Ezrin is a cytoplasmic protein that can exist in multiple conformations that are regulated by phosphorylation at Thr. The phosphorylated, open form of ezrin generally has been considered the active form because it transl...Ezrin is a cytoplasmic protein that can exist in multiple conformations that are regulated by phosphorylation at Thr. The phosphorylated, open form of ezrin generally has been considered the active form because it translocates to the plasma membrane. In contrast, the unphosphorylated, closed form of ezrin is sequestered in the cytoplasm and is considered inactive, although it directly interacts with cytoplasmic RNA binding proteins. Here, we found that the closed form of ezrin is itself an RNA binding protein with biological activity. The abundance of ezrin correlated with that of RBPs in human osteosarcoma samples. Purified recombinant ezrin protein engineered to maintain a closed conformation (rEZRIN-T567A) directly bound RNA, with greatest affinity for guanine-rich sequences and RNA G-quadruplexes (G4 RNAs). Expressing closed ezrin in ezrin-null osteosarcoma cells restored the transcriptomic and proteomic profiles. Closed ezrin bound to endogenous mRNAs associated with pathways related to RNA processing and splicing, DNA maintenance, and cellular metabolism. In zebrafish, expression of closed ezrin rescued the metastatic capability of ezrin-null osteosarcoma xenografts. Our findings demonstrate that the closed conformation of ezrin-previously thought to be inactive-can directly bind RNA, regulate transcription and translation, and contribute to a metastatic phenotype in osteosarcoma cells.
Sensory neurons turn cues from breast cancer cells into a dense stromal barrier against antitumor immunity.Sensory neurons turn cues from breast cancer cells into a dense stromal barrier against antitumor immunity.
Long noncoding RNAs (lncRNAs) constitute a substantial portion of the transcriptome and outnumber protein-coding transcripts in humans. lncRNA molecules are bioactive and control cellular and systemic functions by direct...Long noncoding RNAs (lncRNAs) constitute a substantial portion of the transcriptome and outnumber protein-coding transcripts in humans. lncRNA molecules are bioactive and control cellular and systemic functions by directly or indirectly regulating gene expression at the transcriptional or posttranscriptional levels. Here, we established the use of lncRNA as a modality to treat disease by reengineering three lncRNAs-GAPLINC, MIST, and DRAIR-to treat acute inflammation in mice and human macrophages. For each lncRNA, we established an in vitro transcription synthesis and high-performance liquid chromatography purification workflow and optimized the lncRNA isoforms, 5' cap, 3' poly(A) tail, and chemical base modifications to improve specificity and performance. We also optimized the in vivo delivery of lncRNA with a lipid nanoparticle system that did not induce confounding effects. Using this pipeline and delivery platform, we demonstrated that each lncRNA reduced lipopolysaccharide (LPS)-induced inflammation by specifically regulating distinct subsets of cytokines in cultured mouse macrophages and in mice. GAPLINC and DRAIR reduced the transcription of IL-1β and IL-6, respectively, whereas MIST attenuated TNFα production posttranscriptionally. In addition, we showed that reengineered GAPLINC reduced LPS-induced inflammation in human monocytes, suggesting the clinical potential of this approach. Our engineering approach and findings establish a previously unidentified nucleic acid modality and demonstrate an effective way to reengineer regulatory lncRNAs to treat disease.
Cancer neuroscience is an emerging field at the intersection of oncology, neuroscience, and immunology in which the interactions between cancer cells and neural and immune systems generate extraordinary biological comple...Cancer neuroscience is an emerging field at the intersection of oncology, neuroscience, and immunology in which the interactions between cancer cells and neural and immune systems generate extraordinary biological complexity. Thus, artificial intelligence may be a technological tool that is increasingly necessary to decode nonlinear cancer-neuron networks and to translate this complexity into biological and therapeutic insight.
