Signaling Activity Research Articles

Mitochondrial Dynamics Drive Muscle Stem Cell Progression from Quiescence to Myogenic Differentiation

From quiescence to activation and myogenic differentiation, muscle stem cells (MuSCs) experience drastic alterations in their signaling activity and metabolism. Through balanced cycles of fission and fusion, mitochondria alter their morphology and metabolism, allowing them to affect their decisive role in modulating MuSC activity and fate decisions. This tightly regulated process contributes to MuSC regulation by mediating changes in redox signaling pathways, cell cycle progression, and cell fate decisions. In this review, we discuss the role of mitochondrial dynamics as an integral modulator of MuSC activity, fate, and maintenance. Understanding the influence of mitochondrial dynamics in MuSCs in health and disease will further the development of therapeutics that support MuSC integrity and thus may aid in restoring the regenerative capacity of skeletal muscle.

Notch signaling pathway suppresses mRNA expression of hexokinase 2 under nutrient-poor conditions in U87-MG glioma cells.

Control of nutrient homeostasis plays a central role in cell proliferation/survival during embryonic development and tumor growth. Activation of the Notch signaling pathway, a major contributor to cell-cell interactions, is a potential mechanism for cell adaptation to nutrient-poor conditions. Our previous study also demonstrated that during embryogenesis when nutrients such as glutamine and growth factors are potentially maintained at lower levels, Notch signaling suppresses mRNA expression of hexokinase 2 (hk2), which is a glycolysis-associated gene, in the central nervous system. However, whether and how the genetic regulation of HK2 via Notch signaling contributes to cellular adaptability to nutrient-poor environments remains unknown. In this study, we performed gene expression analysis using a U87-MG human glioma cell line and revealed that under conditions where both glutamine and serum were absent, Notch signaling was activated and HK2 expression was downregulated by Notch signaling. We also found that Notch-mediated HK2 suppression was triggered in a Notch ligand-selective manner. Furthermore, HK2 was shown to inhibit cell proliferation of U87-MG gliomas, which might depend on Notch signaling activity. Together, our findings suggest the involvement of Notch-mediated HK2 suppression in an adaptive mechanism of U87-MG glioma cells to nutrient-poor conditions.

Mechanism of Gastrodin against neurotoxicity based on network pharmacology, molecular docking and experimental verification

BackgroundDisorders of glutamate metabolism and excessive release participat in multiple neuronal pathologies including ischemic stroke (IS), Alzheimer's disease (AD), or Parkinson's disease (PD). Recently, herbal medicines have been widely used and have shown satisfactory results in the treatment of neurological disorders. Gastrodin is a traditional Chinese medicine (TCM) used for the treatment of nerve injuries, spinal cord injuries, and some central nervous system diseases as well. This research examines the neuroprotective effects of Gastrodin against glutamate-induced neurotoxicity in neuronal cells. MethodsThe HERB database was used to explore the active ingredients and target genes of Gastrodia Elata. The STRING database and Cytoscape software were used to screen and construct the Protein-Protein Interaction (PPI). Furthermore, we used molecular docking to predict the potential targets of Gastrodin. The effects of Gastrodin were revealed by western blot, calcium imaging, membrane clamp, CCK8 and flow cytometry. Neuronal oxidative stress and damage were assessed by measuring malondialdehyde (MDA) levels and superoxide dismutase (SOD) activity. Neuronal morphology was examined using Golgi-Cox staining. Finally, animal behavior was examined using novel object recognition and fear conditioning tests. ResultsWe have obtained 22 components such as TM10, TM17, TM25 (Gastrodin), and 281 targets such as AKT, EGFR, and CDK1 through network pharmacology. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed these genes were significantly enriched in protein phosphorylation, protein serine/threonine/tyrosine kinase activity, apoptosis and HIF-1 signaling pathways, etc. A higher affinity between Gastrodin and AKT was revealed by PPI analysis and molecular docking. Further, Gastrodin significantly inhibited Ca2+ influxes and excitatory synaptic transmission in cortical neurons. In addition, Gastrodin effectively alleviated neuron apoptosis, oxidative stress and damage. ConclusionGastrodin has neuroprotective effects against glutamate-induced neurotoxicity.

