Regulation Of Signaling Research Articles

H2AK119ub dynamics controls hair follicle stem cell quiescence.

The transition of stem cells from a quiescent state to an active state is a finely tuned process that requires the dismantling of the quiescence program and the establishment of a cell cycle-promoting transcriptional landscape. Whether epigenetic processes control stem cell states to promote the regeneration of adult tissues remains elusive. In this study, we show that a repressive histone modification, H2AK119ub, is dynamic between quiescent and active hair follicle stem cells (HFSCs) in the adult murine skin. Ablation of H2AK119ub in HFSCs leads to impaired quiescence leading to premature activation and an eventual exhaustion of HFSC pool. Transcriptional and chromatin studies revealed that H2AK119ub directly represses a proliferation promoting transcriptional program in the HFSCs to preserve quiescence. Lastly, we identify that the inhibitory FGF signaling produced by the hair follicle niche keratinocytes maintains H2AK119ub in quiescent HFSCs. Together, these findings reveal that a repressive histone mark, H2AK119ub, is under the dynamic regulation of inhibitory niche signaling to prevent the untimely establishment of an activated state to preserve SC function and longevity.

The Influence of Circadian Rhythms on DNA Damage Repair in Skin Photoaging.

Circadian rhythms, the internal timekeeping systems governing physiological processes, significantly influence skin health, particularly in response to ultraviolet radiation (UVR). Disruptions in circadian rhythms can exacerbate UVR-induced skin damage and increase the risk of skin aging and cancer. This review explores how circadian rhythms affect various aspects of skin physiology and pathology, with a special focus on DNA repair. Circadian regulation ensures optimal DNA repair following UVR-induced damage, reducing mutation accumulation, and enhancing genomic stability. The circadian control over cell proliferation and apoptosis further contributes to skin regeneration and response to UVR. Oxidative stress management is another critical area where circadian rhythms exert influence. Key circadian genes like brain and muscle ARNT-like 1 (BMAL1) and circadian locomotor output cycles kaput (CLOCK) modulate the activity of antioxidant enzymes and signaling pathways to protect cells from oxidative stress. Circadian rhythms also affect inflammatory and immune responses by modulating the inflammatory response and the activity of Langerhans cells and other immune cells in the skin. In summary, circadian rhythms form a complex defense network that manages UVR-induced damage through the precise regulation of DNA damage repair, cell proliferation, apoptosis, inflammatory response, oxidative stress, and hormonal signaling. Understanding these mechanisms provides insights into developing targeted skin protection and improving skin cancer prevention.

RGS22 maintains the physiological function of ependymal cells to prevent hydrocephalus.

Ependymal cells line the wall of cerebral ventricles and ensure the unidirectional cerebrospinal fluid (CSF) flow by beating their motile cilia coordinately. The ependymal denudation or ciliary dysfunction causes hydrocephalus. Here, we report that the deficiency of regulator of G-protein signaling 22 (RGS22) results in severe congenital hydrocephalus in both mice and rats. Interestingly, RGS22 is specifically expressed in ependymal cells within the brain. Using conditional knock-out mice, we further demonstrate that the deletion of Rgs22 exclusively in nervous system is sufficient to induce hydrocephalus. Mechanistically, we show that Rgs22 deficiency leads to the ependymal denudation and impaired ciliogenesis. This phenomenon can be attributed to the excessive activation of lysophosphatidic acid receptor (LPAR) signaling under Rgs22-/- condition, as the LPAR blockade effectively alleviates hydrocephalus in Rgs22-/- rats. Therefore, our findings unveil a previously unrecognized role of RGS22 in the central nervous system, and present RGS22 as a potential diagnostic and therapeutic target for hydrocephalus.