Buscher K, Temprine K, Mays C
… +11 more, Aabed N, Kerk SA, Bell HN, Nieto Carrion JA, S Greenbaum H, Lee ZH, Ponnusamy V, Ramakrishnan SK, Lyssiotis CA, Xue X, Shah YM
The JAK-STAT3 signaling pathway is a key driver of colorectal cancer (CRC) progression. STAT3 is a transcription factor that is canonically activated by cytokines, such as IL-6, in a transient manner because of negative...The JAK-STAT3 signaling pathway is a key driver of colorectal cancer (CRC) progression. STAT3 is a transcription factor that is canonically activated by cytokines, such as IL-6, in a transient manner because of negative feedback mechanisms. However, STAT3 is aberrantly and persistently activated in CRC, promoting tumor cell proliferation and survival. Here, we demonstrated that glucose sustained STAT3 activation independently of cytokine availability. We manipulated glucose metabolism, which showed that both glucose and its downstream metabolite GlcNAc were essential to maintain STAT3 activation. Moreover, cells with high basal STAT3 activity produced proteins that were glycosylated in a glucose-dependent manner and that activated STAT3 in neighboring cells through paracrine signaling. Proteomic analysis identified multiple candidate proteins involved in this process; however, no single protein was sufficient to fully activate STAT3, suggesting that this activation process requires several glycosylated proteins. In a syngeneic mouse model of CRC, inhibition of glycolysis reduced STAT3 activation in tumors, and genetic deletion of STAT3 substantially decreased tumor growth. Together, these findings show how glucose metabolism supports sustained STAT3 activation in CRC, highlighting a potential metabolic vulnerability for therapeutic targeting.
Wnt ligands stimulate β-catenin-dependent (canonical) or β-catenin-independent (noncanonical) signaling, depending on which co-receptors are recruited to the Wnt receptor FZD. Both pathways are initiated by receptor olig...Wnt ligands stimulate β-catenin-dependent (canonical) or β-catenin-independent (noncanonical) signaling, depending on which co-receptors are recruited to the Wnt receptor FZD. Both pathways are initiated by receptor oligomerization into signalosomes and involve a largely overlapping set of downstream effectors. To resolve the assembly of Wnt signalosomes with high spatiotemporal resolution for extended times, we developed single-molecule tracking and localization microscopy based on labeling with reversibly binding nanobodies (rbTALM). We engineered nanobody-tag pairs with finely tuned binding affinities to ensure single-molecule tracking with high fidelity while also permitting continuous exchange of photobleached labels. Multicolor rbTALM imaging enabled simultaneous tracking and super-resolution imaging of three different tagged Wnt co-receptors in the same cell for more than 1 hour at video rate. Time-lapse correlation analyses uncovered cooperative association of canonical (LRP6) and noncanonical (ROR2) Wnt co-receptors with FZD8 into a common, hybrid Wnt signalosome. These findings demonstrate the potential for rbTALM imaging for exploring nanoscale dynamics across millisecond to hour timescales and for deciphering the molecular dynamics that underlie signaling complex formation.
Metabolism not only provides essential substances and energy for cells through catabolism and anabolism but also exerts broader regulatory roles through metabolic enzymes and products that influence gene expression, ther...Metabolism not only provides essential substances and energy for cells through catabolism and anabolism but also exerts broader regulatory roles through metabolic enzymes and products that influence gene expression, thereby maintaining homeostasis. Upon neuronal injury, metabolic changes in both neurons and supporting cells influence neuronal survival and regeneration by regulating energy supply, substrate availability, regeneration-related gene expression, and cell-cell metabolic interactions. Axon regeneration is a key process in neural repair after injury. Beyond the nervous system itself, systemic factors such as diet, exercise, circadian rhythms, and psychological stress also play crucial roles in axon regeneration through interorgan metabolic communication and microbiota-host metabolic cross-talk. In this Review, we summarize advances in understanding metabolic alterations during axon regeneration, with a focus on glycometabolism, lipid metabolism, protein degradation, mitochondrial activity, and systemic factor-driven metabolic cross-talk between nervous and non-nervous systems. We also highlight the therapeutic potential of metabolites themselves, analyze distinct metabolic responses after injury in the peripheral and central nervous systems, and discuss their spatiotemporal dynamics and cell type specificity. Last, we propose that successful neural repair requires the establishment of a systemic pro-regenerative state throughout the entire body.