Insight into structural properties of viral G protein-coupled receptors and their role in the viral infection: IUPHAR Review 41.

G protein-coupled receptors (GPCRs) are pivotal in cellular signalling and drug targeting. Herpesviruses encode GPCRs (vGPCRs) to manipulate cellular signalling, thereby regulating various aspects of the virus life cycle, such as viral spreading and immune evasion.vGPCRs mimic host chemokine receptors, often with broader signalling and high constitutive activity. This review focuses on the recent advancements in structural knowledge about vGPCRs, with an emphasis on molecular mechanisms of action and ligand binding. The structures of US27 and US28 from human cytomegalovirus (HCMV) are compared to their closest human homologue, CX3CR1. Contrasting US27 and US28, the homotrimeric UL78 structure (HCMV) reveals more distance to chemokine receptors. Open reading frame 74 (ORF74; Kaposi's sarcoma-associated herpesvirus) is compared to CXCRs, whereas BILF1 (Epstein-Barr virus) is discussed as a putative lipid receptor. Furthermore, the rolesof vGPCRs in latency and lytic replication, reactivation, dissemination and immune evasion arereviewed, together with their potential as drug targets for virus infections and virus-related diseases.

The ameliorating effects of adipose-derived stromal vascular fraction cells on blue light-induced rat retinal injury via modulation of TLR4 signaling, apoptosis, and glial cell activity.

Blue light (BL)-induced retinal injury has become a very common problem due to over exposure to blue light-emitting sources. This study aimed to investigate the possible ameliorating impact of stromal vascular fraction cells (SVFCs) on BL-induced retinal injury. Forty male albino rats were randomly allocated into four groups. The control group rats were kept in 12-h light/12-h dark. Rats of SVFC-control as the control group, but rats were intravenously injected once by SVFCs. Rats of both the BL-group and BL-SVFC group were exposed to BL for 2 weeks; then rats of the BL-SVFC group were intravenously injected once by SVFCs. Following the BL exposure, rats were kept for 8 weeks. Physical and physiological studies were performed; then retinal tissues were collected for biochemical and histological studies. The BL-group showed physical and physiological changes indicating affection of the visual function. Biochemical marker assessment showed a significant increase in MDA, TLR4 and MYD88 tissue levels with a significant decrease in TAC levels. Histological and ultrastructural assessment showed disruption of the normal histological architecture with retinal pigment epithelium, photoreceptors, and ganglion cell deterioration. A significant increase in NF-κB, caspase-3, and GFAP immunoreactivity was also detected. BL-SVFC group showed a significant improvement in physical, physiological, and biochemical parameters. Retinal tissues revealed amelioration of retinal structural and ultrastructural deterioration and a significant decrease in NF-κB and caspase-3 immunoreactivity with a significant increase in GFAP immunoreaction. This study concluded that SVFCs could ameliorate the BL-induced retinal injury through TLR-4/MYD-88/NF-κB signaling inhibition, regenerative, anti-oxidative, and anti-apoptotic effects.

Anti-Inflammatory and Autophagy Activation Effects of 7-Methylsulfonylheptyl Isothiocyanate Could Suppress Skin Aging: In Vitro Evidence

Skin inflammation, characterized by redness, swelling, and discomfort, is exacerbated by oxidative stress, where compounds like 7-methylsulfonylheptyl isothiocyanate (7-MSI) from cruciferous plants exhibit promising antioxidant and anti-inflammatory properties, though their effects on skin aging and underlying mechanisms involving the NLRP3 inflammasome and autophagy are not fully elucidated. NLRP3 is a crucial inflammasome involved in regulating inflammatory responses, and our study addresses its activation and associated physiological effects. Using biochemical assays such as ELISA, RT-qPCR, Western blotting, confocal microscopy, and RNA interference, we evaluated 7-MSI’s impact on cytokine production, protein expression, and genetic regulation in Raw 264.7 and RAW-ASC cells. 7-MSI significantly reduced TNF-α, IL-1β, IL-6, COX-2, and PGE transcription levels in LPS-stimulated Raw 264.7 cells, indicating potent anti-inflammatory effects. It also inhibited NF-κB signaling and NLRP3 inflammasome activity, demonstrating its role in preventing the nuclear translocation of NF-κB and reducing caspase-1 and IL-1β production. In terms of autophagy, 7-MSI enhanced the expression of Beclin-1, LC3, and Atg12 while reducing phospho-mTOR levels, suggesting an activation of autophagy. Moreover, it effectively decreased ROS production induced by LPS. The interaction between autophagy and inflammasome regulation was further confirmed through experiments showing that interference with autophagy-related genes altered the effects of 7-MSI on cytokine production. Collectively, this study demonstrates that 7-MSI promotes autophagy, including ROS removal, and to suppress inflammation, we suggest the potential use of 7-MSI as a skin care and disease treatment agent.

