Poly(ADP-ribosyl)ation (PARylation) is a transient post-translational modification catalyzed by PARP enzymes and reversed by PARG. PARG inhibition causes sustained PARylation and is being explored as an anticancer strate...Poly(ADP-ribosyl)ation (PARylation) is a transient post-translational modification catalyzed by PARP enzymes and reversed by PARG. PARG inhibition causes sustained PARylation and is being explored as an anticancer strategy, but its cellular consequences remain incompletely understood. Here, we examine how persistent PARylation influences cellular responses to replication stress and DNA damage. We show that sustained PARylation reduces phosphorylated and chromatin-bound RPA most strongly under fork-stalling conditions that progress toward fork collapse. This effect requires PARP1 activity and is restrained by intact ATR-CHK1 signaling, as checkpoint inhibition renders otherwise resistant cells permissive for PARG inhibitor-associated phosphorylated RPA loss from the chromatin. The reduction of RPA phosphorylation is not dependent on BRCA1 and it is not accompanied by increased RAD51 loading. Instead, reduced chromatin-bound RPA coincides with decreased exposed ssDNA. Our results identify a checkpoint-dependent fork-collapse state in which sustained PARylation limits ssDNA and RPA levels.
GATOR1 is an evolutionarily-conserved negative regulator of mTORC1-dependent signal transduction with pathogenic mutations linked to epilepsy, infantile spasms, and autism spectrum disorders. While a biochemical role of...GATOR1 is an evolutionarily-conserved negative regulator of mTORC1-dependent signal transduction with pathogenic mutations linked to epilepsy, infantile spasms, and autism spectrum disorders. While a biochemical role of GATOR1 in amino acid-signaling is established, its cell-type specific contributions within the brain remain poorly defined. Here, we show that loss of GATOR1 function in astrocytic cells disrupts mitochondrial metabolism, with a selective dysfunction of the electron transport chain Complex II leading to elevated reactive oxygen species (ROS) and redox imbalance. These changes are accompanied by compensatory increases in antioxidant regulatory systems including superoxide dismutase, but remain insufficient to ameliorate the increased ROS. GATOR1-deficient astrocytes show metabolic rewiring marked by enhanced expression of glutamate uptake and glutamine synthesis pathways that contribute to the glutamate-glutamine cycle governing neuronal glutamine availability and synaptic homeostasis. In vivo, GATOR1 deficiency results in progressive astrocytic reactivity, seizures, and a reduced lifespan. These findings demonstrate that GATOR1 function is critical to coordinate astrocytic mitochondrial activity and neurotransmitter cycling pathways, establishing a novel link between intracellular amino acid-signaling in astrocytes and excitatory neural network homeostasis.
Cevallos J, Eubanks E, Jung S
… +25 more, Huang Y, Guadagno E, Jaber N, Xia Y, Wang J, Wang H, Suvvari NR, Doshi A, Ravinutala N, Mosera A, Hsu T, Mody J, Sacks B, Narayan A, Smith B, Wang M, Gottapu M, Alnakhala H, Ramalingam N, Tripathi A, Bartels T, Dettmer U, Shi Z, Dai W, Kara E
Alpha-synuclein (αSyn) inclusions are a defining neuropathological feature of Parkinson's disease, but the cellular events that initiate their formation and promote neurotoxicity remain incompletely understood. Aberrant...Alpha-synuclein (αSyn) inclusions are a defining neuropathological feature of Parkinson's disease, but the cellular events that initiate their formation and promote neurotoxicity remain incompletely understood. Aberrant liquid-liquid phase separation has emerged as a potential early step in αSyn dysregulation, yet the physiological triggers and functional consequences of this process are unclear. Here, we show that lipid droplets promote the spontaneous phase separation of wild-type and E46K mutant αSyn into condensates. These condensates sequester lipid droplets and impair their turnover, indicating disruption of cellular lipid homeostasis. Mitochondria in close proximity to αSyn condensates exhibit reduced membrane potential and increased mitophagy. Correlative light and electron microscopy further reveals αSyn oligomers associated with mitochondrial membranes displaying structural abnormalities. Together, these findings identify lipid droplets as drivers of aberrant αSyn phase separation and suggest that lipid droplet-rich condensates contribute to mitochondrial dysfunction and impaired energy homeostasis. Given the enrichment of lipid droplets within neuromelanin-containing dopaminergic neurons of the substantia nigra, this mechanism may be relevant to the selective neuronal vulnerability observed in Parkinson's disease.
