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Influenza A virus (IAV) Matrix 2 protein (M2) is an ion channel, required for efficient viral entry and egress. M2 interacts with the small ubiquitin-like LC3 protein through a cytoplasmic C-terminal LC3-interacting region (LIR). Here, we report that M2 is cleaved by caspases, abolishing the M2-LC3 interaction. A crystal structure of the M2 LIR in complex with LC3 indicates the caspase cleavage tetrapeptide motif ( 82 SAVD 85 ) is an unstructured linear motif that does not overlap with the LIR. IAV mutant expressing a permanently truncated M2, mimicking caspase cleavage, exhibit defects in M2 plasma membrane transport, viral filament formation, and virion production. Our results reveal a dynamic regulation of the M2-LC3 interaction by caspases. This highlights the role of host proteases in regulating IAV exit, relating virion production with host cell state., Competing Interests: Disclosure and competing interests statement. The authors declare no competing interests., (© 2025. The Author(s).)

Intrinsically disordered regions (IDRs) are crucial to homeostatic and organellar remodeling pathways. In reticulophagy/ER-phagy, long cytosolic IDR-containing receptors (e.g. RETREG1/FAM134B) house the LC3-interacting region (LIR) motif to recruit the phagophore. The precise functions of the IDR beyond engaging the autophagic machinery are unclear. Here, we comment on the role of the RETREG1-IDR based on our recent computer modeling and molecular dynamics (MD) simulations. Extensive analysis of the RETREG1-IDR indicates a continuum of conformations between expanded and compact structures, displaying a Janus-like feature. Using an adapted MARTINI model, we find that the IDR ensemble properties vary widely depending on the membrane anchor. IDRs alone are sufficient to promote and sense membrane curvature and can act as entropic tethers. When anchored to the Reticulon homology domain (RHD), they adopt compact collapsed conformations, acting as effector scaffolds that amplify RHD membrane remodeling properties, enhancing receptor-clustering and accelerating spontaneous budding. These findings expand the operational scope of IDRs within reticulophagy, offering fresh insights into a mechanistic understanding of membrane remodeling.

Chemical probes are powerful small-molecule tools in fundamental and translational cancer research. They are highly versatile, complementing genetic technologies in the annotation of protein function, and invaluable in target validation and drug discovery. However, continued improvements are needed to enhance best practices in selection and use of chemical probes. We discuss progress over the last decade, highlight key issues, and indicate a path to generate a high-quality chemical probe for every human protein., Competing Interests: Declaration of interests S.M. and P.W. are current directors of the non-profit Chemical Probes Portal, were instrumental in the creation of the Portal, and are affiliated with the activities of Target 2035. D.S. and P.W. are employees of The Institute of Cancer Research (ICR), which has a commercial interest in a range of drug targets and therapeutics. The ICR operates a Rewards to Discoverers scheme whereby employees of the ICR may receive financial benefits following the commercial licensing of a project. P.W. has been instrumental in the creation/development of canSAR and Probe Miner. P.W. is a consultant/scientific advisory board member for Alterome Therapeutics, Astex Pharmaceuticals, CHARM Therapeutics, CV6 Therapeutics, Epicombi Therapeutics, Merck KGaA, Nextech Invest, Nuvectis Pharma, Storm Therapeutics, and Vividion Therapeutics; holds stock/options in Alterome, CHARM Therapeutics, Chroma Therapeutics (founder), Epicombi Therapeutics, Next Invest, Nuvectis, and Storm; is a non-executive director of Storm and director of Derwentwater Associates; and is a former employee of AstraZeneca. P.W. has received research funding from Astex, BACIT, Cyclacel Pharmaceuticals, Merck KGaA, Nuvectis, Piramed Pharmaceuticals (founder), Sixth Element Capital/CRT Pioneer Fund, Vernalis, and Vivan Therapeutics. P.W. is a member of the advisory board of Cancer Cell., (Copyright © 2025 Elsevier Inc. All rights reserved.)

The majority of clinical degraders utilize an immunomodulatory imide drug (IMiD)-based derivative that directs their target to the E3 ligase receptor cereblon (CRBN); however, identification of IMiD molecular glue substrates has remained underexplored. To tackle this, we design human CRBN constructs, which retain all features for ternary complex formation, while allowing generation of homogenous and cost-efficient expression in E. coli. Extensive profiling of the construct shows it to be the "best of both worlds" in terms of binding activity and ease of production. We next designed the "Enamine focused IMiD library" and demonstrated applicability of the construct to high-throughput screening, identifying binders with high potency, ligand efficiency, and specificity. Finally, we adapt our construct for proof of principle glue screening approaches enabling IMiD cellular interactome determination. Coupled with our IMiD binding landscape the methods described here should serve as valuable tools to assist discovery of next generation CRBN glues., Competing Interests: Declaration of interests Dr Kondratov, Dr Matviyuk, and Dr Tolmachova are current or prior employees of Enamine Ltd., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Functional revascularization is key to stroke recovery and requires remodeling and regeneration of blood vessels around which is located the brain's only stromal compartment. Stromal progenitor cells (SPCs) are critical for tissue regeneration following injury in many organs, yet their identity in the brain remains elusive. Here we show that the perivascular niche of brain SPCs includes pericytes, venular smooth muscle cells and perivascular fibroblasts that together help cerebral microvasculature regenerate following experimental stroke. Ischemic injury triggers amplification of pericytes and perivascular fibroblasts in the infarct region where they associate with endothelial cells inside a reactive astrocyte border. Fate-tracking of Hic1 + SPCs uncovered a transient functional and transcriptional phenotype of stroke-activated pericytes and perivascular fibroblasts. Both populations of these cells remained segregated, displaying distinct angiogenic and fibrogenic profiles. Therefore, pericytes and perivascular fibroblasts are distinct subpopulations of SPCs in the adult brain that coordinate revascularization and scar formation after injury., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s), under exclusive licence to Springer Nature America, Inc.)