Identification and characterization of chemotherapy resistant high-risk neuroblastoma persister cells.

Relapse rates in high-risk neuroblastoma remain exceedingly high. The malignant cells that are responsible for relapse have not been identified, and mechanisms of therapy resistance remain poorly understood. Here, we used single nucleus RNA sequencing and bulk whole genome sequencing to identify and characterize the residual malignant persister cells that survive chemotherapy from a cohort of 20 matched diagnosis and definitive surgery tumor samples from patients treated with high-risk neuroblastoma induction chemotherapy. We show that persister cells share common mechanisms of chemotherapy escape including suppression of MYCN activity and activation of NF-κB signaling, the latter is further enhanced by cell-cell communication between the malignant cells and the tumor microenvironment. Overall, our work dissects the transcriptional landscape of cellular persistence in high-risk neuroblastoma and paves the way to the development of new therapeutic strategies to prevent disease relapse.

Compromised endothelial Wnt/β-catenin signaling mediates the blood-brain barrier disruption and leads to neuroinflammation in endotoxemia.

The blood-brain barrier (BBB) is a critical interface that maintains the central nervous system homeostasis by controlling the exchange of substances between the blood and the brain. Disruption of the BBB plays a vital role in the development of neuroinflammation and neurological dysfunction in sepsis, but the mechanisms by which the BBB becomes disrupted during sepsis are not well understood. Here, we induced endotoxemia, a major type of sepsis, in mice by intraperitoneal injection of lipopolysaccharide (LPS). LPS acutely increased BBB permeability, activated microglia, and heightened inflammatory responses in brain endothelium and parenchyma. Concurrently, LPS or proinflammatory cytokines activated the NF-κB pathway, inhibiting Wnt/β-catenin signaling in brain endothelial cells in vitro and in vivo. Cell culture study revealed that NF-κB p65 directly interacted with β-catenin to suppress Wnt/β-catenin signaling. Pharmacological NF-κB pathway inhibition restored brain endothelial Wnt/β-catenin signaling activity and mitigated BBB disruption and neuroinflammation in septic mice. Furthermore, genetic or pharmacological activation of brain endothelial Wnt/β-catenin signaling substantially alleviated LPS-induced BBB leakage and neuroinflammation, while endothelial conditional ablation of the Wnt7a/7b co-receptor Gpr124 exacerbated the BBB leakage caused by LPS. Mechanistically, Wnt/β-catenin signaling activation rectified the reduced expression levels of tight junction protein ZO-1 and transcytosis suppressor Mfsd2a in brain endothelial cells of mice with endotoxemia, inhibiting both paracellular and transcellular permeability of the BBB. Our findings demonstrate that endotoxemia-associated systemic inflammation decreases endothelial Wnt/β-catenin signaling through activating NF-κB pathway, resulting in acute BBB disruption and neuroinflammation. Targeting the endothelial Wnt/β-catenin signaling may offer a promising therapeutic strategy for preserving BBB integrity and treating neurological dysfunction in sepsis.

Ethanolic Extract of Bergenia purpurascens Exhibits Antimelanogenic Effects in B16F10 Cells through Multiple Mechanisms That Suppress Tyrosinase.