G-protein–coupled estrogen receptor 30 regulation of signaling downstream of protein kinase Cε mediates sex dimorphism in hyaluronan-induced antihyperalgesia

Abstract High molecular weight hyaluronan (HMWH) inhibits hyperalgesia induced by diverse pronociceptive inflammatory mediators and their second messengers, in rats of both sexes. However, the hyperalgesia induced by ligands at 3 pattern recognition receptors, lipopolysaccharide (a toll-like receptor 4 agonist), lipoteichoic acid (a toll-like receptor 2/6 agonist), and nigericin (a NOD-like receptor family, pyrin domain containing 3 activator), and oxaliplatin and paclitaxel chemotherapy–induced peripheral neuropathy are only attenuated in males. After gonadectomy or intrathecal administration of an antisense to G-protein–coupled estrogen receptor 30 (GPER) mRNA, HMWH produces antihyperalgesia in females. In nociceptors cultured from rats that had been treated with oxaliplatin, HMWH reverses nociceptor sensitization from male and GPER antisense–treated female, but not from gonad intact females. G-protein–coupled estrogen receptor–dependent sex dimorphism for HMWH-induced antihyperalgesia was also observed for the prolongation of prostaglandin E2 (PGE2)-induced hyperalgesia in primed nociceptors. While in primed rats, HMWH inhibits early, protein kinase A-dependent hyperalgesia, 30 minutes post PGE2 injection, in both sexes; measured 4 hours post-PGE2, HMWH inhibits the protein kinase Cε (PKCε)-dependent prolongation of PGE2 hyperalgesia only in males and GPER antisense–treated females. In females, hyperalgesia induced by PKCε agonist, ψεRACK, in control but not in primed nociceptors, was inhibited by HMWH. Inhibitors of 2 GPER second messengers, extracellular-regulated kinase 1/2 and nonreceptor tyrosine kinase, also unmasked HMWH antihyperalgesia in females with oxaliplatin chemotherapy–induced peripheral neuropathy, a condition in which nociceptors are primed as well as sensitized. Our results support GPER-dependent sex dimorphism in HMWH-induced antihyperalgesia for pain induced by pattern recognition receptor agonists, and chronic inflammatory and neuropathic pain, mediated by changes in signaling downstream of PKCε in primed nociceptors.

Amylin receptor subunit interactions are modulated by agonists and determine signaling.

Three amylin receptors (AMYRs) mediate the metabolic actions of the peptide hormone amylin and are drug targets for diabetes and obesity. AMY 1 R, AMY 2 R, and AMY 3 R are heterodimers consisting of the G protein-coupled calcitonin receptor (CTR) paired with a RAMP1, -2, or -3 accessory subunit, respectively, which increases amylin potency. Little is known about AMYR subunit interactions and their role in signaling. Here, we show that the AMYRs have distinct basal subunit equilibriums that are modulated by peptide agonists and determine the cAMP signaling phenotype. Using a novel biochemical assay that resolves the AMYR heterodimers and free subunits, we found that the AMY 1/2 R subunit equilibriums favored free CTR and RAMP1/2, and rat amylin and αCGRP agonists promoted subunit association. A stronger CTR-RAMP3 transmembrane domain interface yielded a more stable AMY 3 R, and human and salmon calcitonin agonists promoted AMY 3 R dissociation. Similar changes in subunit association-dissociation were observed in live cell membranes, and G protein coupling and cAMP signaling assays showed how these altered signaling. Our findings reveal regulation of heteromeric GPCR signaling through subunit interaction dynamics.

Multiplexed, image-based pooled screens in primary cells and tissues with PerturbView.

Optical pooled screening (OPS) is a scalable method for linking image-based phenotypes with cellular perturbations. However, it has thus far been restricted to relatively low-plex phenotypic readouts in cancer cell lines in culture due to limitations associated with in situ sequencing of perturbation barcodes. Here, we develop PerturbView, an OPS technology that leverages in vitro transcription to amplify barcodes before in situ sequencing, enabling screens with highly multiplexed phenotypic readouts across diverse systems, including primary cells and tissues. We demonstrate PerturbView in induced pluripotent stem cell-derived neurons, primary immune cells and tumor tissue sections from animal models. In a screen of immune signaling pathways in primary bone marrow-derived macrophages, PerturbView uncovered both known and novel regulators of NF-κB signaling. Furthermore, we combine PerturbView with spatial transcriptomics in tissue sections from a mouse xenograft model, paving the way to in situ screens with rich optical and transcriptomic phenotypes. PerturbView broadens the scope of OPS to a wide range of models and applications.

