Immune cells undergo metabolic reprogramming in response to inflammatory stimuli. The immuneresponsive gene 1 (Irg1) encodes aconitate decarboxylase (ACOD1), which generates itaconate from cis-aconitate in the TCA cycle. Itaconate inhibits succinate dehydrogenase, resulting in succinate accumulation. Stable ACOD1 overexpression in RAW264.7 cells shifted cellular metabolism towards glycolysis, as indicated by enhanced mTOR activation, increased 4E-BP1 phosphorylation, and reduced ATP levels. ACOD1 cells displayed impaired osteoclastogenesis with reduced expression of osteoclast-associated genes and fewer TRAP-positive multinucleated osteoclasts. Unexpectedly, NFATc1 was constitutively present in the nucleus of untreated ACOD1 cells, resulting in residual NFAT activity and induction of inflammatory genes. Upon RANKL stimulation, these pre-activated cells showed delayed osteoclastogenic signalling accompanied by sustained expression of the transcriptional repressors BCL6, MafB, and IRF8. Using a GPR91 antagonist and a Gαq inhibitor, we demonstrate that extracellular succinate activates NFATc1 via GPR91-Gαq signalling. RNA sequencing further revealed that ACOD1 overexpression promotes an innate immune transcriptional program rather than osteoclast differentiation. Together, our findings identify succinate-GPR91 signalling as a regulator of the transition between inflammatory activation and osteoclastogenesis.
The X-ray crystal structure of astacin from the freshwater crayfish Astacus astacus published in 1992 was the second structural prototype of a zinc metalloendopeptidase solved after thermolysin from Bacillus thermoproteolyticus 20 years earlier. Astacin became the founding member of a clan of metalloendopeptidases, for which Wolfram Bode coined the designation 'metzincins'. This review recapitulates the discovery of the metzincin clan, which includes the astacins, MMPs (matrix metalloproteinases), serralysins and adamalysins/ADAMs (A disintegrin and metalloproteinase enzymes). Currently, over a dozen subclans (families) have been assigned including the pappalysins, leishmanolysins, snapalysins, igalysins, helicolysins, and toxylysins.
RNA molecules carry a wide range of chemical modifications that play key roles in regulating their structure, stability, and function. These modifications are especially abundant in small and non-coding RNAs, such as transfer RNAs (tRNA), ribosomal RNA (rRNA), and related fragments, where they influence processes from translation to gene silencing. However, studying these modifications remains challenging due to the short length of these RNAs, their strong secondary structures, and the high density of chemical marks, all of which can interfere with standard sequencing workflows. This review summarizes current strategies for detecting and mapping RNA modifications in the small RNome. We outline the diversity of RNA classes and their characteristic modification patterns, and then discuss key methodological advances. These include demodification-based sequencing approaches for heavily modified RNAs, targeted chemical and enzymatic methods for site-specific mapping, and emerging direct RNA sequencing technologies that allow analysis of native molecules without prior conversion. We also highlight orthogonal validation techniques used to confirm modification identity and improve reliability. Together, these approaches provide complementary insights, but no single method is sufficient on its own. Careful experimental design and validation therefore remain essential for accurate and comprehensive analysis of RNA modifications.
Over the past two decades, advances in omics technologies, combined with classical genetics, have fundamentally reshaped our understanding of glucocorticoid (GC) biology and informed strategies to optimise GC therapy. Traditional models focused on uniform, ligand-dependent gene regulation via the glucocorticoid receptor (GR). In contrast, genome-wide transcriptomic, epigenomic, and proteomic approaches have revealed a far more complex landscape in which GC responses are highly cell-type-specific and context-dependent. High-resolution transcriptomics and single-cell RNA sequencing have uncovered divergent GC-regulated gene networks across immune, metabolic, and structural cell lineages, providing mechanistic insight into both therapeutic efficacy and tissue-specific adverse effects. Epigenomic profiling, including ChIP-seq and ATAC-seq, has shown that chromatin accessibility, enhancer architecture, and GR cistromes vary across cell types and states, thereby shaping transcriptional outcomes. Complementary proteomic studies have defined GR-interacting protein networks that modulate receptor activity. Together, these multi-layered datasets support a systems-level model of GR signalling that integrates chromatin context, transcriptional regulation, and cellular state and begins to explain GC sensitivity, resistance, and context-specific side effects. Emerging multi-omics and single-cell multimodal approaches now enable high-resolution dissection of GC action in complex tissues and disease contexts, advancing precision therapeutic strategies.