Hao Y, Li Z, Lara E
… +24 more, Ramos DM, Santiana M, Jin B, Epstein J, Kowal I, Camacho J, Carmiol N, Park JH, Beylina A, Yang LG, Root JT, Sacks DC, Jarreau P, Weller CA, Klaisner S, Screven LA, Pantazis CB, Nalls MA, Narayan P, Ferrucci L, Singleton AB, Ward ME, Cookson MR, Qi YA
Induced pluripotent stem cell (iPSC)-derived neurons are a powerful tool with which to investigate both neuronal development and neurodegenerative diseases. Here, we applied quantitative proteomic and phosphoproteomic an...Induced pluripotent stem cell (iPSC)-derived neurons are a powerful tool with which to investigate both neuronal development and neurodegenerative diseases. Here, we applied quantitative proteomic and phosphoproteomic analyses to profile the neuronal differentiation of the KOLF2.1J iPSC line, the first reference line of the iPSC Neurodegenerative Disease Initiative (iNDI) project. We developed an automated workflow enabling high-coverage enrichment of proteins and phosphorylated peptides, which revealed molecular signatures during the differentiation of iPSC-derived neurons. Proteomic data highlighted distinct changes in mitochondrial pathways throughout the course of differentiation, whereas phosphoproteomic data revealed specific regulatory dynamics in GTPase-mediated signaling pathways and microtubule proteins. Additionally, phosphosite dynamics were not correlated to changes in protein abundance, particularly in processes related to axon functions and RNA transport. We measured the dynamic changes in kinases that are critical for neuronal development and maturation and developed an interactive web app to visualize the temporal landscape dynamics of protein and phosphosite abundance. By establishing baselines of proteomic and phosphoproteomic profiles for neuronal differentiation, this dataset is a valuable resource for future research into neuronal development and neurodegenerative diseases using this reference iPSC line.
Metabolic control of chromatin and gene expression is emerging as a key mechanism influencing critical neuronal functions. Here, we found that the intermediary metabolite acetate enhanced long-term memory in female mice,...Metabolic control of chromatin and gene expression is emerging as a key mechanism influencing critical neuronal functions. Here, we found that the intermediary metabolite acetate enhanced long-term memory in female mice, which was associated with epigenetic and transcriptional remodeling in the dorsal hippocampus. Acetate-enhanced memory was driven by increased acetylation of the histone variant H2A.Z and increased expression of genes implicated in learning in the female dorsal hippocampus. The effect of acetate on dorsal hippocampal histone modifications and gene expression differed markedly between the sexes during critical windows of memory consolidation and recall, and home cage exposure to acetate without the learning and recall tasks did not recapitulate these effects. These findings elucidate the ways in which acetate exposure enhances memory.
A protein secreted by a mouse commensal fungus stimulates intestinal repair.A protein secreted by a mouse commensal fungus stimulates intestinal repair.
Microglia are the brain's resident immune cells that respond to injury and disease by transitioning between homeostatic and reactive states. These cell state transitions determine whether microglia promote or resolve inf...Microglia are the brain's resident immune cells that respond to injury and disease by transitioning between homeostatic and reactive states. These cell state transitions determine whether microglia promote or resolve inflammation in the central nervous system (CNS). In this study, we explored the role of Ca signaling in regulating broader microglial cell state transitions and identified Orai1 Ca channels as critical regulators of microglial plasticity and neuroinflammatory signaling. Conditional deletion of Orai1 in microglia impaired their ability to adopt reactive, proinflammatory states. Transcriptomic and metabolomic profiling revealed that Orai1 deletion suppressed the expression of proinflammatory genes linked to immunity, inflammation, and cell metabolism. Conversely, Orai1-deficient microglia generated greater amounts of neuroprotective and anti-inflammatory mediators, including BDNF, ARG1, and the mitochondrial metabolite itaconate. In a model of CNS inflammation induced by peripheral lipopolysaccharide (LPS) challenge, microglial Orai1 deletion attenuated microglial and astrocyte reactivity and reduced hippocampal amounts of the proinflammatory cytokines IL-1β and IL-6. Consistent with these cellular changes, microglial Orai1 knockout mice were protected against LPS-induced decreases in motivational behaviors, including impaired reward-seeking and escape behaviors. These findings establish Orai1 channels as key regulators of microglial cell state transitions, linking Ca signaling to neuroinflammation and inflammation-driven behavioral dysfunction.
Adhesion GPCRs are a family of surface receptors involved in many developmental and physiological decisions in every organ system. In this issue of , Bernadyn report insights into the activation mechanism and cellular f...Adhesion GPCRs are a family of surface receptors involved in many developmental and physiological decisions in every organ system. In this issue of , Bernadyn report insights into the activation mechanism and cellular functions of the adhesion GPCR GPR97/ADGRG3.