In order to effectively replicate within a host cell, the Legionella pneumophila bacterium secretes effector enzymes into the cytoplasm in order to manipulate cellular host pathways including host ubiquitination. Some of these effectors, the so-called SidE-family, mediate noncanonical phosphoribosyl serine ubiquitination (PR-ubiquitination) of host substrate proteins, contributing to the recruitment of ER-remodeling proteins and the formation of a Legionella -containing vacuole, which is crucial in the early stages of bacterial infection. PR-ubiquitination is a dynamic process that is reversed by other Legionella effectors called deubiquitinases for PR-ubiquitination (Dups). We recently discovered a reactive allosteric cysteine in close proximity to the catalytic triad of DupA, which can be exploited as a target for covalent probe development. We here report on the synthesis of vinyl-sulfonate and fluoro-sulfonate warhead-containing phosphoribosyl ubiquitin probes, where the Arg42 position of ubiquitin is linked to the C1 of ribose via a native guanidinium group, and compare them to triazole-linked probes. In vitro tests on recombinant DupA and SdeA PDE revealed that these probes are able to capture the enzymes covalently. In a pull-down proteomics experiment, DupA and DupB enzymes are enriched from Legionella -infected cell lysates, highlighting the potential of native Arg-riboside linked probes to capture Legionella effector enzymes in a complex proteome.

Aims: The role of circular RNAs (circRNAs) and their regulation in health and disease are poorly understood. Here, we systematically investigated the temporally resolved transcriptomic expression of circRNAs during differentiation of human induced pluripotent stem cells (iPSC) into vascular endothelial cells (EC) and smooth muscle cells (SMC) and explored their potential as biomarkers for human vascular disease., Methods and Results: Using high-throughput RNA sequencing and a de novo circRNA detection pipeline, we quantified the daily levels of 31,369 circRNAs in a two-week differentiation trajectory from human stem cells to proliferating mesoderm progenitors to quiescent, differentiated EC and SMC. We detected a significant global increase in RNA circularization, with 397 and 214 circRNAs upregulated > 2 fold (adjusted P < 0.05) in mature EC and SMC, compared with undifferentiated progenitor cells. This global increase in circRNAs was associated with upregulation of host genes and their promoters and a parallel downregulation of splicing factors. Underlying this switch, the proliferation-regulating transcription factor MYC decreased as vascular cells matured, and inhibition of MYC led to downregulation of splicing factors such as SRSF1 and SRSF2 and changes in vascular circRNA levels. Examining the identified circRNAs in arterial tissue samples and in peripheral blood mononuclear cells (PBMC) from patients, we found that circRNA levels decreased in atherosclerotic disease, in contrast to their increase during iPSC maturation into EC and SMC. Using machine learning, we determined that a set of circRNAs derived from COL4A1, COL4A2, HSPG2, and YPEL2 discriminated atherosclerotic from healthy tissue with an AUC of 0.79. CircRNAs from HSPG2 and YPEL2 in blood PBMC samples detected atherosclerosis with an AUC of 0.73., Conclusions: Time-resolved transcriptional profiling of linear and circular RNA species revealed that circRNAs provide granular molecular information for disease profiling. The identified circRNAs may serve as blood biomarkers for atherosclerotic vascular disease., (© The Author(s) 2025. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Protein arginine methyltransferase 6 (PRMT6) is a well-characterized epigenetic regulator that methylates histone H3 at arginine 2 (H3R2me2a) in both promoter and enhancer regions, thereby modulating transcriptional initiation. We report here that PRMT6 also regulates gene expression at the post-transcriptional level in the neural pluripotent state and during neuronal differentiation of NT2/D1 cells. PRMT6 knockout causes widespread alternative splicing changes in NT2/D1 cells, most frequently cassette exon alterations. Most of the PRMT6-dependent splicing targets are not transcriptionally affected by the enzyme and regulated in an H3R2me2a-independent manner. However, for a small subset of splicing events, the PRMT6-mediated deposition of H3R2me2a overlaps with the splice site, suggesting a potential dual function in both transcriptional and co-/post-transcriptional regulation. The splicing targets of PRMT6 include ribosomal proteins, splicing factors, and chromatin-modifying enzymes such as PRMT4, DNMT3B, and ASH2L, some of which are associated with differentiation decisions. Taken together, our results in NT2/D1 cells show that PRMT6 exerts predominantly H3R2me2a-independent functions in RNA splicing, which may contribute to pluripotency and neuronal identity., (© 2025 Eudenbach et al.)

Endoplasmic reticulum (ER) plasticity and ER-phagy are intertwined processes essential for maintaining ER dynamics. We investigated the interplay between two isoforms of the ER-phagy receptor FAM134B in regulating ER remodeling in differentiating myoblasts. During myogenesis, the canonical FAM134B1 is degraded, while its isoform FAM134B2 is transcriptionally upregulated. The switch, favoring FAM134B2, is an important regulator of ER morphology during myogenesis. FAM134B2 partial reticulon homology domain, with its rigid conformational characteristics, enables efficient ER reshaping. FAM134B2 action increases in the active phase of differentiation leading to ER restructuring via ER-phagy, which then reverts to physiological levels when myotubes are mature and the ER is reorganized. Knocking out both FAM134B isoforms in myotubes results in an aberrant proteome landscape and the formation of dilated ER structures, both of which are rescued by FAM134B2 re-expression. Our results underscore how the fine-tuning of FAM134B isoforms and ER-phagy orchestrate the ER dynamics during myogenesis providing insights into the molecular mechanisms governing ER homeostasis in muscle cells., Competing Interests: Disclosure and competing interests statement. The authors declare no competing interests., (© 2025. The Author(s).)