The eukaryotic genome is replicated according to a tightly regulated temporal program that ensures each DNA segment is copied once per cell cycle. This program reflects the coordinated action of replication origin licensing, chromatin state, transcriptional activity, and nuclear organisation. While the core mechanisms of licensing and initiation are well characterised, the determinants of origin selection and firing time remain incompletely understood. In budding yeast, origins are defined by specific DNA elements and chromatin features, whereas in metazoans, origin specification is largely sequence-independent and influenced by epigenetic and three-dimensional genome architecture. This review summarises current knowledge of how replication timing is established, regulated, and functionally integrated with chromatin states across eukaryotes, with emphasis on chromatin accessibility, histone modifications and variants, transcriptional regulators, and higher-order genome topology. It also highlights recent genome-wide approaches that map origin licensing, usage, and nascent DNA synthesis at high resolution, revealing dynamic connections between replication, transcription, and nuclear compartmentalisation. Finally, we discuss how disrupted replication timing contributes to replication stress, genome instability, ageing, and cancer. By integrating findings from diverse eukaryotic systems, this provides an updated framework for understanding replication timing as a key layer of genome regulation.
Phospholipid phosphatase-related protein 3 (PLPPR3) is a regulator of neuronal membrane dynamics and cytoskeletal remodeling. In this review, we synthesize current evidence positioning PLPPR3 as a key driver of neuronal filopodia formation, highlighting its roles in lipid signaling, membrane protrusion and actin organization. By consolidating recent molecular, cellular, and functional studies, we propose a mechanistic framework for PLPPR3-dependent filopodia formation and outline its implications for neuronal morphogenesis.
Histidine triad nucleotide-binding protein 1 (HINT1) is a small and evolutionarily conserved HIT family protein with putative tumor suppressor function. It has been implicated in the negative regulation of several transcription factors, but a role for HINT1 in Myocardin-related transcription factor A (MRTF-A)/serum response factor (SRF) signaling has not been described. Here, we examined whether HINT1 functionally interacts with MRTF-A in fibroblasts. HINT1 reduced MRTF-A-dependent reporter activity in a dose-dependent manner, whereas a histidine triad mutant did not. Repression by HINT1 was retained upon deletion of the N-terminal RPEL motifs of MRTF-A, indicating that the effect was not confined to the inhibition of Rho-actin signaling. In parallel, HINT1-WT increased the MRTF-A protein abundance, which required the C-terminal part of MRTF-A. Co-immunoprecipitation experiments revealed the physical association of HINT1 with MRTF-A, whereas the histidine triad mutant or a C-terminally truncated MRTF-A did not show complex formation. Thus, our work suggests that HINT1 acts as a negative regulator of MRTF-A-dependent transcription by binding to the MRTF-A C-terminal part, which appears to require the histidine triad region and is accompanied by a separable increase in MRTF-A abundance.
Our understanding of trypsin, its zymogenicity and its inhibition is intimately intertwined with the history of biochemistry. Early structural studies revealed its close relationship to chymotrypsin, with its active site triad, oxyanion hole and N-terminus involved in a buried salt bridge near the active site. Its complex with basic pancreatic trypsin inhibitor provided a model for how peptide substrates bind to and are cleaved by the proteinase. Analysis of crystals of trypsinogen by Wolfram Bode and Robert Huber revealed that a large region of the zymogen is disordered prior to proteolytic activation, with an associated disruption of the oxyanion hole and substrate binding pockets, highlighting the importance of disorder in protein function. As archetype of numerous therapeutically important serine proteinases, trypsin can also serve as a surrogate for structure-based drug design. Trypsin variants designed for ligand binding studies resulted however in an unexpected plasticity of the mutant proteins that underlines the complexity of protein stability. A trypsin variant selected for peptide ligation (reverse proteolysis) was shown to possess zymogen-like characteristics that proved central to its application in modification of therapeutic proteins. The review pays homage to the seminal works of Bode and Huber and their influence on modern structural biology.
