability.

Keywords:  AiCE; base editor optimization; evolutionary coupling; genome editing; high-fitness mutations; inverse folding; protein evolution; structure-informed constraints

DOI:  https://doi.org/10.1016/j.cell.2025.06.014
Nature. 2025 Jul 09.

The role of metabolism in shaping enzyme structures over 400 million years.

Oliver Lemke, Benjamin Murray Heineike, Sandra Viknander, Nir Cohen, Feiran Li, Jacob Lucas Steenwyk, Leonard Spranger, Federica Agostini, Cory Thomas Lee, Simran Kaur Aulakh, Judith Berman, Antonis Rokas, Jens Nielsen, Toni Ingolf Gossmann, Aleksej Zelezniak, Markus Ralser.

Advances in deep learning and AlphaFold2 have enabled the large-scale prediction of protein structures across species, opening avenues for studying protein function and evolution1. Here we analyse 11,269 predicted and experimentally determined enzyme structures that catalyse 361 metabolic reactions across 225 pathways to investigate metabolic evolution over 400 million years in the Saccharomycotina subphylum2. By linking sequence divergence in structurally conserved regions to a variety of metabolic properties of the enzymes, we reveal that metabolism shapes structural evolution across multiple scales, from species-wide metabolic specialization to network organization and the molecular properties of the enzymes. Although positively selected residues are distributed across various structural elements, enzyme evolution is constrained by reaction mechanisms, interactions with metal ions and inhibitors, metabolic flux variability and biosynthetic cost. Our findings uncover hierarchical patterns of structural evolution, in which structural context dictates amino acid substitution rates, with surface residues evolving most rapidly and small-molecule-binding sites evolving under selective constraints without cost optimization. By integrating structural biology with evolutionary genomics, we establish a model in which enzyme evolution is intrinsically governed by catalytic function and shaped by metabolic niche, network architecture, cost and molecular interactions.

DOI: https://doi.org/10.1038/s41586-025-09205-6
PLoS Genet. 2025 Jul 10. 21(7): e1011794

Nuclear p62/SQSTM1 facilitates ubiquitin-independent proteasomal degradation of BMAL1.

Chenliang Zhang, Quanyou Wu, Huan Zhang, RuiChen Liu, Liping Li.

Brain and muscle arnt-like protein 1(BMAL1) is a critical regulator of circadian rhythm. Although transcriptional regulation of BMAL1 has been extensively studied, the mechanisms governing the stability of BMAL1 at the protein level remain unclear. p62/SQSTM1 is a crucial factor in proteostasis regulation and is involved in both autophagy and the ubiquitin-proteasome system. We demonstrated that p62 promotes proteasomal degradation of BMAL1 within the nucleus, independent of ubiquitination. Additional molecular analyses indicated that p62 functions as a receptor for the 20S proteasome, facilitating the recruitment of BMAL1 to the 20S proteasome for degradation. This mechanism is independent of recently identified p62-driven nuclear biomolecular condensates. We also revealed that remodeling the nuclear accumulation of p62 may represent a potential strategy for targeting BMAL1 to suppress tumor cell growth. In conclusion, our findings revealed a novel mechanism by which nuclear p62 regulates BMAL1 proteostasis.

DOI: https://doi.org/10.1371/journal.pgen.1011794
Autophagy. 2025 Jul 07.

Activation of endogenous PRKN by structural derepression is linked to increased turnover of the E3 ubiquitin ligase.

Fabienne Fiesel, Bernardo Bustillos, Jens Watzlawik, Carol Chen, Martin Berryer, Jiazhen Zhang, Paige Boneski, Caleb Hayes, Jenny Bredenberg, Eric Deneault, Zhipeng You, Narges Abdien, Nathalia Aprahamian, Taylor Goldsmith, Zahra Baninameh, Liam Cocker, Haonan Zhang, Matthew Goldberg, Edward Fon, Jean-François Trempe, Satpal Virdee, Thomas Durcan, Wolfdieter Springer.