Selective macroautophagy/autophagy of the endoplasmic reticulum, known as reticulophagy/ER-phagy, is essential to maintain ER homeostasis. We recently showed that members of the autophagy receptor family RETREG/FAM134 are regulated by phosphorylation-dependent ubiquitination. In an unbiased screen we had identified several kinases downstream of MTOR with profound impact on reticulophagy flux, including ATR and CSNK2/CK2. Inhibition of CSNK2 by SGC-CK2-1 prevented regulatory ubiquitination of RETREG1/FAM134B and RETREG3/FAM134C upon autophagy activation as well as the formation of high-density RETREG1- and RETREG3-clusters. Here we report on additional resource data of global proteomics upon CSNK2 and ATR inhibition, respectively. Our data suggests that the function of CSNK2 is mainly limited to the ER/reticulophagy and Golgi/Golgiphagy, while ATR inhibition by VE-822 affects the vast majority of organelles/selective autophagy pathways. Abbreviation: ATRi: ATR inhibitor VE-822; CSNK2i: CSNK2 inhibitor SGC-CK2-1; ER: endoplasmic reticulum.

The transcription factor p53 plays a key role in the cellular defense against cancer development. It is inactivated in virtually every tumor, and in every second tumor this inactivation is due to a mutation in the TP53 gene. In this perspective, we show that this diverse mutational spectrum is unique among all other cancer-associated proteins and discuss what drives the selection of TP53 mutations in cancer. We highlight that several factors conspire to make the p53 protein particularly vulnerable to inactivation by the mutations that constantly plague our genome. It appears that the TP53 gene has emerged as a victim of its own evolutionary past that shaped its structure and function towards a pluripotent tumor suppressor, but came with an increased structural fragility of its DNA-binding domain. TP53 loss of function - with associated dominant-negative effects - is the main mechanism that will impair TP53 tumor suppressive function, regardless of whether a neomorphic phenotype is associated with some of these variants., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

The leukotriene B4 receptor 2 (BLT2) is a G-protein coupled receptor, which is endogenously activated by 12( S )-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT). BLT2 is gaining attention as a potential therapeutic target involved in various pathologies including diabetic wound healing, ophthalmic diseases, and colitis. However, validation of BLT2 as drug target requires chemical probes and pharmacological tools which will allow for application in vivo. In this work, we present the discovery of a novel chemical probe T-10430 for BLT2 agonism following a scaffold-hopping approach. T-10430 exhibits high potency, good selectivity profile, promising physicochemical and PK properties and can potentially serve as orally applicable pharmacological tool for validation of BLT2 as drug target. Using T-10430 , we demonstrate the beneficial effect of BLT2 activation in mouse model of psoriasis.

The entire RNA life cycle, spanning from transcription to decay, is intricately regulated by RNA-binding proteins (RBPs). To understand their precise functions, it is crucial to identify direct targets, pinpoint their exact binding sites, and unravel the underlying specificity in vivo. Individual-nucleotide resolution UV cross-linking and immunoprecipitation 2 (iCLIP2) is a state-of-the-art technique that enables the identification of RBP-binding sites at single-nucleotide resolution. However, in the field of microbiology, optimized iCLIP protocols compared to mammalian systems are lacking. Here, we present the first microbial iCLIP2 approach using the multi-RRM domain protein Rrm4 from the fungus Ustilago maydis as an example. Key challenges, such as inherently high RNase and protease activity in fungi, were addressed by improving mechanical cell disruption and lysis buffer composition. Our modifications increased the yield of cross-link events and improved the identification of Rrm4-binding sites. Thereby, we were able to pinpoint that Rrm4 binds the stop codons of nuclear-encoded mRNAs of mitochondrial respiratory complexes I, III, and V-revealing an intimate link between endosomal mRNA transport and mitochondrial physiology. Thus, our study using U. maydis as an example might serve as a blueprint for optimizing iCLIP2 procedures in other microorganisms with high RNase/protease conditions., (© 2025 Stoffel et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Glioblastoma (GBM), the most malignant primary brain tumor in adults, has poor prognosis irrespective of therapeutic advances due to its radio-resistance and infiltrative growth into brain tissue. The present study assessed functions and putative druggability of BRCA1-associated ATM activator 1 (BRAT1) as a crucial factor driving key aspects of GBM, including enhanced DNA damage response and tumor migration. By a stable depletion of BRAT1 in GBM and glioma stem-like (GSC) cell lines, we observed a delay in DNA double-strand break repair and increased sensitivity to radiation treatment, corroborated by in vitro and in vivo studies demonstrating impaired tumor growth and invasion. Proteomic and phosphoproteomic analyses further emphasize the role of BRAT1's cell migration and invasion capacity, with a notable proportion of downregulated proteins associated with these processes. In line with the genetic manipulation, we found that treatment with the BRAT1 inhibitor Curcusone D (CurD) significantly reduced GSC migration and invasion in an ex vivo slice culture model, particularly when combined with irradiation, resulting in a synergistic inhibition of tumor growth and infiltration. Our results reveal that BRAT1 contributes to GBM growth and invasion and suggest that therapeutic inhibition of BRAT1 with CurD or similar compounds might constitute a novel approach for anti-GBM directed treatments., Competing Interests: Declarations. Ethical approval: All animal procedures were conducted in accordance with a valid approval (FK-1116) by the Regierungspräsidium Darmstadt. Conflict of interest: The authors declare no conflict of interest., (© 2024. The Author(s).)

The human kinome has tremendous medical potential. In the past decade, mixed-lineage protein kinase 3 (MLK3) has emerged as an interesting and druggable target in oncogenic signaling. The important role of MLK3 has been demonstrated in several types of cancer. In a target hopping example we started with the focal adhesion kinase (FAK) inhibitor PF-431396 ( 10 ), which shows off-target activity toward MLK3. We were able to develop highly active compounds in the single digit nanomolar range for MLK3. Furthermore, we achieved a dramatic shift in selectivity from FAK to MLK3. Here we present a new chemical class of MLK3 inhibitors, including our lead compound 37 with an outstanding IC 50 value of <1 nM in a biochemical MLK3 assay while simultaneously exhibiting kinome-wide selectivity.