Cerebral cavernous malformation (CCM) is a genetic vascular disorder arising from endothelial dysfunction, affecting the microvasculature of the central nervous system. Patients with mutations in any of three CCM genes may suffer from neurological deficits and even hemorrhagic stroke. The three CCM proteins are structurally unrelated and assemble into the CCM complex that forms scaffolds associated with junctional complexes and the actomyosin cytoskeleton. Recent work points to a dual role of the CCM complex in structural scaffolding and mechanosensitive signal transduction. In this review, we highlight recent advances examining the CCM complex's role in organizing various multi-protein interactions and facilitating mechanosensitive signaling that regulates endothelial cell behavior. Importantly, accumulating evidence emphasizes the impact of hemodynamic forces on CCM signaling, pointing to distinct mechanisms in arterial versus venous vessels. Surprisingly, the loss of CCM proteins has vasoprotective effects in arteries but pathological effects in veins and small capillaries. Understanding CCM scaffolds that integrate structural support with mechanosensitive signaling is fundamental to deciphering the molecular mechanisms that underlie physiological versus pathological outcomes within the vasculature. This knowledge advances the development of targeted therapeutic strategies aimed at restoring endothelial integrity and normal vascular function, particularly by modulating signaling pathways influenced by hemodynamic forces.
Neuronal function relies on precise compartmentalization into dendritic, somatic, axonal, and synaptic domains that require specialized cellular architectures. Axons, in particular, can extend over extraordinary distances while preserving stable membrane composition, mechanical integrity, and reliable excitability. A submembranous scaffold composed of spectrin, ankyrin, actin, and associated proteins provides a conserved platform for coupling membrane proteins to cytoskeletal support and organizing membrane domains. In axons, this scaffold assembles into a membrane-associated periodic skeleton (MPS) with near-regular spacing that supports mechanical load, patterns membrane components, and contributes to compartmental boundaries. Comparative genetics indicates that core principles of spectrin-ankyrin organization are ancient, whereas vertebrate evolution expanded spectrin and ankyrin families and enabled specialized excitable domains such as the axon initial segment (AIS) and nodes of Ranvier. Pathogenic variants in spectrin and ankyrin genes disrupt neuronal development and excitability and cause a growing spectrum of neurodevelopmental and neurodegenerative disorders, underscoring scaffold integrity as a key determinant of circuit stability. Here, we examine how conserved spectrin-ankyrin scaffold principles were adapted to neuronal cell-type diversity and domain specialization, with emphasis on axons, synapses, and disease, and we discuss how the membrane-associated periodic skeleton may function not only as a stabilizing framework but also as a nanoscale organizer of neuronal membrane architecture.
Golgi-associated membrane scaffolds, or tethers, have broad roles in membrane-bound protein and lipid trafficking and in maintaining Golgi architecture. Accordingly, they exert strong influence over cellular development, signalling, cargo modification and transport. An ever-expanding group of Golgins and multi-subunit tethering complexes assumes distinct functions in specific Golgi subcompartments in close partnership with Rab and ARL family GTPases. Their dysregulation or mutation impairs glycosylation, vesicle trafficking, and cytoskeletal dynamics, thereby contributing to a spectrum of human pathologies ranging from neurodegenerative disorders (e.g. Alzheimer's and Parkinson's disease) to cancers (e.g. lung, breast, colon) and metabolic defects (impaired insulin secretion and lipid droplet formation). Here, we review these diverse roles across molecular, cellular and organismal physiology.