Müller J, Daunke T, Berner N
… +18 more, Bhandare P, Gebhardt A, Hoffmann L, Diebold M, Vogt M, Hofstetter J, Cruz-Garcia Y, Dudvarski-Stankovic N, Schneider K, Adhikari B, Lamer S, Schlosser A, Büchel G, Sos ML, Küster B, Knapp S, Wilhelm S, Wolf E
The oncogenic kinase AURORA A is essential for mitotic progression, and its catalytic inhibition arrests cells at the G2/M-transition. Unexpectedly, degradation of AURORA A by PROTACs (proteolysis targeting chimeras) ind...The oncogenic kinase AURORA A is essential for mitotic progression, and its catalytic inhibition arrests cells at the G2/M-transition. Unexpectedly, degradation of AURORA A by PROTACs (proteolysis targeting chimeras) induces profound S-phase defects, revealing a non-catalytic scaffolding function of AURORA A. To dissect this function, we profile the AURORA A S-phase interactome and identify multiple RNA-binding proteins not characterized as AURORA A substrates. Among these, the ribonuclease DICER directly associates with AURORA A to form an abundant nuclear complex. RNA degradation shifts AURORA A, DICER, and additional RNA-binding proteins from heavy to light gradient fractions, implicating RNA-dependent complex function. In contrast, PROTAC-mediated depletion of AURORA A alters the gradient migration behavior and chromatin association of the histone methyltransferase SETD2, which is known to prevent spurious transcription. These findings reveal a dual-output model for the S-phase AURORA A complex: First, RNA-binding proteins are recruited to R-loops, which may arise from transcription-replication conflicts. DICER then processes the R-loop, while AURORA A simultaneously recruits SETD2, which facilitates efficient resolution of replicative stress by preventing spurious transcription.
Human outbreaks of West Nile virus (WNV) are an imminent threat in North America, with many annual infections and numerous cases of severe neuroinvasive disease. There are no licensed treatments for WNV disease. Previous...Human outbreaks of West Nile virus (WNV) are an imminent threat in North America, with many annual infections and numerous cases of severe neuroinvasive disease. There are no licensed treatments for WNV disease. Previous research identified WNV-86 as an ultrapotent neutralizing human antibody that binds domain II of the major envelope (E) glycoprotein in mature virions. Here, we report the structure of mature WNV in complex with the Fab of WNV-86, at a resolution of 3.8 Å, solved using cryogenic electron microscopy. Structure-based epitope mapping identifies a new class of E dimer epitope (EDE) antibodies that we designate as EDE3 antibodies. A partial overlap of WNV-86 and pre-membrane protein (prM) binding regions at more than one site ensures selective binding of WNV-86 to mature virions. The structure reveals the quaternary epitope of the neutralizing Fab and supports a model in which engaging both protomers of the dimer likely interferes with fusion-triggering rearrangements. This study identifies critical residues for binding, neutralization, and immune escape and clarifies the promise of this molecule for future immunotherapeutic interventions.
NLRP10 is an atypical member of the NLR family because it lacks a leucine-rich repeat domain at its C-terminus. Here, we show that in human epithelial cells and keratinocytes NLRP10 oligomerises in response to m-3M3FBS a...NLRP10 is an atypical member of the NLR family because it lacks a leucine-rich repeat domain at its C-terminus. Here, we show that in human epithelial cells and keratinocytes NLRP10 oligomerises in response to m-3M3FBS and SC-10 treatment. NLRP10 co-localises with ASC upon overexpression, but ASC nucleation and recruitment are different to NLRP3. While neither ATP hydrolysis nor the pyrin domain is required, the C-terminal tail region is both necessary and sufficient for oligomerisation. The generation of chimeric proteins shows that the tail region of human and mouse NLRP10 has a conserved function in oligomerisation but determines different protein stabilities. Changes in the subcellular localisation of NLRP10 and oligomerisation are dependent on the presence of evolutionarily conserved lysine residues in the tail region, which localise the majority of NLRP10 to lipid droplets. Our study identifies the C-terminal basic tail of NLRP10 as a key regulatory element for oligomerisation and localisation at lipid interfaces. These findings underline differences in NLRP10 activation with respect to other inflammasome-forming NLRPs and suggest a role of lipids in NLRP10 activation.