To understand the function of cells such as neurons within an organism, it can be instrumental to inhibit cellular function, or to remove the cell (type) from the organism, and thus to observe the consequences on organismic and/or circuit function and animal behavior. A range of approaches and tools were developed and used over the past few decades that act either constitutively or acutely and reversibly, in systemic or local fashion. These approaches make use of either drugs or genetically encoded tools. Also, there are acutely acting inhibitory tools that require an exogenous trigger like light. Here, we give an overview of such methods developed and used in the nematode Caenorhabditis elegans., Competing Interests: Conflicts of interest: The authors declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

The transcription factor p63 is expressed in many different isoforms as a result of differential promoter use and splicing. Some of these isoforms have very specific physiological functions in the development and maintenance of epithelial tissues and surveillance of genetic integrity in oocytes. The ASPP family of proteins is involved in modulating the transcriptional activity of the p53 protein family members, including p63. In particular, iASPP plays an important role in the development and differentiation of epithelial tissues. Here we characterize the interaction of iASPP with p63 and show that it binds to the linker region between the DNA binding domain and the oligomerization domain. We further demonstrate that this binding site is removed in a splice variant of p63 where a stretch of five amino acids is replaced with a single alanine residue. This stretch contains a degenerate class II SH3 domain binding motif that is responsible for interaction with iASPP, as well as two positively charged amino acids. Moreover, the concomitant loss of the charged amino acids in the alternatively spliced version decreases the affinity of p63 to its cognate DNA element two- to threefold. mRNAs encoding full-length p63, as well as its alternatively spliced version, are present in all tissues that we investigated, albeit in differing ratios. We speculate that, through the formation of hetero-complexes of both isoforms, the affinity to DNA, as well as the interaction with iASPP, can be fine-tuned in a tissue-specific manner., Competing Interests: Competing interests: The authors declare no competing interests. Ethical statement: All methods were performed in accordance with the relevant guidelines and regulations. As this is an in vitro study without human patients or animals involved no ethics committee approval was required. The human cDNA from ten different tissues was purchased directly from the company Zyagen without any connection to identifiable human patients., (© 2025. The Author(s).)

The Chemical Probes Portal (www.chemicalprobes.org) is a free, public resource, based on expert-reviews, that supports the assessment, selection and use of small-molecule compounds that qualify as chemical probes. These high-quality reagents are essential for exploring the function of individual proteins in complex biological systems, such as cells and organisms, and for validating proteins as potential therapeutic targets. The use of reliable chemical probes accelerates protein annotation in basic biological studies and informs drug discovery. However, the use of low-quality compounds has historically led to erroneous conclusions in biomedical research, and experience shows that failure to follow best practice continues, an issue which the Portal aims to address. Here, we describe the latest updates to the Chemical Probes Portal in both content and functionality. The number of chemical probes and human protein targets covered has increased significantly, with improvements in the processes for obtaining expert reviews and user engagement. Moreover, new functionalities and enhanced tools have been introduced to better support biological researchers in selecting and using the best chemical probes for their studies., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The coordination of chromatin remodeling is essential for DNA accessibility and gene expression control. The highly conserved and ubiquitously expressed SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complex plays a central role in cell type- and context-dependent gene expression. Despite the absence of a defined DNA recognition motif, SWI/SNF binds lineage specific enhancers genome-wide where it actively maintains open chromatin state. It does so while retaining the ability to respond dynamically to cellular signals. However, the mechanisms that guide SWI/SNF to specific genomic targets have remained elusive. Here we demonstrate that trans-acting long non-coding RNAs (lncRNAs) direct the SWI/SNF complex to cell type-specific enhancers. SWI/SNF preferentially binds lncRNAs and these predominantly bind DNA targets in trans. Together they localize to enhancers, many of which are cell type-specific. Knockdown of SWI/SNF- and enhancer-bound lncRNAs causes the genome-wide redistribution of SWI/SNF away from enhancers and a concomitant differential expression of spatially connected target genes. These lncRNA-SWI/SNF-enhancer networks support an enhancer hub model of SWI/SNF genomic targeting. Our findings reveal that lncRNAs competitively recruit SWI/SNF, providing a specific and dynamic layer of control over chromatin accessibility, and reinforcing their role in mediating enhancer activity and gene expression., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