Bergenia purpurascens, renowned for its antibacterial and antioxidant properties, remains relatively unexplored in its impact on melanogenesis. This study delves into the antimelanogenic potential of the ethanol extract derived from B. purpurascens (BPE). Our investigations reveal the robust antioxidant capabilities of the BPE, along with its effective inhibition of mushroom tyrosinase activity. Remarkably, these effects were significantly stronger than those observed with arbutin, the positive control. In vitro assays demonstrate the BPE's efficacy in reducing the melanin content and tyrosinase activity in α-MSH-stimulated B16F10 cells. Immunofluorescence and qPCR analyses further reveal the BPE's ability to inhibit MITF-mediated gene expression levels associated with melanogenesis, including Trp-1, Trp-2, and tyrosinase. These findings suggest that the extract operates through dual mechanisms, suppressing both tyrosinase activity and key transcription factor-mediated downstream signaling. Utilizing UPLC-MS/MS analysis, we identified six key compounds implicated in tyrosinase activity inhibition and melanogenesis suppression. Docking simulations confirm moderate binding affinities between these compounds and tyrosinase. This study highlights the potential of B. purpurascens as a novel natural agent for depigmentation in medicinal and cosmetic applications, elucidating its dual mechanism of action in melanogenesis inhibition.

Plasma Lipidomics Reveals Lipid Signatures of Early Pregnancy in Mares

Understanding the systemic biochemistry of early pregnancy in the mare is essential for developing new diagnostics and identifying causes for pregnancy loss. This study aimed to elucidate the dynamic lipidomic changes occurring during the initial stages of equine pregnancy, with a specific focus on days 7 and 14 post-ovulation. By analysing and comparing the plasma lipid profiles of pregnant and non-pregnant mares, the objective of this study was to identify potential biomarkers for pregnancy and gain insights into the biochemical adaptations essential for supporting maternal recognition of pregnancy and early embryonic development. Employing discovery lipidomics, we analysed plasma samples from pregnant and non-pregnant mares on days 7 and 14 post-conception using the SCIEX ZenoTOF 7600 system. This high-resolution mass spectrometry approach enabled us to comprehensively profile and compare the lipidomes across these critical early gestational timepoints. Our analysis revealed significant lipidomic alterations between pregnant and non-pregnant mares and between days 7 and 14 of pregnancy. Key findings include the upregulation of bile acids, sphingomyelins, phosphatidylinositols, and triglycerides in pregnant mares. These changes suggest enhanced lipid synthesis and mobilization, likely associated with the embryo’s nutritional requirements and the establishment of embryo–maternal interactions. There were significant differences in lipid metabolism between pregnant and non-pregnant mares, with a notable increase in the sterol lipid BA 24:1;O5 in pregnant mares as early as day 7 of gestation, suggesting it as a sensitive biomarker for early pregnancy detection. Notably, the transition from day 7 to day 14 in pregnant mares is characterized by a shift towards lipids indicative of membrane biosynthesis, signalling activity, and preparation for implantation. The study demonstrates the profound lipidomic shifts that occur in early equine pregnancy, highlighting the critical role of lipid metabolism in supporting embryonic development. These findings provide valuable insights into the metabolic adaptations during these period and potential biomarkers for early pregnancy detection in mares.

A comprehensive review on the role of Vitamin A on human health and nutrition

Vitamin A is a crucial nutrient that protects the body from oxidative stress caused by free radicals, which are unstable molecules that can damage cellular components. Its antioxidant properties helps maintaining various physiological functions that depend on its presence, including vision, immune function and skin health. The human body can convert natural forms of vitamin A (retinol, retinal and retinoic acid) and provitamin A (β-carotene) into active forms, which interact with specific molecular targets, influencing gene expression, cell differentiation and cellular processes. This comprehensive review provides an in-depth examination of vitamin A, covering its dietary sources, physiological roles and consequences of deficiency. In this research review explored the metabolic pathways of vitamin A, including absorption, transport and storage, as well as its potential applications, offering a thorough understanding of this essential nutrient. Furthermore, we have decreased the latest research on vitamin A's role in, maintaining healthy vision, immune function, and skin health, treating various diseases and conditions, such as retinal disorders, infectious diseases and cancer, mechanisms of action, including gene regulation, cell signaling, and antioxidant activity, relationships with other nutrients and health outcomes, including vitamin D, iron, and zinc, impact of deficiency on human health, including night blindness, impaired immunity, and skin disorders and emerging research on potential benefits and risks, including its role in chronic diseases and interactions with medications. Key words: Nutrition, retinyl ester, Nyctalopia, Rhodopsin, Vitamin A, β-carotene

Cathepsin L Promotes Pulmonary Hypertension via BMPR2/GSDME-Mediated Pyroptosis.