USP15 inhibits hypoxia-induced IL-6 signaling by deubiquitinating and stabilizing MeCP2.

Methyl-CpG binding protein 2 (MeCP2) is an important X-linked DNA methylation reader and a key heterochromatin organizer. The expression level of MeCP2 is crucial, as indicated by the observation that loss-of-function mutations of MECP2 cause Rett syndrome, whereas an extra copy spanning the MECP2 locus results in MECP2 duplication syndrome, both being progressive neurodevelopmental disorders. Our previous study demonstrated that MeCP2 protein expression is rapidly induced by renal ischemia-reperfusion injury (IRI) and protects the kidney from IRI through transcriptionally repressing the interleukin-6 (IL-6)/signal transducer and activator of transcription 3 signaling pathway. However, the mechanisms underlying the upregulation of MeCP2 have remained elusive. Here, by using two hypoxia cell models, hypoxia and reoxygenation and cobalt chloride stimulation, we confirmed that the removal of lysine 48-linked ubiquitination from MeCP2 prevented its proteasome-dependent degradation under hypoxic conditions. Through unbiased screening based on a deubiquitinating enzymes library, we identified ubiquitin-specific protease 15 (USP15) as a stabilizer of MeCP2. Further studies revealed that USP15 could attenuate hypoxia-induced MeCP2 degradation by cleaving lysine 48-linked ubiquitin chains from MeCP2, primarily targeting its C-terminal domain. Consistently, USP15 inhibited hypoxia-induced signal transducer and activator of transcription 3 activation, resulting in reduced transcription of IL-6 downstream genes. In summary, our study reveals an important role for USP15 in the maintenance of MeCP2 stability and the regulation of IL-6 signaling.

Molecular regulation by H2S of antioxidant and glucose metabolism in cold-sensitive Capsicum

BackgroundCold is an important environmental limiting factor affecting plant yield and quality. Capsicum (chili pepper), a tropical and subtropical vegetable crop, is extremely sensitive to cold. Although H2S is an important signaling regulator in the responses of plant growth and development to abiotic stress, few studies have examined its effects on cold-sensitive capsicum varieties. Through biotechnology methods to enhance the cold resistance of peppers, to provide some reference for pepper breeding, investigated molecular regulation by H2S of responses to cold stress in cold-sensitive capsicum plants, via physiological and transcriptomic analyses.ResultsIn capsicum seedlings, exogenous H2S enhanced relative electrical conductivity (REC) and levels of malondialdehyde (MDA) under cold stress, maintained membrane integrity, increased the activity of enzymatic and non-enzymatic antioxidants, balanced reactive oxygen species levels (O2·− and H2O2), and improved photosynthesis, mitigating the damage caused by cold. In addition, 416 differentially expressed genes (DEGs) were involved in the response to cold stress after H2S treatment. These DEGs were mainly enriched in the ascorbate–glutathione and starch–sucrose metabolic pathways and plant hormone signal-transduction pathways. Exogenous H2S altered the expression of key enzyme-encoding genes such as GST, APX, and MDHAR in the ascorbate–glutathione metabolism pathway, as well as that of regulatory genes for stimulatory hormones (auxin, cytokinins, and gibberellins) and inhibitory hormones (including jasmonate and salicylic acid) in the plant hormone signal-transduction pathway, helping to maintain the energy supply and intracellular metabolic stability under cold stress.ConclusionsThese findings reveal that exogenous H2S improves cold tolerance in cold-sensitive capsicum plants, elucidating the molecular mechanisms underlying its responses to cold stress. This study provides a theoretical basis for exploring and improving cold tolerance in capsicum plants.