The mutational landscape of TP53, a tumor suppressor mutated in about half of all cancers, includes over 2,000 known missense mutations. To fully leverage TP53 mutation status for personalized medicine, a thorough understanding of the functional diversity of these mutations is essential. We conducted a deep mutational scan using saturation genome editing with CRISPR-mediated homology-directed repair to engineer 9,225 TP53 variants in cancer cells. This high-resolution approach, covering 94.5% of all cancer-associated TP53 missense mutations, precisely mapped the impact of individual mutations on tumor cell fitness, surpassing previous deep mutational scan studies in distinguishing benign from pathogenic variants. Our results revealed even subtle loss-of-function phenotypes and identified promising mutants for pharmacological reactivation. Moreover, we uncovered the roles of splicing alterations and nonsense-mediated messenger RNA decay in mutation-driven TP53 dysfunction. These findings underscore the power of saturation genome editing in advancing clinical TP53 variant interpretation for genetic counseling and personalized cancer therapy., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Comparative studies across multiple species provide valuable insights into the evolutionary diversification of developmental strategies. While the fruit fly Drosophila melanogaster has long been the primary insect model organism for understanding molecular genetics and embryonic development, the Mediterranean fruit fly Ceratitis capitata, also known as medfly, presents a promising complementary model for studying developmental biology. With its sequenced genome and a diverse array of molecular techniques, the medfly is well-equipped for study. However, an integrative framework for studying its embryogenesis is currently lacking. In this study, we present a two-level staging system for the medfly based on nine datasets recorded using light sheet fluorescence microscopy. The upper level features of six consecutive embryogenetic events, facilitating comparisons between insect orders, while the lower level consists of seventeen stages, adapted from the fruit fly, allowing for comparisons within the Diptera. We provide detailed descriptions of all identifiable characteristics in multiple formats, including a detailed timetable, comprehensively illustrated figures for all embryogenetic events, glossary-like tables for selected structures and processes, as well as a stage-based quick lookup chart. One remarkable difference between the fruit fly and the medfly is that in the latter, the amnioserosa differentiates and unfolds already during gastrulation. Our staging system, which is based on systematically acquired fluorescence live imaging data, provides standard deviations for each developmental time point and serves as a template for future studies seeking to establish an integrative morphogenic framework for other emerging model insect species., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Strobl et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Mitochondrial proteins are transported and sorted to the matrix or inner mitochondrial membrane by the presequence translocase TIM23. In yeast, this essential and highly conserved machinery is composed of the core subunits Tim23 and Tim17. The architecture, assembly, and regulation of the human TIM23 complex are poorly characterized. The human genome encodes two paralogs, TIMM17A and TIMM17B. Here, we describe an unexpected role of the ovarian cancer immunoreactive antigen domain-containing protein 1 (OCIAD1) and the prohibitin complex in the biogenesis of human TIM23. Prohibitins were required to stabilize both the TIMM17A- and TIMM17B-containing variants of the translocase. Interestingly, OCIAD1 assembled with the prohibitin complex to protect the TIMM17A variant from degradation by the YME1L protease. The expression of OCIAD1 was in turn regulated by the status of the TIM23 complex. We postulate that OCIAD1 together with prohibitins constitute a regulatory axis that differentially regulates variants of human TIM23., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Bacillus anthracis is a spore-forming gram-positive bacterium responsible for anthrax, an infectious disease with a high mortality rate and a target of concern due to bioterrorism and long-term site contamination. The entire surface of vegetative cells in exponential or stationary growth phase is covered in proteinaceous arrays called S-layers, composed of Sap or EA1 protein, respectively. The Sap S-layer represents an important virulence factor and cell envelope support structure whose paracrystalline nature is essential for its function. However, the spatial organization of Sap in its lattice state remains elusive. Here, we employed cryoelectron tomography and subtomogram averaging to obtain a map of the Sap S-layer from tubular polymers that revealed a conformational switch between the postassembly protomers and the previously available X-ray structure of the condensed monomers. To build and validate an atomic model of the lattice within this map, we used a combination of molecular dynamics simulations, X-ray crystallography, cross-linking mass spectrometry, and biophysics in an integrative structural biology approach. The Sap lattice model produced recapitulates a close-to-physiological arrangement, reveals high-resolution details of lattice contacts, and sheds light on the mechanisms underlying the stability of the Sap layer., Competing Interests: Competing interests statement:H.R. and A.F. are named as inventors on patent WO/2019/068677 describing the use of S-layer disrupting nanobodies as antibacterial agents. The other authors declare no competing interests.

The autophagy-lysosome system directs the degradation of a wide variety of cargo and is also involved in tumor progression. Here, we show that the immunity-related GTPase family Q protein (IRGQ), an uncharacterized protein to date, acts in the quality control of major histocompatibility complex class I (MHC class I) molecules. IRGQ directs misfolded MHC class I toward lysosomal degradation through its binding mode to GABARAPL2 and LC3B. In the absence of IRGQ, free MHC class I heavy chains do not only accumulate in the cell but are also transported to the cell surface, thereby promoting an immune response. Mice and human patients suffering from hepatocellular carcinoma show improved survival rates with reduced IRGQ levels due to increased reactivity of CD8+ T cells toward IRGQ knockout tumor cells. Thus, we reveal IRGQ as a regulator of MHC class I quality control, mediating tumor immune evasion., Competing Interests: Declaration of interests J.W.H. is a co-founder and consultant for Caraway Therapeutics (a subsidiary of Merck Inc) and is a scientific advisory board member for Lyterian Therapeutics., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

RNA-binding proteins are essential for gene regulation and the spatial organization of cells. Here, we report that the yeast ribosome biogenesis factor Loc1p is an intrinsically disordered RNA-binding protein with eight repeating positively charged, unstructured nucleic acid binding (PUN) motifs. While a single of these previously undefined motifs stabilizes folded RNAs, multiple copies strongly cooperate to catalyze RNA folding. In the presence of RNA, these multivalent PUN motifs drive phase separation. Proteome-wide searches in pro- and eukaryotes for proteins with similar arrays of PUN motifs reveal a strong enrichment in RNA-mediated processes and DNA remodeling. Thus, PUN motifs are potentially involved in a large variety of RNA- and DNA-related processes by concentrating them in membraneless organelles. The general function and wide distribution of PUN motifs across species suggest that in an ancient 'RNA world' PUN-like motifs may have supported the correct folding of early ribozymes., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