Gentamicin (GM)-induced nephrotoxicity is closely linked to oxidative stress, with few therapeutic alternatives available. Chalcones are structurally versatile compounds with reported antioxidant properties, making them attractive candidates for nephroprotection. The 4'-aminochalcones substituted with p-NO2 (31.25-15.62 µM) or p-N(CH3)2 (7.81 µM) groups demonstrated significant protective effects, partially restoring (∼11 %) HK-2 cell viability after GM-induced injury. Both derivatives increased intracellular glutathione levels, reduced superoxide accumulation, and lowered cytoplasmic ROS concentration. Structural and electronic analyses revealed that the electron-donating group p-N(CH3)2 enhanced antioxidant reactivity, whereas the electron-withdrawing group p-NO2 decreased cytotoxicity. HK-2 cells were exposed to GM (IC50 = 5.0 ± 0.7 mmol/L) for 24 h, followed by treatment with 4'-aminochalcones (250-7.81 µM). Cell viability was assessed by MTT assay. ROS were quantified by flow cytometry using DCFH-DA, while redox balance was evaluated through glutathione content and superoxide accumulation. The electronic properties of chalcone derivatives were further investigated by density functional theory calculations. The 4'-aminochalcones mitigate GM-induced oxidative stress in renal cells, and their antioxidant and cytoprotective activities are strongly influenced by electronic substituents. Electron-donating groups enhance reactivity, whereas electron-withdrawing groups improve safety, underscoring the importance of structure-effect relationships in the design of chalcone-based nephroprotective agents.
Lipid-modified membrane-associated proteins can bind reversibly to cellular membranes, and their steady-state localization reflects a balance between membrane-bound and cytosolic pools. For many small GTPases of the Rho and Rab families, this balance is regulated by GDP dissociation inhibitors (GDIs), which control membrane association by shielding the prenyl group and coupling localization to the nucleotide state. In contrast, Ras proteins were long thought to lack a comparable regulatory system. The prenyl-binding protein PDE6D has emerged as a GDI-like factor for prenylated Ras proteins. Here, we discuss the role of PDE6D in KRAS trafficking and spatial organization, and examine its potential as a target for pharmacological inhibition of oncogenic KRAS signaling.
Tricomplex inhibitors (TCIs) are a novel class of direct Ras inhibitors that target the GTP-bound Ras(on) state through recruitment of Cyclophilin A. Daraxonrasib (RMC-6236) is a pan-Ras TCI that was recently shown to restore GTPase activity of G12-mutant Ras proteins. Structural analysis of a pan-Ras TCI bound to K-Ras(GDP-AlF3) reveals a transition-state arrangement of Tyr32 and Gln61 that closely resembles endogenous GTPase-GAP complexes. This includes a closed Switch-I conformation engaging the cis-GTPase machinery in a manner analogous to non-arginine-finger GAPs such as RanGAP. These observations position pan-Ras TCIs as pharmacologic GAP mimetics. The GTPase-promoting activity of daraxonrasib suggests synergy with Switch-II pocket K-Ras inhibitors, including the approved GDP-state selective K-Ras G12C inhibitor adagrasib (MRTX-849), whose engagement of K-Ras(GTP) is kinetically constrained by slow endogenous hydrolysis of the mutant GTPase. We demonstrate that daraxonrasib sensitizes K-Ras(GTP) to adagrasib labeling in both recombinant protein and cellular contexts. In K-Ras G12C and G12D mutant cell lines, combinations of daraxonrasib with adagrasib or HRS-4642 (MRTX-1133 analog) yield more rapid K-Ras engagement, rapid p-ERK suppression, and significant Loewe synergy scores in viability assays. These findings establish GAP mimetics as rational and potent combination partners for Switch-II pocket inhibitors. The synergistic combination has potential to deepen and prolong pathway suppression while enabling dose reductions that may mitigate on-target toxicity and resistance.
Adherens junctions are cellular contact sites that organize epithelial tissues and play well-characterized roles in the coordination of cell collectives. Intercellular contacts are mediated by cadherin- or nectin-based adhesion and intracellularly linked to the actin cytoskeleton via the molecular scaffolds catenin and afadin. In this review, we discuss the mechanisms and roles of these molecular scaffolds for cellular morphogenesis and collective cell behaviour with a focus on neural tissue patterning. We discuss the molecular mechanisms in the conceptual framework of two often opposing, but complementary demands on adherens junctions in developing neural tissues: stability through 'supracellular' cytoskeletal linkage across cells versus local, dynamically adhesive cellular interactions in morphogenesis. Molecular scaffolds mediate localization, mechanosensitive adhesion and the regulation of cytoskeleton tension in both mechanistic contexts. These complementary mechanisms allow for collective behaviour that has predominantly been characterized for the patterning of tissues consisting of cell bodies, but was recently shown to also underlie the patterning of an epithelial-like tissue made entirely of neuronal growth cones. Molecular scaffolding of adherens junctions thereby contributes to patterning mechanisms that may apply to diverse tissue types.