  Loss-of-function mutations in the PINK1 and PRKN genes are the most common cause of early-onset Parkinson disease (PD). The encoded enzymatic pair selectively identifies, labels, and targets damaged mitochondria for degradation via the macroautophagy/autophagy-lysosome system (mitophagy). This pathway is cytoprotective and efforts to activate mitophagy are pursued as therapeutic avenues to combat PD and other neurodegenerative disorders. When mitochondria are damaged, the ubiquitin kinase PINK1 accumulates and recruits PRKN from the cytosol to activate the E3 ubiquitin ligase from its auto-inhibited conformation. We have previously designed several mutations that effectively derepress the structure of PRKN and activate its enzymatic functions in vitro. However, it remained unclear how these PRKN-activating mutations would perform endogenously in cultured neurons or in vivo in the brain. Here, we gene-edited neural progenitor cells and induced pluripotent stem cells to express PRKN-activating mutations in dopaminergic cultures. All tested PRKN-activating mutations indeed enhanced the enzymatic activity of PRKN in the absence of exogenous stress, but their hyperactivity was linked to their own PINK1-dependent degradation. Strikingly, in vivo in a mouse model expressing an equivalent activating mutation, we find the same relationship between PRKN enzymatic activity and protein stability. We conclude that PRKN degradation is the consequence of its structural derepression and enzymatic activation, thus resulting only in a temporary gain of activity. Our findings imply that pharmacological activation of endogenous PRKN will lead to increased turnover and suggest that additional considerations might be necessary to achieve sustained E3 ubiquitin ligase activity for disease treatment.

Keywords:  Autophagy; PINK1; Parkin; mitophagy; parkinson’s disease

DOI:  https://doi.org/10.1080/15548627.2025.2531025
Am J Physiol Cell Physiol. 2025 Jul 10.

Cdk5rap3-mediated regulation of lysosomal and ER membrane proteins is pivotal for the survival and function of pancreatic acinar cells.

Yonghong Huang, Feng Zhou, Xin Xu, Yaqun Wang, Michaela Quintero, Siyang Liu, Cong Liu, Guangxun Zhu, Yafei Cai, Zheng Dong, Roni Bollag, Guangyu Wu, Maria Sabbatini, Honglin Li.

  The acinus is the functional unit of the exocrine pancreas that produces and secretes a large quantity of digestive enzymes. Damage and dysfunction of pancreatic acinar cells (PACs) may lead to malnutrition, pancreatitis and other pathological conditions. Cdk5rap3, a multi-functional protein, is essential for animal development and normal physiology of multiple organs and tissues. Interestingly, the recent studies suggest its involvement in ER-phagy, a lysosomal degradation of the subdomains of the Endoplasmic Reticulum (ER). Herein, we attempted to investigate its physiological function in pancreatic acinar cells. We found that Cdk5rap3 deficient PACs contained fewer zymogen granules and underwent acinar to ductal metaplasia (ADM) and apoptosis, thereby resulting in significant loss of acinar compartment. Interestingly, Cdk5rap3 ablation led to the increase of lysosomal hydrolase cathepsin B and lysosomal protein LAMP1, indicating its novel function in regulation of lysosomal homeostasis and activity. Elevated cathepsin B activity may lead to aberrant activation of trypsinogen and apoptosis of Cdk5rap3 deficient acinar cells, while the increase of lysosomal proteins may enhance lysosomal activity that in turn promotes ADM. Furthermore, Cdk5rap3 knockout led to substantial changes of rough ER structure and significant increase of selective ER membrane proteins including CLIMP63. Our results from both mouse tissues and tissue culture cells strongly suggest that Cdk5rap3 plays a pivotal role in regulating homeostasis of the lysosome and the ER that is essential for the survival and physiological function of pancreatic acinar cells.

Keywords:  Cdk5rap3; ER-phagy; endoplasmic reticulum; lysosome; pancreatic acinar cell

DOI:  https://doi.org/10.1152/ajpcell.00284.2025
Cell Rep. 2025 Jul 08. pii: S2211-1247(25)00743-0. [Epub ahead of print]44(7): 115972

TBK1 phagosomal recruitment enhances antifungal immunity via positive feedback regulation with SRC.