Sahnine M, Crochet M, Tachon S
… +16 more, Goussard S, Pénard E, Scandola C, Duchateau M, Giai Gianetto Q, Salles A, Moneron G, Swistak L, Charles-Orszag A, Mallet A, Matondo M, Tinevez JY, Bonazzi D, Sartori-Rupp A, Duménil G, Obino D
Cells experience external forces that deform the plasma membrane to which they adapt by reorganizing their actin cytoskeleton. Here, using the extracellular bacterium Neisseria meningitidis as a model system, we explore...Cells experience external forces that deform the plasma membrane to which they adapt by reorganizing their actin cytoskeleton. Here, using the extracellular bacterium Neisseria meningitidis as a model system, we explore how this bacterium reorganizes the cortical actin cytoskeleton subsequently to mechanical membrane deformations. Meningococci trigger the formation of tubular cellular plasma membrane protrusions by a previously described adhesion-driven process known as one-dimensional wetting. Cryo-electron tomography reveals that in epithelial cells such a deformation of the plasma membrane leads to the formation of F-actin bundles. In contrast, in endothelial cells a branched F-actin network is formed. By combining high resolution photonic microscopy approaches with genetic and drug perturbations in endothelial cells, we demonstrate that Arp2/3 activity is necessary for forming this branched network. We demonstrate the role of the nucleating-promoting factor N-WASP downstream of Cdc42. Proteomic analyses reveal the contribution of the small GTPase Arf1. Taken together, our results delineate an Arf1-Cdc42-N-WASP-Arp2/3 pathway that links mechanical plasma membrane deformation to the subsequent reorganization of a cortical branched F-actin network in endothelial cells.
Cells frequently undergo spontaneous morphogenesis, yet the underlying mechanisms remain incompletely understood. While actin filaments are central to cell morphogenesis and are typically regulated by biochemical signali...Cells frequently undergo spontaneous morphogenesis, yet the underlying mechanisms remain incompletely understood. While actin filaments are central to cell morphogenesis and are typically regulated by biochemical signaling, cells can form protrusions even without clear external cues, suggesting the existence of intrinsic mechanisms. Here, we report that actin filament assemblies undergo directional movement driven by their directional polymerization and disassembly. These filament assemblies move as discrete "particles" and exhibit random yet directional motion. Since this motion resembles that of self-propelled "particles" rather than the previously reported reaction-diffusion "waves", we have termed them Self-propelled Treadmilling Actin filaments (SpTAs). SpTA arrival at the cell periphery drives membrane protrusion by orienting their polymerizing ends outwards. Furthermore, SpTAs spontaneously accumulate at cell protrusions, guided by membrane curvature. This SpTA accumulation, further boosts the growth and expansion of protrusions, driving cellular polarization for migration. Our findings establish that the assembly of actin filaments as a novel class of biological active particle, and provides new insight into how molecular-scale motion orchestrates complex higher-order organization.
A major goal in epitranscriptomics is to define the diversity of modified nucleotides in mRNA. Most candidate sites are discovered using transcriptome-wide mapping methods that rely on antibody enrichment or modification...A major goal in epitranscriptomics is to define the diversity of modified nucleotides in mRNA. Most candidate sites are discovered using transcriptome-wide mapping methods that rely on antibody enrichment or modification-induced misincorporations during cDNA synthesis. However, these maps are rarely verified biochemically, and in some cases have yielded conflicting conclusions, most notably for N-methyladenosine (mA). Initial transcriptome-wide maps reported thousands of internal mA sites while later studies disputed these findings and showed that these sites lacked the expected mA-induced misincorporations. More recently, an evolved reverse transcriptase that detects mA was developed to resolve this issue and was used to map sites that again suggested that internal mA might be widespread. These divergent results underscore the need for biochemical testing of proposed sites. Here, we describe protocols for SCARPET (site-specific cleavage and radiolabeling followed by purification, exonuclease digestion, and thin-layer chromatography) to rapidly and efficiently detect modified nucleotides in RNA. Using SCARPET, we show that newly mapped internal mA sites are not mA, but instead contain inosine from A-to-I editing. However, N⁷-methylguanosine (mG) sites, which are also controversial, were confirmed to occur at high stoichiometry. These results confirm that view that mA is extremely rare and establish SCARPET as a robust strategy for validating putative modification sites.
