bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Glycolytic Metabolite Methylglyoxal Covalently Inactivates the NLRP3 Inflammasome
Authors: Stanton Caroline , Buasakdi Chavin , Sun Jie , Levitan Ian , Bora Prerona , Kutseikin Sergei , Wiseman R. Luke , Bollong Michael J. 
Published: April 2024

SummaryThe NLRP3 inflammasome promotes inflammation in disease, yet the full repertoire of mechanisms regulating its activity are not well delineated. Among established regulatory mechanisms, covalent modification of NLRP3 has emerged as a common route for pharmacological inactivation of this protein. Here, we show that inhibition of the glycolytic enzyme PGK1 results in the accumulation of methylglyoxal, a reactive metabolite whose increased levels decrease NLRP3 assembly and inflammatory signaling in cells. We find that methylglyoxal inactivates NLRP3 via a non-enzymatic, covalent crosslinking-based mechanism, promoting inter- and intra-protein MICA posttranslational linkages within NLRP3. This work establishes NLRP3 as capable of sensing a host of electrophilic chemicals, both exogenous small molecules and endogenous reactive metabolites, and suggests a mechanism by which glycolytic flux can moderate the activation status of a central inflammatory signaling pathway.
Multiplexed, multimodal profiling of the intracellular activity, interactions, and druggability of protein variants using LABEL-seq
Authors: Simon Jessica J. , Fowler Douglas M. , Maly Dustin J. 
Published: April 2024

AbstractMultiplexed assays of variant effect are powerful tools for assessing the impact of protein sequence variation, but are limited to measuring a single protein property and often rely on indirect readouts of intracellular protein function. Here, we developed LAbeling with Barcodes and Enrichment for biochemicaL analysis by sequencing (LABEL-seq), a platform for the multimodal profiling of thousands of protein variants in cultured human cells. Multimodal measurement of ∼20,000 variant effects for ∼1,600 BRaf variants using LABEL-seq revealed that variation at positions that are frequently mutated in cancer had minimal effects on folding and intracellular abundance but could dramatically alter activity, protein-protein interactions, and druggability. Integrative analysis of our multimodal measurements identified networks of positions with similar roles in regulating BRaf’s signaling properties and enabled predictive modeling of variant effects on complex processes such as cell proliferation and small molecule-promoted degradation. LABEL-seq provides a scalable approach for the direct measurement of multiple biochemical effects of protein variants in their native cellular context, yielding insight into protein function, disease mechanisms, and druggability.
Cross-family transfer of the Arabidopsis cell-surface immune receptor LORE to tomato confers sensing of 3-hydroxylated fatty acids and enhanced disease resistance
Authors: Eschrig Sabine , Kahlon Parvinderdeep S. , Agius Carlos , Holzer Andrea , Hückelhoven Ralph , Schwechheimer Claus , Ranf Stefanie 
Published: April 2024

ABSTRACTPlant pathogens pose a high risk of yield losses and threaten food security. Technological and scientific advances have improved our understanding of the molecular processes underlying host-pathogen interactions, which paves the way for new strategies in crop disease management beyond the limits of conventional breeding. Cross-family transfer of immune receptor genes is one such strategy that takes advantage of common plant immune signaling pathways to improve disease resistance in crops. Sensing of microbe- or host damage-associated molecular patterns (MAMPs/DAMPs) by plasma membrane-resident pattern recognition receptors (PRR) activates pattern-triggered immunity (PTI) and restricts the spread of a broad spectrum of pathogens in the host plant. In the model plantArabidopsis thaliana, the S-domain receptor-like kinase LIPOOLIGOSACCHARIDE-SPECIFIC REDUCED ELICITATION (AtLORE, SD1-29) functions as PRR, which senses medium chain-length 3-hydroxylated fatty acids (mc-3-OH-FAs), such as 3-OH-C10:0, and 3-hydroxyalkanoates (HAAs) of microbial origin to activate PTI. In this study, we show that ectopic expression of the Brassicaceae-specific PRRAtLORE in the solanaceous crop speciesSolanum lycopersicumcv. M82 leads to the gain of 3-OH-C10:0 immune sensing without altering plant development.AtLORE-transgenic tomato shows enhanced resistance againstPseudomonas syringaepv.tomatoDC3000 andAlternaria solaniNL03003. Applying 3-OH-C10:0 to the soil before infection induces resistance against the oomycete pathogenPhytophthora infestansPi100 and further enhances resistance toA. solaniNL03003. Our study proposes a potential application ofAtLORE-transgenic crop plants and mc-3-OH-FAs as resistance-inducing bio-stimulants in disease management.
Glycogen phase separation drives macromolecular rearrangement and asymmetric division inE. coli
Authors: Thappeta Yashna , Cañas-Duarte Silvia J. , Kallem Till , Fragasso Alessio , Xiang Yingjie , Gray William , Lee Cheyenne , Cegelski Lynette , Jacobs-Wagner Christine 
Published: April 2024