Yiting Feng, Xiaochen Cheng, Yuanzhe Yang, Chuanjin Liu, Yu Shi, Xiaopeng Qi, Wei Zhao, Bingyu Liu, Tian Chen, Chengjiang Gao.

  TANK-binding kinase 1 (TBK1) is a versatile serine/threonine protein kinase that is mainly recognized for its canonical role in antiviral immunity through interferon induction. Here, we report a previously uncharacterized function for TBK1 in antifungal defense. Using proteomic analysis, we identified TBK1 as a phagosome-directed protein after fungal infection, and biochemical analysis revealed that TBK1 is directly recruited to the phagosome by SHP2 in a process driven by SRC-mediated phosphorylation. This recruitment facilitates TBK1 aggregation and trans-autophosphorylation at the phagosome. Activated TBK1 then phosphorylates SRC at serine 17, a prerequisite for the full activation of SRC, thereby establishing a robust positive feedback loop among SRC, SHP2, and TBK1. Consistently, SRC-mediated antifungal signaling and production of proinflammatory cytokines and chemokines were significantly impaired in mouse bone marrow-derived macrophages (BMDMs) lacking TBK1. Myeloid Tbk1-deficient mice exhibited greater susceptibility to systemic Candida albicans infection. Overall, our findings reveal a critical role for TBK1 in antifungal immunity and highlight its potential as a therapeutic target for combating fungal pathogens.

Keywords:  Antifungal immunity; CP: Cell biology; CP: Immunology; SHP2; SRC; TBK1; phagosome

DOI:  https://doi.org/10.1016/j.celrep.2025.115972
bioRxiv. 2025 Jul 03. pii: 2025.07.03.663068. [Epub ahead of print]

Human protein interactome structure prediction at scale with Boltz-2.

Alexander M Ille, Christopher Markosian, Stephen K Burley, Renata Pasqualini, Wadih Arap.

In humans, protein-protein interactions mediate numerous biological processes and are central to both normal physiology and disease. Extensive research efforts have aimed to elucidate the human protein interactome, and comprehensive databases now catalog these interactions at scale. However, structural coverage of the human protein interactome is limited and remains challenging to resolve through experimental methodology alone. Recent advances in artificial intelligence/machine learning (AI/ML)-based approaches for protein interaction structure prediction present opportunities for large-scale structural characterization of the human interactome. One such model, Boltz-2, which is capable of predicting the structures of protein complexes, may serve this objective. Here, we present de novo computed models of 1,394 binary human protein interaction structures predicted using Boltz-2. These structural predictions were based on biochemically determined interaction data sourced from the IntAct database, together with corresponding protein sequences obtained from UniProt. We then assessed the predicted interaction structures through various confidence metrics, which consider both overall structure and the interaction interface. These analyses indicated that prediction confidence tended to be greater for smaller complexes, while increased multiple sequence alignment (MSA) depth tended to improve prediction confidence. Additionally, we performed a limited evaluation against experimentally determined human protein complex structures not included in the Boltz-2 training regimen, which indicated prediction accuracy consistent with AlphaFold3. This work demonstrates the utility of Boltz-2 for in silico structural modeling of the human protein interactome, while highlighting both strengths and limitations. Ultimately, such modeling is expected to yield broad structural insights with relevance across multiple domains of biomedical research.

DOI: https://doi.org/10.1101/2025.07.03.663068
bioRxiv. 2025 Jul 04. pii: 2025.07.01.662506. [Epub ahead of print]

mRNA interactions promote cotranslational association of heteromeric membrane proteins.

Lisandra Flores-Aldama, Annabelle S Hoth, Lucas de Carvalho, Ian Seim, Fang Liu, Amy S Gladfelter, Gail A Robertson.