8490 The mRNA for the Nonsyndromic Deafness Gene Atp2b2 Is Reduced Following Developmental Hypothyroidism

Abstract Disclosure: E.A. Gregersen: None. R.P. Peeters: None. D. Forrest: None. D.S. Sharlin: None. Thyroid hormone (TH) is essential for auditory system development. In rodent models, low levels of TH during the early postnatal period delay cochlear tissue remodeling and alter ion channel function required for normal auditory function. TH receptors are ligand-dependent transcription factors that regulate gene expression. Considering this, auditory deficits observed in hypothyroidism are thought to be largely the result of altered expression of specific TH target genes. However, few TH-regulated genes in the developing cochlea have been reported. TH is reported to impact calcium signaling in several tissues, but whether regulators of calcium signaling are altered following developmental hypothyroidism in the cochlea is largely unexplored. In this report, we investigated the spatial and temporal expression of the ATPase, Ca++ transporting, plasma membrane 2 (Atp2b2) gene in a mouse model of developmental hypothyroidism. Timed-pregnant dams were treated with thyroid gland inhibitors to induce developmental TH insufficiency from embryonic day 12.5 (E12.5) through post-natal day 15 (P15). Untreated timed-pregnant dams served as euthyroid controls. Control and experimental cochleae were collected over a developmental period ranging from embryonic day 18.5 (E18.5) through P15. Cochleae were then processed to detect Atp2b2 mRNA by in situ hybridization or quantitative real-time PCR. In situ hybridization showed that Atp2b2 localizes to inner and outer hair cells as expected, but Atp2b2 was also observed at lower levels in the greater epithelial ridge and spiral ganglion. Quantification of Atp2b2 mRNA revealed an effect of postnatal day and treatment on Atp2b2. In euthyroid animals, Atp2b2 expression gradually increased until P10, then decreased slightly by P15. In hypothyroid animals, Atp2b2 mRNA was consistently decreased (2-2.5 fold). Hypothyroidism similarly impacted expression regardless of sex. The reduction in Atp2b2 mRNA expression in the hypothyroid animals suggests that Atp2b2 is developmentally regulated in part by thyroid hormone signaling. This study characterizes the relationship between TH and Atp2b2 expression during early cochlear development while providing insight into abnormal cochlear development due to hypothyroidism. Presentation: 6/3/2024

Nitric oxide participates in sucrose-TOR signaling during meristem activation in Arabidopsis thaliana.

This study provides evidence about the relationship between Target of Rapamycin (TOR) kinase and the signal molecule nitric oxide (NO) in plants. We showed that sucrose (SUC)-mediated TOR activation of root apical meristem (RAM) requires NO and that NO, in turn, participates in the regulation of TOR signaling. Nitric oxide (NO) constitutes a signal molecule that regulates important target proteins related to growth and development and also contributes to metabolic reprogramming that occurs under adverse conditions. Taking into account the important role of NO and its relationship with Target of Rapamycin (TOR) signaling in animals, we wondered about the putative link between both pathways in plants. With this aim, we studied a TOR-dependent process which is the reactivation of the root apical meristem (RAM) in Arabidopsis thaliana. We used pharmacological and genetic tools to evaluate the relationship between NO and TOR on the sugar induction of RAM, using SNP as NO donor, cPTIO as NO scavenger and the nitrate reductase (NR) mutant nia2. The results showed that sucrose (SUC)-mediated TOR activation of the RAM requires NO and that NO, in turn, participates in the regulation of TOR signaling. Interestingly, TOR activation induced by sugar increased the NO levels. We also observed that NO could mediate the repression of SnRK1 activity by SUC. By computational prediction we found putative S-nitrosylation sites in the TOR complex proteins and the catalytic subunit of SnRK1, SnRK1.1. The present work demonstrates for the first time a link between NO and TOR revealing the complex interplay between the two pathways in plants.