Pulmonary hypertension (PH) is a fatal progressive disease characterized by pulmonary endothelial injury and occlusive pulmonary vascular remodeling. Lysosomal protease cathepsin L degrades essential molecules to participate in the human pathophysiological process. BMPR2 (bone morphogenetic protein type II receptor) deficiency, an important cause of PH, results from mutational inactivation or excessive lysosomal degradation and induces caspase-3-mediated cell death. Given recent evidence that pyroptosis, as a new form of programmed cell death, is induced by caspase-3-dependent GSDME (gasdermin E) cleavage, we hypothesized that cathepsin L might promote PH through BMPR2/caspase-3/GSDME axis-mediated pyroptosis. Cathepsin L expression was evaluated in the lungs and plasma of patients with pulmonary arterial hypertension. The role of cathepsin L in the progression of PH and vascular remodeling was assessed in vivo. Small interfering RNA, specific inhibitors, and lentiviruses were used to explore the mechanisms of cathepsin L on human pulmonary arterial endothelial cell dysfunction. Cathepsin L expression is elevated in pulmonary artery endothelium from patients with idiopathic pulmonary arterial hypertension and experimental PH models. Genetic ablation of cathepsin L in PH rats relieved right ventricular systolic pressure, pulmonary vascular remodeling, and right ventricular hypertrophy, also restoring endothelial integrity. Mechanistically, cathepsin L promotes caspase-3/GSDME-mediated endothelial cell pyroptosis and represses BMPR2 signaling activity. Cathepsin L degrades BMPR2 via the lysosomal pathway, and restoring BMPR2 signaling prevents the pro-pyroptotic role of cathepsin L in PAECs and experimental PH models. These results show for the first time that cathepsin L promotes the development of PH by degrading BMPR2 to induce caspase-3/GSDME-mediated endothelial pyroptosis.

Osteoclastic ATP6AP2 maintains β-catenin levels to prevent hyper-osteoclastic activation and trabecular bone-loss.

Osteoclast (OC) formation and bone resorption are regulated by several factors, including V-ATPase, Wnt/β-Catenin, and RANKL/RANK signaling. ATP6AP2, also known as the prorenin receptor (PRR), is an accessory subunit of V-ATPase and a regulator of Wnt/β-Catenin signaling. While the V-ATPase subunit ATP6AP1 is essential for osteoclast formation and function, the role of ATP6AP2 in OC-lineage cells is less clear. Here, we provide evidence that ATP6AP2 plays a negative role in osteoclastogenesis and function, contrasting with the positive role of ATP6AP1. Mice with conditional knockout (cKO) of ATP6AP2 in OCs (Atp6ap2LysM) exhibit trabecular bone loss, likely due to the increased osteoclastogenesis and activity, since bone formation rates are comparable to control mice. In vitro assays using bone marrow macrophages (BMMs) show that Atp6ap2LysM cultures have more RANKL-induced TRAP+ OC-like cells and increased bone resorptive activity. Further studies reveal that while RANKL signaling and V-ATPase activity are normal, in ATP6AP2 KO OCs, but not BMMs, have reduced basal levels of Wnt/β-Catenin pathway proteins, such as LRP5/6 and β-Catenin, compared to controls. Wnt3A treatment induces β-Catenin and suppresses osteoclast formation in both control and ATP6AP2 KO OC-lineage cells, indicating that Wnt/β-Catenin signaling negatively regulates OC-formation and operates independently of ATP6AP2. Overall, these results suggest that ATP6AP2 is critical for maintaining basal levels of LRP5/6 receptors and β-Catenin in osteoclasts, thus acting as a negative regulator of osteoclastogenesis and activation.

