Fragment-based inhibitor design is an established and widely used approach in drug discovery pipelines. Despite several examples of drugs originating from this approach, the identification of fragments still suffers from...Fragment-based inhibitor design is an established and widely used approach in drug discovery pipelines. Despite several examples of drugs originating from this approach, the identification of fragments still suffers from issues with solubility, reactivity, cost and worldwide accessibility. Here, we design a low-cost minimal fragment library (LoCoFrag100) for crystallographic screening, with an average cLogP of 0.03 (median 0.23) and an average of £20/g for each compound, facilitating assembly in any laboratory. Formatted in a 10 × 10 matrix to minimize Tanimoto similarity in the 20 cocktails, we demonstrate its applicability on three structurally distinct enzymes involved in microbial cell wall synthesis. Hit rates range from 1 to 6% among these enzymes, with three fragments suggesting avenues for inhibitor exploration. Impact Statement LoCoFrag100 is a low-cost, easily accessible fragment library that enables rapid survey of target ligandability in any laboratory, providing evidence to prioritise targets for follow-up research.
The t(10;11)(p13;q14-21) PICALM::MLLT10 chromosomal translocation results in the production of the CALM-AF10 fusion oncoprotein and is a driver mutation in both acute myeloid and T-lymphoblastic leukemia. PICALM::MLLT10...The t(10;11)(p13;q14-21) PICALM::MLLT10 chromosomal translocation results in the production of the CALM-AF10 fusion oncoprotein and is a driver mutation in both acute myeloid and T-lymphoblastic leukemia. PICALM::MLLT10 translocated leukemia is primarily an epigenetically driven disease. Global hypomethylation results in genomic instability, while focal H3K79 hypermethylation at target genes induces cell proliferation and blocks differentiation. Nucleocytoplasmic shuttling of CALM-AF10 and its protein partners and impaired endocytosis at the plasma membrane further influence the leukemic phenotype. Leukemias characterized by PICALM::MLLT10 have historically been recognized to portend a poor prognosis; however, insights from larger patient cohorts provide refinement to the prognostic relevance of this chromosomal translocation, highlighting chemotherapy resistance in this leukemic subtype. In addition, a deeper biological understanding of the disease hints at potential therapeutic targets. This approach is demonstrated in the recent promising results achieved utilizing venetoclax, a BCL2 inhibitor, in patients with PICALM::MLLT10 acute leukemia. Herein, we provide updates on the pathophysiology, clinical presentation, prognosis, and treatment of PICALM::MLLT10 acute leukemia.
Gastric cancer research has rapidly progressed due to interdisciplinary advances in stem cell biology and bioengineering. Gastric organoid models, particularly those derived from adult stem cells, have emerged as powerfu...Gastric cancer research has rapidly progressed due to interdisciplinary advances in stem cell biology and bioengineering. Gastric organoid models, particularly those derived from adult stem cells, have emerged as powerful tools that recapitulate the cellular complexity of the human stomach. This review highlights the development of various gastric organoid platforms, with a specific focus on the convergence of engineering strategies to overcome the limitations of conventional organoid systems. We explore how CRISPR-based functional genomics, matrix innovations, co-culture systems, microphysiological systems (MPS), and big data integration are collectively enhancing organoid models. Furthermore, we examine how artificial intelligence may refine the clinical relevance and precision of gastric organoid models. By assessing both current capabilities and future directions, this review offers a perspective on how gastric organoid systems may reflect human physiology more accurately and improve therapeutic outcomes.
Glycosaminoglycan assembly on proteoglycans involves a common tetrasaccharide linker that starts with xylose attached to a serine on the protein. Defective linker biosynthesis caused by a missense mutation of human UDP-x...Glycosaminoglycan assembly on proteoglycans involves a common tetrasaccharide linker that starts with xylose attached to a serine on the protein. Defective linker biosynthesis caused by a missense mutation of human UDP-xylose synthase (hUXS1) is associated with connective tissue disorders characterized by skeletal abnormality and short stature. The Ile181Asn variant of hUXS1 was reported as inactive in releasing UDP-xylose from UDP-glucuronic acid. Here, we show that Ile181Asn-hUXS1 exhibited catalytic properties similar to the wild-type enzyme but featured a significant decrease in stability, expressed in melting temperature lowered from 48.2 °C to 35.2 °C. At 37 °C, Ile181Asn-hUXS1 was ~10-fold less stable and more prone to precipitation than wild-type hUXS1. The loss of function in Ile181Asn-hUXS1 is thus explained by instability, consistent with molecular dynamics simulations predicting structural destabilization. Impact statement The Ile181Asn variant of human UDP-xylose synthase (hUXS1), associated with a short-stature genetic syndrome, has previously been reported as inactive. We show here with experiments and molecular simulations that hUXS1 malfunction arises from structural instability rather than from a catalytic defect.