Deciphering the prognostic and therapeutic significance of BAG1 and BAG2 for predicting distinct survival outcome and effects on liposarcoma

Liposarcoma (LPS) is the second most common kind of soft tissue sarcoma, and a heterogeneous malignant tumor derived from adipose tissue. Up to now, the prognostic value of BAG1 or BAG2 in LPS has not been defined yet. Expression profiling data of LPS patients were collected from TCGA and GEO database. Survival curves were plotted to verify the outcome differences of patients based on BAG1 or BAG2 expression. Univariate and multivariate Cox regression models were used to analyze the prognostic ability of BAG1 or BAG2. Chaperone’s regulators BAG1 and BAG2 were identified as prognostic biomarkers for LPS patients, which exhibited distinct expression patterns and survival outcome prediction performances. Patients with high BAG2 expression and/or low BAG1 expression had worse prognosis. Enrichment analysis showed that BAG1 was involved in negative regulation of TGF-β signaling. Low expression of BAG1 was associated with high abundance of regulatory T cells (Tregs). The 2-gene signature model further confirmed the improved risk assessment performance of BAG1 and BAG2: high risk patients displayed poor prognosis. BAG1 and BAG2 are supposed to be potential prognostic biomarkers for LPS and have impacts on liposarcomagenesis and immune infiltration in distinctive manners, which may function as potential therapy targets (BAG1 agonists/BAG2 inhibitors) for LPS.

A regulatory module mediating temperature control of cell-cell communication facilitates tree bud dormancy release.

The control of cell-cell communication via plasmodesmata (PD) plays a key role in plant development. In tree buds, low-temperature conditions (LT) induce a switch in plasmodesmata from a closed to an open state, which restores cell-to-cell communication in the shoot apex and releases dormancy. Using genetic and cell-biological approaches, we have identified a previously uncharacterized transcription factor, Low-temperature-Induced MADS-box 1 (LIM1), as an LT-induced, direct upstream activator of the gibberellic acid (GA) pathway. The LIM1-GA module mediates low temperature-induced plasmodesmata opening, by negatively regulating callose accumulation to promote dormancy release. LIM1 also activates expression of FT1 (FLOWERING LOCUS T), another LT-induced factor, with LIM1-FT1 forming a coherent feedforward loop converging on low-temperature regulation of gibberellin signaling in dormancy release. Mathematical modeling and experimental validation suggest that negative feedback regulation of LIM1 by gibberellin could play a crucial role in maintaining the robust temporal regulation of bud responses to low temperature. These results reveal genetic factors linking temperature control of cell-cell communication with regulation of seasonally-aligned growth crucial for adaptation of trees.

A20 intrinsically influences human effector T-cell survival and function by regulating both NF-κB and JNK signaling.

A20 is a dual-function ubiquitin-editing enzyme that maintains immune homeostasis by restraining inflammation. Although A20 serves a similar negative feedback function for T-cell receptor (TCR) signaling, the molecular mechanisms utilized and their ultimate impact on human T-cell function remain unclear. TCR engagement triggers the assembly of the CARD11-BCL10-MALT1 (CBM) protein complex, a signaling platform that governs the activation of downstream transcription factors including NF-κB and c-Jun/AP-1. Utilizing WT and A20 knockout Jurkat T cells, we found that A20 is required to negatively regulate NF-κB and JNK. Utilizing a novel set of A20 mutants in NF-κB and AP-1-driven reporter systems, we discovered the ZnF7 domain is crucial for negative regulatory capacity, while deubiquitinase activity is dispensable. Successful inactivation of A20 in human primary effector T cells congruently conferred sustained NF-κB and JNK signaling, including enhanced upregulation of activation markers, and increased secretion of several cytokines including IL-9. Finally, loss of A20 in primary human T cells resulted in decreased sensitivity to restimulation-induced cell death and increased sensitivity to cytokine withdrawal-induced death. These findings demonstrate the importance of A20 in maintaining T-cell homeostasis via negative regulation of both NF-κB and JNK signaling.

M6A/YTHDF2-mediated mRNA decay targets TGF-β signaling to suppress the quiescence acquisition of early postnatal mouse hippocampal NSCs.