SPROUTY2, a Negative Feedback Regulator of Receptor Tyrosine Kinase Signaling, Associated with Neurodevelopmental Disorders: Current Knowledge and Future Perspectives

Growth-factor-induced cell signaling plays a crucial role in development; however, negative regulation of this signaling pathway is important for sustaining homeostasis and preventing diseases. SPROUTY2 (SPRY2) is a potent negative regulator of receptor tyrosine kinase (RTK) signaling that binds to GRB2 during RTK activation and inhibits the GRB2-SOS complex, which inhibits RAS activation and attenuates the downstream RAS/ERK signaling cascade. SPRY was formerly discovered in Drosophila but was later discovered in higher eukaryotes and was found to be connected to many developmental abnormalities. In several experimental scenarios, increased SPRY2 protein levels have been observed to be involved in both peripheral and central nervous system neuronal regeneration and degeneration. SPRY2 is a desirable pharmaceutical target for improving intracellular signaling activity, particularly in the RAS/ERK pathway, in targeted cells because of its increased expression under pathological conditions. However, the role of SPRY2 in brain-derived neurotrophic factor (BDNF) signaling, a major signaling pathway involved in nervous system development, has not been well studied yet. Recent research using a variety of small-animal models suggests that SPRY2 has substantial therapeutic promise for treating a range of neurological conditions. This is explained by its function as an intracellular ERK signaling pathway inhibitor, which is connected to a variety of neuronal activities. By modifying this route, SPRY2 may open the door to novel therapeutic approaches for these difficult-to-treat illnesses. This review integrates an in-depth analysis of the structure of SPRY2, the role of its major interactive partners in RTK signaling cascades, and their possible mechanisms of action. Furthermore, this review highlights the possible role of SPRY2 in neurodevelopmental disorders, as well as its future therapeutic implications.

Two-Photon Microscopy to Measure Calcium Signaling in the Living Brain.

Two-photon microscopy enables imaging of calcium signaling at cellular or subcellular resolution up to hundreds of microns deep in the living brain. Changes in the brightness of fluorescent calcium indicators provide a readout of calcium levels over time, affording information about neuronal activity and/or calcium-dependent subcellular signaling. Here, we describe a protocol for repeated two-photon imaging of calcium signals in mice expressing a genetically encoded calcium indicator that have been implanted with a chronic cranial window.

Cocaine taking and craving produce distinct transcriptional profiles in dopamine neurons.

Dopamine (DA) signaling plays an essential role in reward valence attribution and in encoding the reinforcing properties of natural and artificial rewards. The adaptive responses from midbrain dopamine neurons to artificial rewards such as drugs of abuse are therefore important for understanding the development of substance use disorders. Drug-induced changes in gene expression are one such adaptation that can determine the activity of dopamine signaling in projection regions of the brain reward system. One of the major challenges to obtaining this understanding involves the complex cellular makeup of the brain, where each neuron population can be defined by a distinct transcriptional profile. To bridge this gap, we have adapted a virus-based method for labeling and capture of dopamine nuclei, coupled with nuclear RNA-sequencing, to study the transcriptional adaptations, specifically, of dopamine neurons in the ventral tegmental area (VTA) during cocaine taking and cocaine craving, using a mouse model of cocaine intravenous self-administration (IVSA). Our results show significant changes in gene expression across non-drug operant training, cocaine taking, and cocaine craving, highlighted by an enrichment of repressive epigenetic modifying enzyme gene expression during cocaine craving. Immunohistochemical validation further revealed an increase of H3K9me3 deposition in DA neurons during cocaine craving. These results demonstrate that cocaine-induced transcriptional adaptations in dopamine neurons vary by phase of self-administration and underscore the utility of this approach for identifying relevant phase-specific molecular targets to study the behavioral course of substance use disorders.

Tumor suppressor miR-317 and lncRNA Peony are expressed from a polycistronic non-coding RNA locus that regulates germline differentiation and testis morphology.

This research focuses on investigating the impact of non-coding RNAs on stem cell biology and differentiation processes. We found that miR-317 plays a role in germline stem cell progeny differentiation. miR-317 and its neighbor, the lncRNA Peony, originate and are co-expressed from a singular polycistronic non-coding RNA locus. Alternative polyadenylation is implicated in regulation of their differential expression. While the increased expression of the lncRNA Peony results in the disruption of the muscle sheath covering the testis, the absence of miR-317 leads to the emergence of germline tumors in young flies. The deficiency of miR-317 increases Notch signaling activity in the somatic cyst cells, which drives germline tumorigenesis. Germline tumors also arise from upregulation of several predicted targets of miR-317 , among which are regulators of the Notch pathway. This implicates miR-317 as a novel tumor suppressor that modulates Notch signaling strength.