ABSTRACTBacteria often experience nutrient limitation in nature and the laboratory. While exponential and stationary growth phases are well characterized in the model bacteriumEscherichia coli, little is known about what transpires inside individual cells during the transition between these two phases. Through quantitative cell imaging, we found that the position of nucleoids and cell division sites becomes increasingly asymmetric during transition phase. These asymmetries were coupled with spatial reorganization of proteins, ribosomes, and RNAs to nucleoid-centric localizations. Results from live-cell imaging experiments, complemented with genetic and13C whole-cell nuclear magnetic resonance spectroscopy studies, show that preferential accumulation of the storage polymer glycogen at the old cell pole leads to the observed rearrangements and asymmetric divisions. In vitro experiments suggest that these phenotypes are likely due to the propensity of glycogen to phase separate in crowded environments, as glycogen condensates exclude fluorescent proteins under physiological crowding conditions. Glycogen-associated differences in cell sizes between strains and future daughter cells suggest that glycogen phase separation allows cells to store large glucose reserves without counting them as cytoplasmic space.
Transcriptomic Profiling of Thyroid Eye Disease Orbital Fat Demonstrates Differences in Adipogenicity and IGF-1R Pathway
Authors: Kim Dong Won , Kim Soohyun , Han Jeong , Belday Karan , Li Emily , Mahoney Nicholas , Blackshaw Seth , Rajaii Fatemeh 
Published: April 2024

AbstractDespite recent advances in the treatment of thyroid eye disease (TED), significant gaps remain in our understanding of the underlying molecular mechanisms, particularly concerning the insulin-like growth factor-1 receptor (IGF-1R) pathway. To dissect the pathophysiology of TED, we utilized single nucleus RNA-Seq to analyze orbital fat specimens from both TED patients and matched controls. The analysis demonstrated a marked increase in the proportion of fibroblasts transitioning to adipogenesis in the orbital fat of TED patients compared to controls. This was associated with diverse alterations in immune cell composition. Significant alterations in the IGF-1R signaling pathway were noted between TED specimens and controls, indicating a potential pathological mechanism driven by IGF-1R signaling abnormalities. Additionally, our data show that linsitinib, a small molecule inhibitor of IGF-1R, effectively reduces adipogenesis in TED orbital fibroblastsin vitro, suggesting its potential utility as a therapeutic agent. Our findings reveal that beyond immune dysfunction, abnormal IGF-1R signaling leading to enhanced adipogenesis is a crucial pathogenic mechanism in TED.
VezA/vezatin facilitates proper assembly of the dynactin complex in vivo
Authors: Zhang Jun , Qiu Rongde , Xie Sean , Rasmussen Megan , Xiang Xin 
Published: April 2024

AbstractCytoplasmic dynein-mediated intracellular transport needs the multi-component dynactin complex for cargo binding and motor activation. However, cellular factors involved in dynactin assembly remain unexplored. Here we found inAspergillus nidulansthat the vezatin homolog VezA is important for dynactin assembly. VezA affects the microtubule plus-end accumulation of dynein before cargo binding and cargo adapter-mediated dynein activation, two processes that both need dynactin. The dynactin complex contains multiple components including an Arp1 (actin-related protein 1) mini-filament associated with a pointed-end sub-complex. VezA physically interacts with dynactin either directly or indirectly via the Arp1 mini-filament and its pointed-end sub-complex. Loss of VezA causes a defect in dynactin integrity, most likely by affecting the connection between the Arp1 mini-filament and its pointed-end sub-complex. Using various dynactin mutants, we further revealed that assembly of the dynactin complex must be highly coordinated. Together, these results shed important new light on dynactin assembly in vivo.
A novel iPSC-based model of ICF syndrome subtype 2 recapitulates the molecular phenotype of ZBTB24 deficiency
Authors: Lullo Vincenzo , Cecere Francesco , Batti Saveria , Allegretti Sara , Morone Barbara , Fioriniello Salvatore , Pisapia Laura , Genesio Rita , Ragione Floriana Della , Giardino Giuliana , Pignata Claudio , Riccio Andrea , Matarazzo Maria R. , Strazzullo Maria 
Published: April 2024