Quiescence acquisition of proliferating neural stem cells (NSCs) is required to establish the adult NSC pool. The underlying molecular mechanisms are not well understood. Here, we showed that conditional deletion of the m6A reader Ythdf2, which promotes mRNA decay, in proliferating NSCs in the early postnatal mouse hippocampus elevated quiescence acquisition in a cell-autonomous fashion with decreased neurogenesis. Multimodal profiling of m6A modification, YTHDF2 binding, and mRNA decay in hippocampal NSCs identified shared targets in multiple transforming growth factor β (TGF-β)-signaling-pathway components, including TGF-β ligands, maturation factors, receptors, transcription regulators, and signaling regulators. Functionally, Ythdf2 deletion led to TGF-β-signaling activation in NSCs, suppression of which rescued elevated quiescence acquisition of proliferating hippocampal NSCs. Our study reveals the dynamic nature and critical roles of mRNA decay in establishing the quiescent adult hippocampal NSC pool and uncovers a distinct mode of epitranscriptomic control via co-regulation of multiple components of the same signaling pathway.

New insights into the regulation of GIPR signalling.

New insights into the regulation of GIPR signalling.

Regulation of PI3K signaling in cancer metabolism and PI3K-targeting therapy.

The phosphatidylinositol-3-kinase (PI3K) signaling plays a key role in various cellular functions and is frequently activated in cancer, making it an attractive therapeutic target. The PI3K signaling pathway influencing glucose metabolism, lipid synthesis, nucleotide production, and protein synthesis, all of which contribute to cancer cell proliferation and survival. It enhances glucose uptake through the activation of glucose transporters and glycolysis, while also promoting lipid synthesis via downstream factors like mTORC1. This pathway boosts nucleotide synthesis by regulating transcription factors like MYC, activating key enzymes for purine and pyrimidine production. Additionally, due to its essential role in cancer cell growth, the PI3K pathway is a key target for anticancer therapies. However, treatment using PI3K inhibitors alone has limitations, including drug resistance and significant side effects such as hyperglycemia, fatigue, and liver dysfunction. Clinical trials have led to the development of isoform-specific PI3K inhibitors to reduce toxicity. Combining PI3K inhibitors with other treatments, such as hormone therapy or surgery, may improve efficacy and minimize side effects. Further research is needed to fully understand the mechanisms of PI3K inhibitors and improve individualized treatment approaches. In this review, we introduce the characteristic of three classes of PI3Ks, discuss the regulation of cancer metabolism including the control of glucose uptake, glycolysis, de novo lipid synthesis, nucleotide synthesis and protein synthesis, and review the current statuses of different PI3K inhibitors therapy.

Linkage between Fuz and Gpr161 genes regulates sonic hedgehog signaling during mouse neural tube development.

Sonic hedgehog (Shh) signaling regulates embryonic morphogenesis utilizing the primary cilium, the cell's antenna, which acts as a signaling hub. Fuz, an effector of planar cell polarity signaling, regulates Shh signaling by facilitating cilia formation, and the G protein-coupled receptor 161 (Gpr161) is a negative regulator of Shh signaling. The range of phenotypic malformations observed in mice bearing mutations in either of the genes encoding these proteins is similar; however, their functional relationship has not been previously explored. This study identified the genetic and biochemical linkage between Fuz and Gpr161 in mouse neural tube development. Fuz was found to be genetically epistatic to Gpr161 with respect to regulation of Shh signaling in mouse neural tube development. The Fuz protein biochemically interacts with Gpr161, and Fuz regulates Gpr161-mediated ciliary localization, a process that might utilize β-arrestin 2. Our study characterizes a previously unappreciated Gpr161-Fuz axis that regulates Shh signaling during mouse neural tube development.