Heteromeric membrane proteins play crucial physiological roles, yet how they are formed remains poorly understood. Heteromeric hERG1a/1b ion channels, essential for maintaining normal cardiac rhythm, assemble via cotranslational association of their encoding mRNAs. We hypothesized that direct hERG1a and 1b mRNA interactions facilitate this process. Using fluorescence colocalization and free energy of binding predictions, we found that hERG1a and 1b mRNAs form specific heterotypic condensates in vitro , suggesting direct interactions. When hERG1a mRNA was altered by synonymous mutations predicted to reduce its structural diversity and ability to interact with other mRNAs, overlap with hERG1b was dramatically diminished both in vitro and in cells, indicating weakened interactions. Reducing hERG1a structural diversity also influenced its translational complexes, defined by overlapping between fluorescently labeled mRNA and encoded protein (centroids within 400 nm). Whereas most of the wild-type hERG1a mRNA translates within heterotypic complexes, likely reflecting the biogenesis of hERG1a/1b heteromeric assemblies, reducing hERG1a structural diversity yielded more homotypic translational condensates and fewer hERG1a/1b heterotypic ones. This result suggests that the strength of mRNA interactions impact ion channel biogenesis. Further analysis of the heterotypic translational complexes revealed two distinct classes: a) simultaneous translation of both subunits and b) sequential association of fully translated hERG1b with translating hERG1a. Notably, reducing hERG1a structural diversity and interactions with 1b shifted translation toward the sequential mode. These findings identify a new role of mRNA sequence, structure, and interactions in orchestrating the cotranslational association of important heteromeric membrane proteins.
SIGNIFICANCE: The physiological diversity of membrane protein complexes arises in part from the mixing of related yet functionally distinct subunits such as those forming "heteromeric" ion channels. How this mixing is achieved is not well understood. Here, we show that subunits composing heteromeric hERG1a/1b ion channels, crucial for normal cardiac function, predominantly associate in complexes where both subunits are simultaneously translated. This cotranslation is promoted by the association of the encoding mRNAs. Silent mutations disrupting hERG1a and 1b mRNA association, without altering the encoded protein, diminish simultaneous translation in favor of hERG1a translational complexes alone or in association with fully synthesized hERG1b. This study highlights the significant impact of RNA sequence, structure, and association on the formation of heteromeric membrane proteins.

DOI: https://doi.org/10.1101/2025.07.01.662506
FASEB Bioadv. 2025 Jul;7(7): e70031

Endoplasmic Reticulum Stress and Unfolded Protein Response Sensor ERN1 Regulates Organic Dust Induction of Lung Inflammation.

Shilpa Kusampudi, Velmurugan Meganathan, Vijay Boggaram.

  Inhalation of organic dust increases the risk for respiratory symptoms and respiratory diseases, with chronic inflammation playing a major role in their development. Previously, we reported that organic dust induction of inflammatory mediators in bronchial epithelial cells is mediated through increase of intracellular reactive oxygen species (ROS) and activation of NFκB and Stat3. Oxidative stress caused by increased ROS has been linked to the activation of endoplasmic reticulum (ER) stress and unfolded protein response (UPR). UPR modulates immune responses and plays key roles in the development of acute and chronic diseases. Herein, we hypothesized that organic dust-induced ER stress-UPR regulates airway epithelial cell inflammatory responses. We found that poultry organic dust extract (referred to as dust extract) increased the expression of ER stress/UPR sensor ERN1 in Beas2B bronchial epithelial cells. Dust extract was also found to increase ERN1 protein levels in mouse lungs with ERN1 immunostaining detected predominantly in the bronchial epithelium. Additionally, dust extract increased Ser724 ERN1 phosphorylation in the mouse bronchial epithelium indicating activation. Chemical inhibition and mRNA knockdown studies revealed that TLR2/TLR4-Myd88-ROS-NFκB/Stat3 pathway mediates ERN1 induction. ERN1 chemical inhibitors, KIRA6 and APY29, and ERN1 mRNA knockdown reduced the induction of IL6, CXCL8, and pro IL1β. KIRA6 inhibited dust extract stimulation of NFκB-p65, Stat3, Jun and MAPK 8/9 phosphorylation. Our studies have shown that ER stress and ERN1 are new players in the control of organic dust induced lung inflammation. Cross-regulation between members of cell signaling cascade, TLR2-TLR4/MyD88/ROS/ERN1/NFκB/Stat3 may fine tune immune and inflammatory responses elicited by organic dust.

Keywords:  endoplasmic reticulum stress; inflammation; lung; oxidative stress; unfolded protein response

DOI:  https://doi.org/10.1096/fba.2025-00069