AbstractImmunodeficiency, Centromeric instability and Facial anomalies (ICF) syndrome is a rare genetic disorder characterized by variable immunodeficiency. More than half of the affected individuals show mild to severe intellectual disability at early onset. This disorder is genetically heterogeneous andZBTB24is the causative gene of the subtype 2, accounting for about 30% of the ICF cases. ZBTB24 is a multifaceted transcription factor belonging to the Zinc-finger and BTB domain-containing protein family, which are key regulators of developmental processes. Aberrant DNA methylation is the main molecular hallmark of ICF syndrome. The functional link between ZBTB24 deficiency and DNA methylation errors is still elusive. Here, we generated a novel ICF2 disease model by deriving induced pluripotent stem cells (iPSCs) from peripheral CD34+-blood cells of a patient homozygous for the p.Cys408Gly mutation, the most frequent missense mutation in ICF2 patients and which is associated with a broad clinical spectrum. The mutation affects a conserved cysteine of the ZBTB24 zinc-finger domain, perturbing its function as transcriptional activator.ICF2-iPSCs recapitulate the methylation defects associated with ZBTB24 deficiency, including centromeric hypomethylation. We validated that the mutated ZBTB24 protein loses its ability to directly activate expression ofCDCA7and other target genes in the patient-derived iPSCs. Upon hematopoietic differentiation, ICF2-iPSCs showed decreased vitality and a lower percentage of CD34+/CD43+/CD45+progenitors. Overall, the ICF2-iPSC model is highly relevant to explore the role of ZBTB24 in DNA methylation homeostasis and provides a tool to investigate the early molecular events linking ZBTB24 deficiency to the ICF2 clinical phenotype.
BNST GluN2D-containing NMDARs contribute to ethanol intake but not negative affective behaviors in female mice
Authors: Doyle Marie A. , Salimando Gregory J. , Altemus Megan E. , Badt Justin K. , Bedenbaugh Michelle N. , Vardy Alexander S. , Adank Danielle N. , Park Anika S. , Winder Danny G. 
Published: April 2024

AbstractAlcohol use disorder (AUD) is a chronic, relapsing disease, highly comorbid with anxiety and depression. The bed nucleus of the stria terminalis (BNST), andCrh+ neurons in this region are thought to play a key role in chronic ethanol-induced increases in volitional ethanol intake. This role has been hypothesized to be driven by emergent BNST-dependent negative affective behaviors. Indeed, we report here that in female mice undergoing a home cage chronic drinking forced abstinence model (CDFA), excitatory transmission undergoes time-dependent upregulation in BNSTCrh+ cells. Excitatory NMDA receptors (NMDARs) are a major target of ethanol, and chronic ethanol exposure has been shown to regulate NMDAR function and expression. GluN2D subunit-containing NMDARs have emerged as a target of interest due to their limited distribution and potential roles in affective behavior. We find that knockdown of dorsal BNST (dBNST) GluN2D expression significantly decreases ethanol intake in female, but not male, mice. While BNSTGrin2bexpression was significantly increased in protracted abstinence following CDFA, no differences inGrin2dexpression were observed in dBNST or specifically in dBNSTCrh+ neurons. Finally, to determine the impact of GluN2D expression on negative affective behaviors, open field, elevated zero maze, and forced swim tasks were used to measure anxiety- and depressive-like behaviors in constitutive and conditional BNST GluN2D knockout mice. Surprisingly, we find that deletion of GluN2D fails to alter negative affect in ethanol-naïve female mice. Together, these data suggest a role for BNST GluN2D-containing NMDARs in ethanol drinking behaviors but not abstinence from ethanol, highlighting potential sex differences and behavioral specificity in the context of AUD behaviors. Overall, these data further suggest roles for BNST synaptic signaling in volitional ethanol intake that are partially independent of actions on affective behavior.
N-terminal tags impair the ability of Lamin A to provide structural support to the nucleus
Authors: Odell Jacob , Lammerding Jan 
Published: April 2024