N 6 -Methyladenosine (m6A) is the predominant internal RNA modification in eukaryotic messenger RNAs (mRNAs) and plays a crucial role in mRNA stability. Here, using human cells, we reveal that m6A sites in the coding sequence (CDS) trigger CDS-m6A decay (CMD), a pathway that is distinct from previously reported m6A-dependent degradation mechanisms. Importantly, CDS m6A sites act considerably faster and more efficiently than those in the 3' untranslated region, which to date have been considered the main effectors. Mechanistically, CMD depends on translation, whereby m6A deposition in the CDS triggers ribosome pausing and transcript destabilization. The subsequent decay involves the translocation of the CMD target transcripts to processing bodies (P-bodies) and recruitment of the m6A reader protein YT521-B homology domain family protein 2 (YTHDF2). Our findings highlight CMD as a previously unknown pathway, which is particularly important for controlling the expression of developmental regulators and retrogenes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Human 5-lipoxygenase (5-LO) is the key enzyme in the biosynthesis of leukotrienes, mediators of the innate immune system that also play an important role in inflammatory diseases and cancer. In this study, we present compounds, containing a Michael-reactive cyanoacrylate moiety as potent inhibitors of 5-LO. Representatives of the tyrosine kinase inhibitor family called tyrphostins, structurally related to known 5-LO inhibitors, were screened for their 5-LO inhibitory properties using recombinant human 5-LO, intact human PMNL (polymorphonuclear leukocytes), and PMNL homogenates. Their mode of action was characterized by the addition of glutathione, using a fourfold cysteine 5-LO mutant and mass spectrometry analysis. SAR studies revealed several members of the tyrphostin family containing a Michael-reactive cyanoacrylate to efficiently inhibit 5-LO. We identified degrasyn (IC 50 0.11 µM), tyrphostin A9 (IC 50 0.8 µM), AG879 (IC 50 78 nM), and AG556 (IC 50 64 nM) as potent 5-LO inhibitors. Mass spectrometry analysis revealed that degrasyn and AG556 covalently bound to up to four cysteines, including C416 and/or C418 which surround the substrate entry site. Furthermore, the 5-LO inhibitory effect of degrasyn was remarkably impaired by the addition of glutathione or by the mutation of cysteines to serines at the surface of 5-LO. We successfully identified several tyrphostins as potent inhibitors of human 5-LO. Degrasyn and AG556 were able to covalently bind to 5-LO via their cyanoacrylate moiety. This provides a promising mechanism for targeting 5-LO by Michael acceptors, leading to new therapeutic opportunities in the field of inflammation and cancer., Competing Interests: Declarations. Competing Interests: The authors declare no competing interests., (© 2024. The Author(s).)

Veins have emerged as the origin of all other endothelial cell subtypes needed to expand vascular networks during developmental and pathological neoangiogenesis. Here, we uncover the role of the angioneurin Fibronectin Leucine Rich Transmembrane protein (FLRT) 2 in central nervous system (CNS) vascular development in the mouse. Early postnatal FLRT2 deletion reveals specific defects in retinal veins, impacting endothelial cell proliferation, sprouting and polarity that result in reduced tip cells at the vascular front. FLRT2 interacts with VE-cadherin and together with the endocytic adaptor protein Numb contribute to the modulation of adherens junction morphology in both retina and cerebral cortex in vivo. Utilizing expansion microscopy, we visualize the altered dynamic distribution of VE-cadherin in tissue of FLRT2 endothelial mutants. Additionally, FLRT2 in cortical vessels regulates the crosstalk between adherens and tight junctions, influencing blood-brain barrier development. Our findings position FLRT2 as a vein-specific regulator of CNS vascular development., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Recent successes in developing small molecule degraders that act through the ubiquitin system have spurred efforts to extend this technology to other mechanisms, including the autophagosomal-lysosomal pathway. Therefore, reports of autophagosome tethering compounds (ATTECs) have received considerable attention from the drug development community. ATTECs are based on the recruitment of targets to LC3/GABARAP, a family of ubiquitin-like proteins that presumably bind to the autophagosome membrane and tether cargo-loaded autophagy receptors into the autophagosome. In this work, we rigorously tested the target engagement of the reported ATTECs to validate the existing LC3/GABARAP ligands. Surprisingly, we were unable to detect interaction with their designated target LC3 using a diversity of biophysical methods. Intrigued by the idea of developing ATTECs, we evaluated the ligandability of LC3/GABARAP by in silico docking and large-scale crystallographic fragment screening. Data based on approximately 1000 crystal structures revealed that most fragments bound to the HP2 but not to the HP1 pocket within the LIR docking site, suggesting a favorable ligandability of HP2. Through this study, we identified diverse validated LC3/GABARAP ligands and fragments as starting points for chemical probe and ATTEC development., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Antibiotic efflux plays a key role for the multidrug resistance in Gram-negative bacteria 1-3 . Multidrug efflux pumps of the resistance nodulation and cell division (RND) superfamily function as part of cell envelope spanning systems and provide resistance to diverse antibiotics 4,5 . Here, we identify two phylogenetic clusters of RND proteins with conserved binding pocket residues. Based on the characterisation of one representative of each cluster, K. pneumoniae OqxB and E. coli AcrB, we show that the transfer of a single conserved residue between both clusters alters the resistance against a panel of structurally unrelated drugs. The substitution is not only associated with changes in the binding pocket architecture, but also alters the equilibrium between the conformational states of the transport cycle. We show that AcrB and OqxB adopt fundamentally different apo states that suggest different mechanisms of initial substrate binding and might determine the differences between the substrate preferences of both pumps. The observed conformational heterogeneity between different RND clusters is suggested to be phylogenetically conserved and might play a role for the diversification of the resistance phenotype between homologous RND multidrug efflux pumps., Competing Interests: Competing interests: The authors declare no competing interests.