KRas4b-Calmodulin Interaction with Membrane Surfaces: Role of Headgroup, Acyl Chain, and Electrostatics.

KRas4b is a small plasma membrane-bound G-protein that regulates signal transduction pathways. The interaction of KRas4b with the plasma membrane is governed by both its basic C-terminus, which is farnesylated and methylated, and the lipid composition of the membrane itself. The signaling activity of KRas4b is intricately related to its interaction with various binding partners at the plasma membrane, underlining the critical role played by the lipid environment. The calcium-binding protein calmodulin binds farnesylated KRas4b and plays an important role in the dynamic spatial cycle of KRas4b trafficking in the cell. We utilize Biolayer Interferometry to assay the role of lipid headgroup, chain length, and electrostatics in the dissociation kinetics of fully post-translationally modified KRas4b from Nanodisc bilayers with defined lipid compositions. Our results suggest that calmodulin promotes the dissociation of KRas4b from an anionic membrane, with a comparatively slower displacement of KRas4b from PIP2 relative to PS containing bilayers. In addition to this headgroup dependence, KRas4b dissociation appears to be slower from Nanodiscs wherein the lipid composition contains mismatched, unsaturated acyl chains as compared to lipids with a matched acyl chain length. These findings contribute to understanding the role of the lipid composition in the binding of KRas4b and release from lipid bilayers, showing that the overall charge of the bilayer, the identity of the headgroups present, and the length and saturation of the acyl chains play key roles in KRas4b release from the membrane, potentially providing insights in targeting Ras-membrane interactions for therapeutic interventions.

6846 Rare Variants in SEMA3A in Individuals with Constitutional Delay of Puberty (CDP)

Abstract Disclosure: W. He: None. E.C. Schafer: None. R. Brauner: None. A. Delaney: None. L. Dunkel: None. R. Grinspon: None. J.E. Hall: None. J.N. Hirschhorn: None. S. Howard: None. A. Latronico: None. A.A. Jorge: None. K. McElreavey: None. V. Mericq: None. P.M. Merino: None. M.R. Palmert: None. R.A. Rey: None. R.C. Rezende: None. S.B. Seminara: None. N. Shaw: None. T. Delayed Puberty Genetics Consortium: None. Y. Chan: None. Background: Constitutional delay of puberty (CDP), also known as self-limited delayed puberty, refers to late onset of puberty with relatively normal progression afterward. In contrast, idiopathic hypogonadotropic hypogonadism (IHH) results in a long-lasting defect in reproductive endocrine activity. Both CDP and IHH are highly heritable and are sometimes observed in different family members in the same pedigree. Previous work by our group and others has suggested genetic overlap between the two conditions. In this study, we aimed to identify specific IHH-related genes that are also genetic causes of CDP. Methods: The Delayed Puberty Consortium assembled exome data for 233 individuals of diverse ancestries with CDP, with onset of puberty between 12 and 18 y in girls and between 13.5 and 18 y in boys. For control data, we used exome sequencing data from gnomAD v2.1.1 (n=125,748). After data harmonization and quality control, we used a modification of the “TRAPD” method to perform gene-based burden testing for 63 genes associated with IHH. We analyzed rare variants (minor allele frequency < 0.01) with high impact (premature stop, frameshift, start-lost, stop-lost) in all individuals across ancestries (n=233), as allele frequencies for high-impact variants are unlikely to vary widely across populations. We also analyzed rare variants with moderate impact (missense, inframe insertions/deletions) predicted to be deleterious by the Primate AI algorithm (score >0.6) in non-Finnish European (NFE) individuals with CDP (n=99), then meta-analyzed the results for high- and moderate-impact variants. We considered p-values < 7.9 x 10-4 (0.05 with Bonferroni adjustment for 63 genes) significant. Results: Of the 63 genes analyzed, SEMA3A (which encodes semaphorin-3A) exhibited significant enrichment for moderate-impact variants in the NFE CDP cohort compared to controls (variant frequency in CDP vs control: 0.064 vs 0.0076, p = 1.3 x 10-4) and in the meta-analysis of both high- and moderate-impact variants (p = 3.5 x 10-6). Novel SEMA3A variants identified exclusively in CDP individuals but not in controls were a frameshift variant, p.F546fsX2, and a missense variant, I252R. Also, three missense variants in SEMA3A were observed at a higher frequency in NFE CDP individuals compared with controls: p.R66T, p.V461A, and p.T717I. No other IHH genes showed significant enrichment of rare variants in the CDP cohort. Conclusion: Rare variants in SEMA3A contribute to both CDP and IHH. While functional studies are required to assess the effects of these specific variants on protein function, previous in vitro studies of similar SEMA3A variants demonstrated defects in secretion or signaling activity. Subsequent studies employing individual-level ancestry-matched controls and larger CDP cohorts are planned to identify additional genes that contribute to CDP. Presentation: 6/1/2024