AbstractLamins are intermediate filament proteins that contribute to numerous cellular functions, including nuclear morphology and mechanical stability. The N-terminal head domain of lamin is critical for higher order filament assembly and function, yet the effects of commonly used N-terminal tags on lamin function remain largely unexplored. Here, we systematically studied the effect of two differently sized tags on Lamin A (LaA) function in a mammalian cell model engineered to allow for precise control of expression of tagged lamin proteins. Untagged, FLAG-tagged, and GFP-tagged LaA completely rescued nuclear shape defects when expressed at similar levels in lamin A/C-deficient (Lmna−/−) MEFs, and all LaA constructs prevented increased nuclear envelope (NE) ruptures in these cells. N-terminal tags, however, altered the nuclear localization of LaA and impaired the ability of LaA to restore nuclear deformability and to recruit Emerin to the nuclear membrane inLmna−/−MEFs. Our finding that tags impede some LaA functions but not others may explain the partial loss of function phenotypes when tagged lamins are expressed in model organisms and should caution researchers using tagged lamins to study the nucleus.
Spatiotemporal proteomic profiling of cellular responses to NLRP3 agonists
Authors: Hollingsworth L. Robert , Veeraraghavan Priya , Paulo Joao A. , Harper J. Wade 
Published: April 2024

ABSTRACTNucleotide-binding domain and leucine-rich repeat pyrin-domain containing protein 3 (NLRP3) is an innate immune sensor that forms an inflammasome in response to various cellular stressors. Gain-of-function mutations in NLRP3 cause autoinflammatory diseases and NLRP3 signalling itself exacerbates the pathogenesis of many other human diseases. Despite considerable therapeutic interest, the primary drivers of NLRP3 activation remain controversial due to the diverse array of signals that are integrated through NLRP3. Here, we mapped subcellular proteome changes to lysosomes, mitochondrion, EEA1-positive endosomes, and Golgi caused by the NLRP3 inflammasome agonists nigericin and CL097. We identified several common disruptions to retrograde trafficking pathways, including COPI and Shiga toxin-related transport, in line with recent studies. We further characterized mouse NLRP3 trafficking throughout its activation using temporal proximity proteomics, which supports a recent model of NLRP3 recruitment to endosomes during inflammasome activation. Collectively, these findings provide additional granularity to our understanding of the molecular events driving NLRP3 activation and serve as a valuable resource for cell biological research. We have made our proteomics data accessible through an open-access Shiny browser to facilitate future research within the community, available at:https://harperlab.connect.hms.harvard.edu/inflame/. We will display anonymous peer review for this manuscript onpubpub.org(https://harperlab.pubpub.org/pub/nlrp3/) rather than a traditional journal. Moreover, we invite community feedback on the pubpub version of this manuscript, and we will address criticisms accordingly.
Two dynamic, N-terminal regions are required for function in Ribosomal RNA Adenine Dimethylase family members
Authors: McGaha Danielle A. , Collins Alexandrea , Ajisafe Luqman O. , Perdigao Calvin C. , Bondrowski Jordan L. , Fetsch Karen , Dunkle Jack A. 
Published: April 2024

AbstractThe Ribosomal RNA Adenine Dimethylase (RRAD) family of enzymes facilitate ribosome maturation in all organisms by dimethylating two nucleotides of small subunit rRNA. Prominent members of this family are the human DIMT1 and bacterial KsgA enzymes. A sub-group of RRAD enzymes, named erythromycin resistance methyltransferases (Erm) dimethylate a specific nucleotide in large subunit rRNA to confer antibiotic resistance. How these enzymes regulate methylation so that it only occurs on the specific substrate is not fully understood. While performing random mutagenesis on the catalytic domain of ErmE, we discovered that mutants in an N-terminal region of the protein that is disordered in the ErmE crystal structure are associated with a loss of antibiotic resistance. By subjecting site-directed mutants of ErmE and KsgA to phenotypic and in vitro assays we found that the N-terminal region is critical for activity in RRAD enzymes: the N-terminal basic region promotes rRNA binding and the conserved motif likely assists in juxtaposing the adenosine substrate and the SAM cofactor. Our results and emerging structural data suggest this dynamic, N-terminal region of RRAD enzymes becomes ordered upon rRNA binding forming a cap on the active site required for methylation.
Roles of Tubulin Concentration during Prometaphase and Ran-GTP during Anaphase ofC. elegansmeiosis
Authors: Gong Ting , McNally Karen L. , Konanoor Siri , Peraza Alma , Bailey Cynthia , Redemann Stefanie , McNally Francis J. 
Published: April 2024