Recording and Interpretation of Active Calcium Transients in Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Calcium plays a pivotal role in the excitation-contraction coupling process in cardiomyocytes, a critical multi-parametric event leading to rhythmic contraction. Over the past few decades, calcium signaling in cardiomyocytes has been extensively studied in cardiovascular sciences. However, a standard methodology is needed not only to trace the calcium within cells but also to remove signal processing biases and to accurately interpret the features of calcium transient signals in relation to cardiomyocyte electrophysiology. This article outlines the use of genetically encoded calcium indicator (GCaMP) human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to record calcium transients. These cells express a green fluorescent signal when calcium binds to intracellular calmodulin, a key regulator of calcium signaling. The extraction and processing of calcium transient waveforms are performed using ImageJ and MATLAB software. Key features of these waveforms are then identified and categorized based on their physiological relevance to cardiomyocyte function. Additionally, this work includes a Support Protocol for the successful replating of cardiomyocytes onto non-traditional culture platforms, such as metallic sensors and polymer-based substrates, to facilitate data multiplexing. The three Basic Protocols outlined here provide a comprehensive approach for maintaining, expanding, and differentiating the GCaMP hiPSCs, video recording of calcium transients, and the subsequent signal extraction, preprocessing, analysis, and data visualization. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Maintenance, expansion, and differentiation of genetically encoded calcium indicator hiPSCs Support Protocol: Replating GCaMP hiPSC-CMs for stimulation and multielectrode array studies Basic Protocol 2: Video recording from calcium transients of GCaMP hiPSC-CMs Basic Protocol 3: Signal extraction, preprocessing, analysis, and data visualization.

Regulatory role of Heparan sulfate in leptin signaling

Leptin, a hormone mainly secreted by adipocytes, has attracted significant attention since its discovery in 1994. Initially known for its role in appetite suppression and energy regulation, leptin is now recognized for its influence on various physiological processes, including immune response, bone formation, and reproduction. It exerts its effects by binding to receptors and initiating an intracellular signaling cascade. Heparan sulfate (HS) is known to regulate the intracellular signaling of various ligands. HS is present as the glycan portion of HSPGs on cell surfaces and in intercellular spaces, with diverse structures due to extensive sulfation and epimerization. Although HS chains on HSPGs are involved in many physiological processes, the detailed effects of HS chains on leptin signaling are not well understood.This study examined the role of HS chains on HSPGs in leptin signaling using Neuro2A cells expressing the full-length leptin receptor (LepR). We showed that cell surface HS was essential for efficient leptin signaling. Enzymatic degradation of HS significantly reduced leptin-induced phosphorylation of downstream molecules, such as signal transducer and activator of transcription 3 and p44/p42 Mitogen-activated protein kinase. In addition, HS regulated LepR expression and internalization, as treatment with HS-degrading enzymes decreased cell surface LepR. HS was also found to exhibit a weak interaction with LepR. Enzymatic removal of HS enhanced the interaction between LepR and low-density lipoprotein receptor-related protein 1, suggesting that HS negatively regulates this interaction. In conclusion, HS plays a significant role in modulating LepR availability on the cell surface, thereby influencing leptin signaling. These findings provide new insights into the complex regulation of leptin signaling and highlight potential therapeutic targets for metabolic disorders and obesity.

Heterologous expression and biochemical characterization of the recombinant nucleoside triphosphate diphosphohydrolase 2 (LbNTPDase2) from Leishmania (Viannia) braziliensis.

Leishmania braziliensis is a pathogenic protozoan parasite that causes American Tegumentary Leishmaniasis (ATL), an important tropical neglected disease. ENTPDases are nucleotidases that hydrolyze intracellular and/or extracellular nucleotides. ENTPDases are known as regulators of purinergic signalling induced by extracellular nucleotides. Leishmania species have two isoforms of ENTPDase, and, particularly, ENTPDase2 seems to be involved in infectivity and virulence. In this study, we conducted the heterologous expression and biochemical characterization of the recombinant ENTPDase2 of L. braziliensis (rLbNTPDase2). Our results show that this enzyme is a canonical ENTPDase with apyrase activity, capable of hydrolysing triphosphate and diphosphate nucleotides, and it is dependent on divalent cations (calcium or magnesium). Substrate specificity was characterized as UDP>GDP>ADP>GTP>ATP=UTP. The enzyme showed optimal activity at a neutral to basic pH and was partially inhibited by suramin and DIDS. Furthermore, the low apparent Km for ADP suggests that the enzyme may play a role in adenosine-mediated signalling. The biochemical characterization of this enzyme can open new avenues for using LbNTPDase2 as a drug target.