The folate biosynthesis activity of the human microbiome provides reduced folate metabolites that are readily absorbed from the gastrointestinal (GI) tract. The bacterial folate biosynthesis enzyme dihydropteroate syntha...The folate biosynthesis activity of the human microbiome provides reduced folate metabolites that are readily absorbed from the gastrointestinal (GI) tract. The bacterial folate biosynthesis enzyme dihydropteroate synthase (DHPS), which adds p-aminobenzoate (pABA) to an activated pterin precursor, is an important antibiotic target. Both the broad-spectrum p-aminobenzenesulfonamide antibiotics, and the drug p-aminosalicylate (PAS, 2-hydroxy-pABA) with high selectivity for Mycobacterium tuberculosis, are competitive DHPS substrates. The adducts formed from these drugs, DHP-sulfonamides (sulfapterins) and 2'-hydroxyfolate metabolites, respectively, have been reported to exhibit antifolate activity in studies of microorganisms. The presence of these DHP-adducts and their effects on the host organism are largely undetermined; however, their close structural relationship to dihydrofolate (DHF) suggests that they are likely to mediate some side effects reported for these antibiotics. Naturally occurring pABA analogs that probably function similar to DHPS-targeted antibiotics have been identified in carrots and bacteria. Impact statement pABA analogs represent an important class of antibiotics, that are converted into dihydrofolate analogs by organisms present in the human microbiome. These analogs may mediate reported side-effects associated with these antibiotics. Several naturally occurring pABA mimics have been identified that are likely to exhibit antibiotic activity.
The tumor suppressor p53 is normally maintained at low levels through MDM2-mediated degradation; however, this regulation becomes ineffective upon DNA damage, leading to p53 phosphorylation and accumulation. This study s...The tumor suppressor p53 is normally maintained at low levels through MDM2-mediated degradation; however, this regulation becomes ineffective upon DNA damage, leading to p53 phosphorylation and accumulation. This study shows that E6-associated protein (E6AP) provides an alternative regulatory pathway during genotoxic stress. Unlike MDM2, E6AP can effectively decrease p53 levels in HepG2 cells exposed to DNA-damaging agents, such as etoposide. Additionally, E6AP specifically targets p53 phosphorylated at serine-15, promoting its proteasomal degradation, whereas MDM2 cannot. This phosphorylation-dependent regulation by E6AP helps maintain p53 at appropriate levels during mild DNA damage, preventing excessive accumulation that could threaten cell survival, while still allowing for necessary stress responses. Impact statement The mechanism by which p53 is negatively regulated under genotoxic stress is largely unknown. E6-associated protein (E6AP), unlike MDM2, downregulates p53 levels following exposure to etoposide. E6AP specifically targets p53 phosphorylated at Ser-15. This mechanism prevents excessive accumulation of p53 that could otherwise reach lethal levels.
RNA sequences with the potential to form G-quadruplexes (rGQs) are widespread but largely unfolded in cells by unknown mechanisms. rGQ folding status is a critical regulator of RNA splicing and translation. We show that...RNA sequences with the potential to form G-quadruplexes (rGQs) are widespread but largely unfolded in cells by unknown mechanisms. rGQ folding status is a critical regulator of RNA splicing and translation. We show that rGQs can be unfolded by SR proteins, SR-related proteins, and other Arg-rich proteins, including SRSF1, SRSF3, SRSF9, U1-70K, and U2AF1. The length and composition of Arg-rich regions are key determinants of this activity: Arg residues are the primary drivers, while acidic residues attenuate the unfolding activity. To unfold ARPC2 rGQ, at least 13 Arg residues are required. Our findings identify Arg-rich proteins as previously unrecognized, helicase-independent regulators of rGQ structures, with potential broad impacts on RNA processing that merit further investigation.