Asymmetric division is crucial for oocyte and embryo development. During meiosis I, the spindle migrates to the subcortex, and F-actin accumulates to form the actin cap, which supports polar body extrusion. While the CDC...Asymmetric division is crucial for oocyte and embryo development. During meiosis I, the spindle migrates to the subcortex, and F-actin accumulates to form the actin cap, which supports polar body extrusion. While the CDC42-NWASP-ARP2/3 pathway, regulated by Ran-GTP, is known to promote actin cap formation, how Ran-GTP controls cortical polarization and the precise function of the actin cap remain unclear. Here, we identify Depdc7 as a novel actin cap-localized protein essential for actin cap formation and first polar body extrusion. DEPDC7 is recruited to the oocyte membrane by Ran-GTP and acts as a scaffold to promote cortical actin assembly. Strikingly, inhibiting CDK1 activity partially rescues the extrusion defect in Depdc7-depleted oocytes. These findings indicate that both CDK1 activity and actin cap formation are essential for polar body extrusion and function synergistically. Our study refines the molecular mechanism of actin cap assembly and highlights DEPDC7 as a key regulator of oocyte maturation and female fertility.
Brown adipose tissue (BAT) counteracts obesity-related metabolic dysfunction through both thermogenic and non-thermogenic means. However, substantial evidence indicates that obesity negatively affects BAT mitochondrial m...Brown adipose tissue (BAT) counteracts obesity-related metabolic dysfunction through both thermogenic and non-thermogenic means. However, substantial evidence indicates that obesity negatively affects BAT mitochondrial morphology and oxidative capacity, impairing systemic energy homeostasis. Motivated by this apparent contradiction, we investigate the relationship between obesity and mitochondrial dynamics, as the underlying mechanisms remain incompletely understood. Here, we identify E4BP4 as a transcriptional repressor that prevents obesity-induced mitochondrial fragmentation and oxidative dysfunction by inhibiting ceramide synthesis in brown fat. Specifically, E4BP4 interacts with PRDM16 to repress Cers6 mRNA expression and consequently reduces C16:0 ceramide levels by binding to a 65 kb upstream enhancer region of the Cers6 gene. Notably, the preservation of mitochondrial integrity in BAT by E4BP4 gain-of-function improves systemic glucose homeostasis, independent of weight loss. Collectively, our findings establish E4BP4 as a molecular safeguard against obesity-induced mitochondrial fragmentation and oxidative dysfunction, primarily by suppressing ceramide synthesis in brown fat.
Patterning of mammalian endoderm into lung and thyroid lineages depends upon a correct early expression of a homeobox domain-containing transcription factor, Nkx2-1. However, the gene networks distinguishing the differen...Patterning of mammalian endoderm into lung and thyroid lineages depends upon a correct early expression of a homeobox domain-containing transcription factor, Nkx2-1. However, the gene networks distinguishing the differentiation of those lineages remain largely unknown. In this work, by using mouse stem cell lines, scRNA-seq, and transcriptomic and chromatin accessibility profiling, we show that Foxe1 knockout impairs Nkx2-1+ cell differentiation and maturation into thyroid follicular-like cells. Concomitantly, a subset of Foxe1 null/Nkx2-1+ cells follows a lung epithelial differentiation program and form lung-like organoids harboring cells transcriptionally similar to mouse fetal lung types. Chromatin analyses reveal that, while accessibility at the Pax8 locus is reduced, loci associated with lung programs are in an open configuration, indicating that lung fate can be adopted without additional chromatin remodeling. These findings demonstrate that Foxe1 loss destabilizes thyroid commitment but also creates a permissive state in which Nkx2-1+ foregut progenitors can adopt an alternative lung fate. Our study illustrates how the interplay between transcription factors and chromatin context governs lineage decisions in vitro and provides a platform to investigate mechanisms underlying organ specification and plasticity.