12330 Rare Variants in SEMA3A in Individuals with Constitutional Delay of Puberty (CDP)

Abstract Disclosure: W. He: None. E.C. Schafer: None. R. Brauner: None. A. Delaney: None. L. Dunkel: None. R. Grinspon: None. J.E. Hall: None. J.N. Hirschhorn: None. S. Howard: None. A. Latronico: None. A.A. Jorge: None. K. McElreavey: None. V. Mericq: None. P.M. Merino: None. M.R. Palmert: None. R.A. Rey: None. R.C. Rezende: None. S.B. Seminara: None. N. Shaw: None. T. Delayed Puberty Genetics Consortium: None. Y. Chan: None. Background: Constitutional delay of puberty (CDP), also known as self-limited delayed puberty, refers to late onset of puberty with relatively normal progression afterward. In contrast, idiopathic hypogonadotropic hypogonadism (IHH) results in a long-lasting defect in reproductive endocrine activity. Both CDP and IHH are highly heritable and are sometimes observed in different family members in the same pedigree. Previous work by our group and others has suggested genetic overlap between the two conditions. In this study, we aimed to identify specific IHH-related genes that are also genetic causes of CDP. Methods: The Delayed Puberty Consortium assembled exome data for 233 individuals of diverse ancestries with CDP, with onset of puberty between 12 and 18 y in girls and between 13.5 and 18 y in boys. For control data, we used exome sequencing data from gnomAD v2.1.1 (n=125,748). After data harmonization and quality control, we used a modification of the “TRAPD” method to perform gene-based burden testing for 63 genes associated with IHH. We analyzed rare variants (minor allele frequency < 0.01) with high impact (premature stop, frameshift, start-lost, stop-lost) in all individuals across ancestries (n=233), as allele frequencies for high-impact variants are unlikely to vary widely across populations. We also analyzed rare variants with moderate impact (missense, inframe insertions/deletions) predicted to be deleterious by the Primate AI algorithm (score >0.6) in non-Finnish European (NFE) individuals with CDP (n=99), then meta-analyzed the results for high- and moderate-impact variants. We considered p-values < 7.9 x 10-4 (0.05 with Bonferroni adjustment for 63 genes) significant. Results: Of the 63 genes analyzed, SEMA3A (which encodes semaphorin-3A) exhibited significant enrichment for moderate-impact variants in the NFE CDP cohort compared to controls (variant frequency in CDP vs control: 0.064 vs 0.0076, p = 1.3 x 10-4) and in the meta-analysis of both high- and moderate-impact variants (p = 3.5 x 10-6). Novel SEMA3A variants identified exclusively in CDP individuals but not in controls were a frameshift variant, p.F546fsX2, and a missense variant, I252R. Also, three missense variants in SEMA3A were observed at a higher frequency in NFE CDP individuals compared with controls: p.R66T, p.V461A, and p.T717I. No other IHH genes showed significant enrichment of rare variants in the CDP cohort. Conclusion: Rare variants in SEMA3A contribute to both CDP and IHH. While functional studies are required to assess the effects of these specific variants on protein function, previous in vitro studies of similar SEMA3A variants demonstrated defects in secretion or signaling activity. Subsequent studies employing individual-level ancestry-matched controls and larger CDP cohorts are planned to identify additional genes that contribute to CDP. Presentation: 6/1/2024