FEBS Letters最新文献

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.

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 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 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.

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.

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.

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 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 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 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.