Mutations in splicing factor 3B subunit 1 ( SF3B1 ) frequently occur in patients with chronic lymphocytic leukemia (CLL) and myelodysplastic syndromes (MDSs). These mutations have different effects on the disease prognosis with beneficial effect in MDS and worse prognosis in CLL patients. A full-length transcriptome approach can expand our knowledge on SF3B1 mutation effects on RNA splicing and its contribution to patient survival and treatment options. We applied long-read transcriptome sequencing (LRTS) to 44 MDS and CLL patients, as well as two pairs of isogenic cell lines with and without SF3B1 mutations, and found >60% of novel isoforms. Splicing alterations were largely shared between cancer types and specifically affected the usage of introns and 3' splice sites. Our data highlighted a constrained window at canonical 3' splice sites in which dynamic splice-site switches occurred in SF3B1 -mutated patients. Using transcriptome-wide RNA-binding maps and molecular dynamics simulations, we showed multimodal SF3B1 binding at 3' splice sites and predicted reduced RNA binding at the second binding pocket of SF3B1 K700E Our work presents the hitherto most-complete LRTS study of the SF3B1 mutation in CLL and MDS and provides a resource to study aberrant splicing in cancer. Moreover, we showed that different disease prognosises result most likely from the different cell types expanded during carcinogenesis rather than different mechanisms of action of the mutated SF3B1. These results have important implications for understanding the role of SF3B1 mutations in hematological malignancies and other related diseases., (© 2024 Pacholewska et al.; Published by Cold Spring Harbor Laboratory Press.)

Human organoids have been proposed to be powerful tools mimicking the physiopathological processes of the organs of origin. Recently, human pancreatic organoids (hPOs) have gained increasing attention due to potential theragnostic and regenerative medicine applications. However, the cellular components of hPOs have not been defined precisely. In this work, we finely characterized these structures, focusing first on morphology and identity-defining molecular features under long-term culture conditions. Next, we focused our attention on hPOs cell type composition using single-cell RNA sequencing founding a complex heterogeneity in ductal components, ranging from progenitor components to terminally differentiated ducts. Furthermore, an extensive comparison of human pancreatic organoids with previously reported transcriptomics signature of human and mouse pancreatic ductal populations, confirmed the functional pancreatic duct subpopulation heterogeneity. Finally, we showed that pancreatic organoid cells follow a precise developmental trajectory and utilize diverse signalling mechanisms, including EGF and SPP1, to facilitate cell-cell communication and maturation. Together our results offer an in-depth description of human pancreatic organoids providing a strong foundation for future in vitro diagnostic and translational studies of pancreatic health and disease., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Aβ plaques are a main feature of Alzheimer's disease, and pathological alterations especially in their microenvironment have recently come into focus. However, a holistic imaging approach unveiling these changes and their biochemical nature is still lacking. In this context, we leverage confocal Raman microscopy as unbiased tool for non-destructive, label-free differentiation of progressive biomolecular changes in the Aβ plaque microenvironment in brain tissue of a murine model of cerebral amyloidosis. By developing a detailed approach, overcoming many challenges of chemical imaging, we identify spatially-resolved molecular signatures of disease-associated structures. Specifically, our study reveals nuclear condensation, indicating cellular degeneration, and increased levels of cytochrome c, showing mitochondrial dysfunction, in the vicinity of Aβ plaques. Further, we observe severe accumulation of especially unsaturated lipids. Thus, our study contributes to a comprehensive understanding of disease progression in the Aβ plaque microenvironment, underscoring the prospective of Raman imaging in neurodegenerative disorder research., Competing Interests: Competing interests The authors declare no competing interests. Ethics Animal experiments and husbandry were approved by the animal welfare committee of the Johann Wolfgang Goethe University (ethical committee) and the Regierungspraesidium Darmstadt (board); protocol numbers FR1001/FR2018/FR2010., (© 2024. The Author(s).)

RNA splicing enables the functional adaptation of cells to changing contexts. Impaired splicing has been associated with diseases, including retinitis pigmentosa, but the underlying molecular mechanisms and cellular responses remain poorly understood. In this work, we report that deficiency of ubiquitin-specific protease 39 (USP39) in human cell lines, zebrafish larvae, and mice led to impaired spliceosome assembly and a cytotoxic splicing profile characterized by the use of cryptic 5' splice sites. Disruptive cryptic variants evaded messenger RNA (mRNA) surveillance pathways and were translated into misfolded proteins, which caused proteotoxic aggregates, endoplasmic reticulum (ER) stress, and, ultimately, cell death. The detrimental consequence of splicing-induced proteotoxicity could be mitigated by up-regulating the ubiquitin-proteasome system and selective autophagy. Our findings provide insight into the molecular pathogenesis of spliceosome-associated diseases.

Neuronal communication involves small-molecule transmitters, gap junctions, and neuropeptides. While neurons often express multiple neuropeptides, our understanding of the coordination of their actions and their mutual interactions remains limited. Here, we demonstrate that two neuropeptides, NLP-10 and FLP-1, released from the same interneuron pair, AVKL/R, exert antagonistic effects on locomotion speed in Caenorhabditis elegans. NLP-10 accelerates locomotion by activating the G protein-coupled receptor NPR-35 on premotor interneurons that promote forward movement. Notably, we establish that NLP-10 is crucial for the aversive response to mechanical and noxious light stimuli. Conversely, AVK-derived FLP-1 slows down locomotion by suppressing the secretion of NLP-10 from AVK, through autocrine feedback via activation of its receptor DMSR-7 in AVK neurons. Our findings suggest that peptidergic autocrine motifs, exemplified by the interaction between NLP-10 and FLP-1, might represent a widespread mechanism in nervous systems across species. These mutual functional interactions among peptidergic co-transmitters could fine-tune brain activity., (© 2024. The Author(s).)

Induction of Atg8-family protein (LC3/GABARAP proteins in human) interactions with target proteins of interest by proximity-inducing small molecules offers the possibility for novel targeted protein degradation approaches. However, despite intensive screening campaigns during the last 5 years, no potent ligands for LC3/GABARAPs have been developed, rendering this approach largely unexplored and unsuitable for therapeutic exploitation. In this Viewpoint, we analyze the reported attempts identifying LC3/GABARAP inhibitors and provide our own point of view why no potent inhibitors have been found. Additionally, we designate reasonable directions for the identification of potent and probably selective LC3/GABARAP inhibitors for alternative therapeutic applications., (© 2023 The Authors. Journal of Cellular Biochemistry published by Wiley Periodicals LLC.)