AbstractIn many animal species, the oocyte meiotic spindle, which is required for chromosome segregation, forms without centrosomes. In some systems, Ran-GEF on chromatin initiates spindle assembly. We found that inC. elegansoocytes, endogenously-tagged Ran-GEF dissociates from chromatin during spindle assembly but re-associates during meiotic anaphase. Meiotic spindle assembly occurred after auxin-induced degradation of Ran-GEF but anaphase I was faster than controls and extrusion of the first polar body frequently failed. In search of a possible alternative pathway for spindle assembly, we found that soluble tubulin concentrates in the nuclear volume during germinal vesicle breakdown. We found that the concentration of soluble tubulin in the metaphase spindle region is enclosed by ER sheets which exclude cytoplasmic organelles including mitochondria and yolk granules. Measurement of the volume occupied by yolk granules and mitochondria indicated that volume exclusion would be sufficient to explain the concentration of tubulin in the spindle volume. We suggest that this concentration of soluble tubulin may be a redundant mechanism promoting spindle assembly near chromosomes.
Parallel pheromone, metabolite, and lipid analyses reveal patterns associated with early life transitions and ovary activation in honey bee (Apis mellifera) queens
Authors: McAfee Alison , Magaña Armando Alcazar , Foster Leonard J. , Hoover Shelley E. 
Published: April 2024

AbstractEusocial insects exist in a state of reproductive conflict in which workers forgo reproduction in favor of helping relatives, typically queens, rear offspring. The honest signal hypothesis posits that queens emit pheromonal signals that convey information about their fecundity, which workers use to make decisions around investing in direct vs. indirect fitness and queen acceptance. We evaluated this idea using liquid chromatography-tandem mass spectrometry to measure honey bee queen retinue pheromone (QRP) components in relation to queen age, laying status, and likelihood of acceptance using a protocol that enables QRP to be measured concurrently with metabolomic and lipidomic analyses. We found that older mated queens (>1 month) were more readily accepted by colonies than younger queens (10-12 d), regardless of the queen’s prior laying status. This is despite non-laying queens having significantly smaller ovaries at the time of introduction. Older queens produced higher levels of the QRP components 9(R)-HDA, LEA, and HVA compared to younger queens, with HVA also positively correlating with ovary mass. However, these data suggest that ovary mass is not an influential fertility metric for worker decision-making; therefore, the relationship between HVA and ovary mass is merely an honest signal of a non-influential metric. Metabolomic and lipidomic analyses showed that samples cluster strongly according to queen age and mating status, but not ovary mass. These data also reveal some of the first hints of the importance of prostanoids in queen maturation, along with many other physiological changes that occur in the first month of a queen’s life.Significance statementInsect pheromones have historically been evaluated using gas chromatography-mass spectrometry, a technique that is incompatible with parallel lipidomics and metabolomics inquests. Here, we repurposed an established two-phase extraction protocol and optimized a liquid chromatography-tandem mass spectrometry method to acquire pheromone, metabolite, and lipid data concurrently from a single sample. We applied this technique to interrogate the honest signal hypothesis, which relates queen pheromone profiles to reproductive quality, but the approach is broadly applicable to any question in which simultaneous determination of complex pheromone profiles and lipidomics or metabolomics data is asset. Such applications may help uncover new pheromones and reveal relationships between pheromones, hormones, and physiology in diverse biological systems.
Point contact-restricted cAMP signaling control ephrin-A5-induced axon repulsion
Authors: Bécret J , Michaud C , Assali A , Chenais NAL , Kankadze I , Gomez-Bravo C , Roche F , Couvet S , Fassier C , Nicol X 
Published: April 2024

AbstractSignal transduction downstream of axon guidance molecules is essential to steer developing axons. Second messengers including cAMP are key molecules shared by a multitude of signaling pathways and are required for a wide range of cellular processes including axon pathfinding. Yet, how these signaling molecules achieve specificity for each of their downstream pathways remains elusive. Subcellular compartmentation emerged as a flexible strategy to reach such a specificity. Here, we show that point contact-restricted cAMP signals control ephrin-A5-evoked axon repulsion in vitro by modulating Focal Adhesion Kinase phosphorylation and the assembly and disassembly rate of point contacts. Consistently, preventing point contact-specific cAMP signals, in developing retinal ganglion cells in vivo alters the refinement of their terminal axonal arbor in the brain. Altogether, our study identifies point contacts as a compartment containing a local cAMP signal required for ephrin-A5-dependent axon guidance and highlights the crucial role of such subcellularly restricted second messenger signals in the wiring of neuronal circuits.