Polycomb group (PcG) genes are epigenetic silencers that maintain transcriptional repression of target genes essential for normal development. However, their roles in adult multipotent stem cell lineages remain poorly un...Polycomb group (PcG) genes are epigenetic silencers that maintain transcriptional repression of target genes essential for normal development. However, their roles in adult multipotent stem cell lineages remain poorly understood. Here, we show that simultaneous loss of the PRC1 component Psc and its homolog Su(z)2 in intestinal stem cells (ISCs) of the adult Drosophila midgut leads to tumor formation composed of proliferative, undifferentiated cells. Strikingly, these tumor cells do not activate proliferation-associated pathways, including JAK/STAT, Ras/MAPK, and Wnt, nor do they activate Notch or JAK/STAT signaling, which are essential for ISC differentiation. Transcriptomic and chromatin accessibility profiling reveal widespread downregulation of ISC and progenitor cell identity genes and ectopic activation of neural lineage genes. Among these, chinmo is aberrantly upregulated and required for tumor overgrowth. Notably, loss of other PRC1 components does not recapitulate the tumor phenotype, suggesting that the tumor-suppressive role of Psc and Su(z)2 is independent of canonical PRC1 function. Together, our findings uncover a noncanonical, context-specific tumor-suppressive role for Psc and Su(z)2 in preserving ISC identity and restricting lineage deviation.
Proteases are enzymes that catalyze the hydrolysis of peptide bonds in proteins for their functional modification or degradation. Members of the Dipeptidyl Peptidase IV (DPPIV) family are exopeptidases that cleave dipept...Proteases are enzymes that catalyze the hydrolysis of peptide bonds in proteins for their functional modification or degradation. Members of the Dipeptidyl Peptidase IV (DPPIV) family are exopeptidases that cleave dipeptides off the N-termini of their substrate peptides, typically after proline or alanine. Recently, we showed that human DPP4 and Caenorhabditis elegans DPF-3 have a larger target repertoire in vitro, permitting cleavage after additional amino acids. Here, we use terminal amine isotopic labeling of substrates (TAILS) to identify DPF-3 targets in vivo and observe cleavage of MEP-1 after threonine, confirming a broader substrate specificity of DPF-3 also in vivo. Demonstrating physiological relevance, we show that rendering MEP-1 resistant to cleavage disrupts its stability, leading to developmental abnormalities such as defective gonadal migration and reproductive issues. Collectively, our findings highlight a previously unappreciated complexity in the substrate specificity of DPPIV family proteases and suggest that their physiological roles may extend beyond what is currently known.
Muscle fibrosis is a major driver of progression in diverse myopathies, yet the conserved molecular mediators of this process in humans remain poorly defined. Here, we identify collagen VI as a common regeneration-impair...Muscle fibrosis is a major driver of progression in diverse myopathies, yet the conserved molecular mediators of this process in humans remain poorly defined. Here, we identify collagen VI as a common regeneration-impairing extracellular matrix (ECM) component across three distinct human myopathies: Duchenne Muscular Dystrophy (DMD), Oculopharyngeal Muscular Dystrophy (OPMD), and Inclusion Body Myositis (IBM). Proteomic profiling of fibrotic biopsies reveals consistent upregulation of collagen VI and laminin γ1, alongside disease-specific alterations. Fibroadipogenic progenitors (FAPs) are the predominant source of these ECM components, including collagen VI and laminin γ1. Functionally, xenotransplantation of patient-derived FAPs into regenerating mouse muscle induces localized collagen deposition, myofiber atrophy, and depletion of Pax7⁺ muscle stem cells. Mechanistic assays demonstrate that FAP-derived collagen VI is sufficient to impair myogenic fusion, while silencing COL6 in patient FAPs restores fusion capacity, directly linking pathological collagen VI deposition to regeneration failure. Our findings uncover collagen VI as a conserved effector of fibrosis and stem cell niche disruption in human myopathies, positioning it as a potential therapeutic target across genetically and clinically distinct muscle diseases.