Selective autophagy receptors (SARs) are central to cellular homeostatic and organellar recycling pathways. Over the last two decades, more than 30 SARs have been discovered and validated using a variety of experimental approaches ranging from cell biology to biochemistry, including high-throughput imaging and screening methods. Yet, the extent of selective autophagy pathways operating under various cellular contexts, for example, under basal and starvation conditions, remains unresolved. Currently, our knowledge of all known SARs and their associated cargo components is fragmentary and limited by experimental data with varying degrees of resolution. Here, we use classical predictive and modeling approaches to integrate high-quality autophagosome content profiling data with disparate datasets. We identify a global set of potential SARs and their associated cargo components active under basal autophagy, starvation-induced, and proteasome-inhibition conditions. We provide a detailed account of cellular components, biochemical pathways, and molecular processes that are degraded via autophagy. Our analysis yields a catalog of new potential SARs that satisfy the characteristics of bonafide, well-characterized SARs. We categorize them by the subcellular compartments they emerge from and classify them based on their likely mode of action. Our structural modeling validates a large subset of predicted interactions with the human ATG8 family of proteins and shows characteristic, conserved LC3-interacting region (LIR)-LIR docking site (LDS) and ubiquitin-interacting motif (UIM)-UIM docking site (UDS) binding modes. Our analysis also revealed the most abundant cargo molecules targeted by these new SARs. Our findings expand the repertoire of SARs and provide unprecedented details into the global autophagic state of HeLa cells. Taken together, our findings provide motivation for the design of new experiments, testing the role of these novel factors in selective autophagy., (© 2023 The Authors. Journal of Cellular Biochemistry published by Wiley Periodicals LLC.)

The multidomain protein BAG3 exerts pleiotropic oncogenic functions in many tumor entities including glioblastoma (GBM). Here, we compared BAG3 protein-protein interactions in either adherently cultured or stem-like cultured U251 GBM cells. In line with BAG3's putative role in regulating stem-like properties, identified interactors in sphere-cultured cells included different stem cell markers (SOX2, OLIG2, and NES), while interactomes of adherent BAG3-proficient cells indicated a shift toward involvement of BAG3 in regulation of cilium assembly (ACTR3 and ARL3). Applying a set of BAG3 deletion constructs we could demonstrate that none of the domains except the WW domain are required for suppression of cilia formation by full-length BAG3 in U251 and U343 cells. In line with the established regulation of the Hippo pathway by this domain, we could show that the WW mutant fails to rescue YAP1 nuclear translocation. BAG3 depletion reduced activation of a YAP1/AURKA signaling pathway and induction of PLK1. Collectively, our findings point to a complex interaction network of BAG3 with several pathways regulating cilia homeostasis, involving processes related to ciliogenesis and cilium degradation., (© 2024 The Authors. BioFactors published by Wiley Periodicals LLC on behalf of International Union of Biochemistry and Molecular Biology.)

Nitrogen-containing heterocycles are present in most approved drugs, reflecting the significance of their synthetic strategies. By utilizing oxadiazolone as a nitrenoid (nitrene-like) precursor, we have developed a general strategy for the annulation with nucleophilic heterocycles to access various polycyclic aminoheterocycles. We have discovered that 2-pyrryl-substituted substrates undergo a rearrangement, which indicates a spirocyclization-migration pathway. Given their fluorescence and biological activity as kinase hinge binders, these fragment-like aminoheterocycles represent potential starting points for applications in chemical biology and drug discovery.

Cortical formins, pivotal for the assembly of linear actin filaments beneath the membrane, exert only minor effects on unconfined cell migration of weakly and moderately adherent cells. However, their impact on migration and mechanostability of highly adherent cells remains poorly understood. Here, we demonstrate that loss of cortical actin filaments generated by the formins mDia1 and mDia3 drastically compromises cell migration and mechanics in highly adherent fibroblasts. Biophysical analysis of the mechanical properties of the mutant cells revealed a markedly softened cell cortex in the poorly adherent state. Unexpectedly, in the highly adherent state, associated with a hyperstretched morphology with exaggerated focal adhesions and prominent high-strain stress fibers, they exhibited even higher cortical tension compared to control. Notably, misguidance of intracellular forces, frequently accompanied by stress-fiber rupture, culminated in the formation of tension- and contractility-induced macroapertures, which was instantly followed by excessive lamellipodial protrusion at the periphery, providing critical insights into mechanotransduction of mechanically stressed and highly adherent cells.

Intrinsically disordered regions (IDRs) play a pivotal role in organellar remodeling. They transduce signals across membranes, scaffold signaling complexes, and mediate vesicular traffic. Their functions are regulated by constraining conformational ensembles through specific intra- and intermolecular interactions, physical tethering, and posttranslational modifications. The endoplasmic reticulum (ER)-phagy receptor FAM134B/RETREG1, known for its reticulon homology domain (RHD), includes a substantial C-terminal IDR housing the LC3 interacting motif. Beyond engaging the autophagic machinery, the function of the FAM134B-IDR is unclear. Here, we investigate the characteristics of the FAM134B-IDR by extensive modeling and molecular dynamics simulations. We present detailed structural models for the IDR, mapping its conformational landscape in solution and membrane-anchored configurations. Our analysis reveals that depending on the membrane anchor, the IDRs collapse onto the membrane and induce positive membrane curvature to varying degrees. The charge patterns underlying this Janus-like behavior are conserved across other ER-phagy receptors. We found that IDRs alone are sufficient to sense curvature. When combined with RHDs, they intensify membrane remodeling and drive efficient protein clustering, leading to faster budding, thereby amplifying RHD remodeling functions. Our simulations provide a perspective on IDRs of FAM134B, their Janus-like membrane interactions, and the resulting modulatory functions during large-scale ER remodeling., Competing Interests: Competing interests statement:The authors declare no competing interest.