The Dot/Icm type IV secretion system (T4SS) is essential for Legionella pneumophila infection, but its in situ architecture and mechanism remain incompletely understood. Using cryo-electron tomography, we performed subto...The Dot/Icm type IV secretion system (T4SS) is essential for Legionella pneumophila infection, but its in situ architecture and mechanism remain incompletely understood. Using cryo-electron tomography, we performed subtomogram averaging and 3D classification to resolve structural heterogeneity within the complex. We identified multiple assembly states of the inner membrane complex, including a fully assembled form with a hexamer-of-dimers DotO ATPase and symmetry mismatches between subcomplexes. A composite in situ model revealed a central channel above the inner membrane, likely used for substrate secretion. Imaging of infected macrophages showed T4SSs tethered to host vacuoles and extracellular vesicle release, suggesting additional effector delivery routes. These findings provide insight into Dot/Icm T4SS structure and infection-related dynamics.
A newly developed noninvasive Oncomagnetic device (OMD) causes selective cytotoxicity in glioblastoma and diffuse intrinsic pontine glioma cells, providing a novel, non-toxic approach to anticancer therapy. Here, we repo...A newly developed noninvasive Oncomagnetic device (OMD) causes selective cytotoxicity in glioblastoma and diffuse intrinsic pontine glioma cells, providing a novel, non-toxic approach to anticancer therapy. Here, we report results in cultured glioma cells and in a syngeneic mouse model indicating that the immediate intracellular target mechanism of action of the spinning oscillating magnetic field (sOMF) produced by this device is reactive oxygen species-dependent persistent inhibition of mitochondrial complex I. Steps downstream of this mechanism involve the production of oxidative stress, DNA damage, G1 phase cell cycle arrest, and caspase-dependent apoptosis. We also show that sOMF does not produce these effects in normal human astrocytes and astroglial cells. These data provide a rationale for safe clinical use of OMD.
Mitochondrial protein Slm35 is linked to TOR1 signaling, mitophagy, and stress response in Saccharomyces cerevisiae. Nonetheless, little is known about its regulation or how it affects stress adaptation. In this work, we...Mitochondrial protein Slm35 is linked to TOR1 signaling, mitophagy, and stress response in Saccharomyces cerevisiae. Nonetheless, little is known about its regulation or how it affects stress adaptation. In this work, we identified stress-related transcription factor binding sites and two upstream open reading frames (uORFs) in the 5'-UTR of SLM35. Using transcriptional reporters, we showed that the transcription factor Gis1 represses SLM35 transcription; however, Slm35 protein levels increased under oxidative stress and in early stationary phase, suggesting post-transcriptional regulation. Site-directed mutagenesis revealed that one uORF negatively regulates translation, with its disruption leading to altered Slm35 levels and a reproducible increase in mitophagy flux. These findings reveal multilayered control of SLM35 expression and underscore the role of uORF-mediated translation in mitochondrial stress responses. Impact statement This study shows that SLM35, encoding a mitochondrial protein, is controlled through multiple regulatory layers, combining transcriptional repression by stress-responsive factors with uORF-mediated translational regulation. By linking these mechanisms to mitophagy, the work provides new insight into mitochondrial quality control under stress.
Phosphoinositides, comprising less than 10% of membrane lipids, function as 'lipid codes' within cellular compartments through seven species formed by myo-inositol headgroup phosphorylation. This review examines their di...Phosphoinositides, comprising less than 10% of membrane lipids, function as 'lipid codes' within cellular compartments through seven species formed by myo-inositol headgroup phosphorylation. This review examines their diverse roles in endocytic transport, encompassing endocytosis, endosomal sorting, degradation, and recycling, as well as specialized mechanisms, such as caveolin-mediated endocytosis. The review also investigates the involvement of specific kinases and phosphatases in these processes. Additionally, it discusses the impact of technological advancements, such as fluorescent biosensors, super-resolution microscopy, optogenetics, and synthetic biology, on elucidating phosphoinositide dynamics during endocytic trafficking. Perturbations in phosphoinositide metabolism have been associated with human diseases, including cancer and neurodegenerative disorders. Exploring these pathways may unveil potential therapeutic targets, with subsequent research focusing on their spatiotemporal regulation, tissue-specific metabolism, the synergistic effects of phosphoinositides with other lipids, and the incorporation of systems biology to bridge basic cell biology with translational medicine.