Alfred Wittinghofer and his group of structural biologists and biochemists laid the groundwork for developing drugs that target RAS, from the first structure of a RAS protein in its active state, through to a deep understanding of RAS function and regulation. Their fundamental discoveries include the mechanisms of hydrolysis and nucleotide exchange, effector engagement and subcellular localization. In this review, I share some of the highlights of years of a friendship and collaboration for the many years during which Fred's group led the field and helped understand and defeat the "beating heart of signal transduction".
The serine protease KLK7 contributes to several skin disorders and tumorigenesis, making it an attractive drug target. Owing to structural similarities in the S1 binding pocket between human KLK7 and certain trypsin-like serine proteases harboring an Ala at position 190, compounds containing a chlorine-substituted benzylamide as P1 group were screened for KLK7 inhibition. Further optimization yielded the substrate analog inhibitor Bzls-d-hTyr-Pro-2-aminomethyl-5-chloro-benzylamide (K i value 29.3 nM), which, however, had insufficient selectivity against the tested clotting proteases. Guided by a published KLK7 crystal structure, a second, more selective non-peptide inhibitor series was synthesized. The best derivatives (K i values <100 nM) contain a chlorine-substituted aromatic P1 group and additionally address the non-prime region of KLK7. Murine Klk7 is poorly inhibited by these compounds; however, mutating threonine 190 in the S1 pocket to alanine restored the inhibitory potency. The new inhibitors effectively block KLK7-mediated functions, including chemokine cleavage and moesin gene upregulation, without cytotoxicity towards murine ovarian cancer cells. Moreover, tumors derived from ovarian cancer cells overexpressing either wild-type or mutated Klk7 reduced overall survival in mice compared to vector control cells. Together, these findings establish a robust inhibitor-enzyme system to evaluate human KLK7 inhibitors in a preclinical mouse model.
Recent studies have identified Aβ peptides in human gut epithelial cells, along with several amyloid-forming proteins and peptides in the gut lumen. These findings suggest that Aβ or other amyloid-like molecules originating from the gut may contribute to the involvement of the gastrointestinal system in the development of Alzheimer's disease (AD) pathology. Modulating the aggregation behaviour of Aβ and other amyloid forming peptides/proteins present in the gut may represent novel strategy to mitigate AD pathology. This study explores the use of Milk-derived Amyloid-like Protein Aggregates (MAPA) to inhibit Aβ(40) aggregation in vitro. MAPA's inhibitory effects were assessed using amyloid dye-binding assays (Thioflavin T, Congo Red, and ANS) and transmission electron microscopy. Toxicity assays showed that the MAPA significantly reduced Aβ(40)-induced neuronal death. Fluorescence quenching suggest MAPA physically interacts with Aβ(40) to prevent its aggregation. By blocking Aβ aggregation and reducing its neurotoxicity, MAPA presents a promising organic strategy to counteract AD progression influenced by gut factors. These findings open new avenues for AD prevention and the disease management, especially via dietary interventions targeting the gastro-intestinal system.
More than a century after Alois Alzheimer's neuropathological description, the mechanisms driving Alzheimer's disease (AD) remain only partially understood, and the failure of most clinical trials underscores the need to identify and target alternative pathogenic pathways. Recent genetic, biochemical, and cellular studies support the view that AD is characterized by early alterations in the endolysosomal system and implicate multiple endocytic scaffold proteins as key drivers of AD progression. In this review, we summarize the current knowledge of five endocytic scaffold proteins, CALM, AP-2, BIN1, CD2AP, and ITSN1, which have been identified as AD risk factors by genome-wide association studies. We describe how, under physiological conditions, they couple membrane remodeling to intracellular signaling, whereas in AD they influence amyloid precursor protein trafficking, amyloid-β (Aβ) generation, tau pathology, and synaptic integrity. Finally, we propose a model in which cell type-specific and age-dependent dysfunction of endocytic scaffolds defines a pathogenic hotspot of proteostasis failure and offers new entry points for therapeutic intervention.