Inositol phosphates (InsPs) represent a conserved class of water-soluble signaling molecules found in all eukaryotes. Their biosynthesis involves a tightly regulated enzymatic network, with inositol polyphosphate multiki...Inositol phosphates (InsPs) represent a conserved class of water-soluble signaling molecules found in all eukaryotes. Their biosynthesis involves a tightly regulated enzymatic network, with inositol polyphosphate multikinase (IPMK) functioning as a pivotal catalytic hub. IPMK exhibits broad substrate specificity, phosphorylating various InsPs and phosphatidylinositol 4,5-bisphosphate. Beyond its enzymatic activity, IPMK also modulates key signaling pathways through noncatalytic mechanisms, including direct interactions with protein partners. This review highlights the functional attributes of IPMK, its diverse roles in cellular physiology and disease, and outlines current challenges and future directions in IPMK research.
Transglutaminase 2 (TG2) exhibits various protein-modifying catalytic and protein-protein interaction properties and is highly expressed in endothelial cells. To provide insight into its endothelial functions, the TG2 in...Transglutaminase 2 (TG2) exhibits various protein-modifying catalytic and protein-protein interaction properties and is highly expressed in endothelial cells. To provide insight into its endothelial functions, the TG2 interactome was identified in HUVECs using biotinylated recombinant TG2 and affinity chromatography. Subsequently, endogenous TG2-silenced and a triple Flag-tagged transgenic TG2-expressing HUVEC line was created, allowing isolation of intracellularly assembled TG2-interacting proteins. Conformation-dependency of TG2's interactome was also determined. RNA-binding proteins associated with TG2 were the most enriched gene ontology terms in all experiments, with a 42% overlap between the TG2 interactome and known RNA-binding proteins in HUVECs. Consistent with TG2's recently described RNA-binding ability, our findings reveal its potential role in post-transcriptional regulation at a central hub within the RNA-binding protein network.
The maintenance of protein homeostasis is a fundamental premise for the survival of all life. The synthesis, folding, localization, and degradation of thousands of proteins must be organized according to various conditio...The maintenance of protein homeostasis is a fundamental premise for the survival of all life. The synthesis, folding, localization, and degradation of thousands of proteins must be organized according to various conditions. To ensure such a stable and functional proteome, the proteostasis network evolved. Dedicated to this, the fourth School on Proteostasis, a co-funded EMBO|FEBS Lecture Course in memory of Susan Lindquist, took place in Espoo, Finland on 16-19 September 2025, with 59 early career researchers (PhD students or postdoctoral fellows), 18 leading scientists, and two editors attending and discussing the current state of the field. From basic principles to the latest therapeutic developments, this meeting provided a comprehensive overview of proteostasis. This report summarizes the lecture course and highlights selected presentations.
In eukaryotes, nuclear size scales with cell size, maintaining a constant nucleocytoplasmic volume ratio, known as the N/C ratio. Although nucleocytoplasmic transport plays a crucial role in nuclear size control, the und...In eukaryotes, nuclear size scales with cell size, maintaining a constant nucleocytoplasmic volume ratio, known as the N/C ratio. Although nucleocytoplasmic transport plays a crucial role in nuclear size control, the underlying mechanisms remain elusive. Here, we investigated the impact of overexpression of a nuclear export signal (NES) fused with GFP (NES-GFP) in fission yeast on nuclear size. The overexpression of NES-GFP disrupts nuclear export, leading to the nuclear accumulation of cargo proteins and the formation of intranuclear microtubule bundles, thereby increasing the nuclear volume to cell volume (N/C) ratio dependent on nuclear import and microtubule nucleation. Enhanced formation of intranuclear microtubule bundles in cells overexpressing NES-GFP further accelerates nuclear expansion. We propose that membrane tension in the nucleus plays an important role in nuclear size control.
Elevated levels of extrachromosomal DNAs (ecDNAs) are associated with poor prognoses of many cancer types. These large circular DNAs typically harbour oncogenes and regulatory elements which, together with high levels of...Elevated levels of extrachromosomal DNAs (ecDNAs) are associated with poor prognoses of many cancer types. These large circular DNAs typically harbour oncogenes and regulatory elements which, together with high levels of ecDNA transcription, confer a growth advantage to cancer cells. Replication of ecDNAs, followed by their unequal distribution at mitosis, further promotes rapid cancer evolution. In contrast to ecDNAs, the role of circular DNA by-products from V(D)J recombination in cancer development has largely been overlooked. Developing lymphocytes generate millions of excised signal circles (ESCs) each day through gene rearrangement at the immunoglobulin and T-cell receptor loci. Despite their similar size to ecDNAs, ESCs were long assumed to be inert and lost during cell division. However, it is now known that ESCs potently trigger genome instability when complexed with recombinase proteins. Not only this, but new data show that just like ecDNAs, ESCs replicate and persist, with high levels strongly correlating with poor prognosis of B-cell precursor acute lymphoblastic leukaemia (BCP-ALL). Despite these striking similarities, the properties of ESCs and ecDNAs are seldom linked. Here, we provide the first comparative review of ecDNAs and ESCs, and highlight the reasons why these molecules are more closely related than once assumed.
Parenthood has been linked to lower retention of women in academia, yet the specific challenges faced by mothers remain underexplored. We interviewed nine mothers across different career stages in the life sciences to qu...Parenthood has been linked to lower retention of women in academia, yet the specific challenges faced by mothers remain underexplored. We interviewed nine mothers across different career stages in the life sciences to qualitatively examine how motherhood intersects with academic milestones. Participants described challenges unique to academia, including dependence on supervisors, publication pressure, financial strain, and limited awareness of available institutional resources. They also shared strategies that supported their success, such as transparent communication with supervisors, designating student-parent coordinators, and creating peer communities for mothers in academia. By highlighting shared barriers and effective solutions, this work underscores the need for structural and cultural reforms to better support mothers in academia and retain talented scientists in STEM fields.
Almost all organisms on earth undergo rhythmic physiological and behavioral changes over the course of day. These rhythms are fundamental in most organisms and are referred to as circadian rhythms. The molecular mechanis...Almost all organisms on earth undergo rhythmic physiological and behavioral changes over the course of day. These rhythms are fundamental in most organisms and are referred to as circadian rhythms. The molecular mechanisms regulating these changes have evolved significantly in different kingdoms of life and they engage in crosstalk with most cellular functions. These molecular mechanisms have been studied for a long time using different model organisms and carefully designed experiments. In the past two decades or so, with advances in high throughput technologies and access to ever increasing computational power, the molecular mechanisms regulating circadian rhythms are being explored at multiple spatial and temporal scales. In this review, we introduce diverse regulatory mechanisms of circadian rhythms. We then focus on the proteins involved in circadian regulation, their structures, complexes and dynamics. This is followed by a review of computational methods such as structural modeling, integrative modeling and molecular simulations as applied to understanding the clock proteins in different organisms and insights obtained from the same. Finally, we highlight the limitations and future prospects of these methods in understanding the circadian regulation.
ETS family transcription factors can mediate mutant p53 functions, but there has been no comprehensive analysis of p53 interaction across the ETS family. By comparing direct mutant p53 binding between 26 ETS proteins, we...ETS family transcription factors can mediate mutant p53 functions, but there has been no comprehensive analysis of p53 interaction across the ETS family. By comparing direct mutant p53 binding between 26 ETS proteins, we found that all bound mutant p53, but relative binding differed significantly. The ETS DNA binding domain provided a common interaction interface, but strong binding required an alternate interaction domain highlighted by a PXXPP motif found in five ETS proteins. Genome-wide mapping found that the ETS protein ERG mediated some mutant p53 DNA binding in prostate cancer cells. Lastly, ETS proteins that interact strongly with mutant p53 tended to be upregulated in p53 mutant ovarian cancer. These results identify multiple ETS family members that could mediate mutant p53 function in cancer. Impact statement The mechanisms behind gain-of-function mutant p53 remain unclear. Here we identify distinct domains and a novel motif that can mediate binding of mutant p53 to multiple different ETS family transcription factors.
During gene expression, ribosome stalling frequently occurs and can lead to detrimental effects on cellular homeostasis. Several quality control mechanisms, including ribosome-associated quality control (RQC) and nonfunc...During gene expression, ribosome stalling frequently occurs and can lead to detrimental effects on cellular homeostasis. Several quality control mechanisms, including ribosome-associated quality control (RQC) and nonfunctional ribosomal RNA decay (NRD), have been identified to resolve these aberrant translation events. While the molecular mechanisms of each pathway have been extensively characterized, the mechanisms underlying the mutual regulation of the expression of pathway factors remain to be elucidated. Here, we employed a series of knockout mouse and human cell lines to investigate the crosstalk between translational quality control factors. Our findings revealed that the E3 ubiquitin ligase LTN1 suppresses expression of the E3 ubiquitin ligase RNF10 in a manner dependent on the RING domain of LTN1. This discovery offers new insights into the coordination of translational surveillance pathways.