Most adhesion G protein-coupled receptors (AGPCRs) are activated by the intramolecular binding of a tethered agonist, which is exposed by shear force-induced dissociation of the N- and C-terminal fragments of AGPCRs. The...Most adhesion G protein-coupled receptors (AGPCRs) are activated by the intramolecular binding of a tethered agonist, which is exposed by shear force-induced dissociation of the N- and C-terminal fragments of AGPCRs. The decrypted tethered agonist binds to its orthosteric site in the C-terminal fragment to stabilize the active state of the AGPCR. Corticosteroids have been proposed to be orthosteric agonists for GPR97/ADGRG3. Here, we showed that GPR97/ADGRG3 is activated by the canonical tethered agonist mechanism. In cell-based luciferase reporter assays and receptor/G protein reconstitution assays, GPR97/ADGRG3 was stimulated by agonist peptidomimetics and by 3-acetoxydihydrodeoxygeduin (3-α-DOG), a partial agonist of the ADGRG subfamily, but not by corticosteroids. We showed that GPR97 was a promiscuous AGPCR that coupled to G, G, and G but not to G. GPR97 is abundant in neutrophils, which undergo cell shape changes and polarization and migrate upon activation. GPR97 activation by tethered agonist peptidomimetics or 3-α-DOG robustly stimulated cAMP production, polarization, and chemotaxis in human and mouse neutrophils. These effects in mouse neutrophils required GPR97 and were not mimicked by the corticosteroid beclomethasone in either cell type. Together, our results demonstrate that GPR97/ADGRG3 uses a tethered agonist mechanism to activate G protein signaling and induce neutrophil polarization and migration.
CD25 is a subunit of the interleukin-2 (IL-2) receptor on T cells and natural killer (NK) cells. Acute leukemias with oncogenic tyrosine kinases often include CD25 leukemia subpopulations, which portend poor clinical out...CD25 is a subunit of the interleukin-2 (IL-2) receptor on T cells and natural killer (NK) cells. Acute leukemias with oncogenic tyrosine kinases often include CD25 leukemia subpopulations, which portend poor clinical outcomes for patients; however, acute leukemia cells do not respond to IL-2. Here, we identified CD25 and its phosphorylation by protein kinase Cδ (PKCδ) as central elements of a feedback loop that stabilized fluctuations in oncogenic tyrosine kinase signaling in acute lymphoblastic and myeloid leukemia. Genetic ablation of in murine and patient-derived xenograft (PDX) models of acute leukemias reduced clonal fitness, colony formation, and leukemia-initiation capacity in serial transplant recipients. Oncogenic tyrosine kinase signaling in leukemia cells stimulated NF-κB-mediated expression, whereas PKCδ-mediated phosphorylation of CD25 suppressed oncogenic tyrosine kinase signaling through inhibitory phosphatases, such as PTPN6. Interactome analyses and mass spectrometry-based global phosphoproteomic analyses showed that deletion abolished the phosphatase activity of PTPN6, resulting in enhanced activation of tyrosine kinases and NF-κB. Four injections of a CD25 antibody-drug conjugate induced complete remission in mice transplanted with PDX refractory leukemia. These findings highlight the dependency of tyrosine kinase-driven leukemias on robust feedback control and the role of PKCδ and CD25 in assembling its components.
Triple-negative breast cancers (TNBCs) lack targeted therapeutics that can inhibit their growth and progression. The long intergenic noncoding RNA LINC01133 promotes TNBC pathogenesis by increasing the abundance of proli...Triple-negative breast cancers (TNBCs) lack targeted therapeutics that can inhibit their growth and progression. The long intergenic noncoding RNA LINC01133 promotes TNBC pathogenesis by increasing the abundance of proline-rich protein 5 (PRR5), an mTORC2 component that activates the kinase AKT in a PI3K-independent, mTORC2-dependent manner. Here, however, we found that TNBC cell proliferation was incompletely sensitive to AKT inhibitors alone because PRR5 also stimulated the mitogen-activated protein kinase (MAPK) cascade in an mTORC2-dependent manner. PRR5 associated with and prevented the ubiquitin-dependent proteasomal degradation of IQGAP1, an adaptor protein that promotes activation of the MAP kinase ERK. ERK signaling was essential for LINC01133-mediated TNBC proliferation in two- and three-dimensional cultures, and ERK inhibitors synergized with AKT blockade to suppress LINC01133-induced TNBC cell growth. Furthermore, PRR5 abundance was particularly enriched and correlated with that of phosphorylated ERK in samples from patients with TNBC. Our results highlight cross-talk between mTORC2 and ERK signaling downstream of LINC01133 and PRR5 that may be therapeutically targeted to treat TNBC.
Inhibiting the deacetylase SIRT2 increases the activity of the kinase Lck to enhance T cell receptor signaling.Inhibiting the deacetylase SIRT2 increases the activity of the kinase Lck to enhance T cell receptor signaling.
Most of the lymphocytes in the human pregnant uterus are natural killer (NK) cells. Here, we showed that soluble HLA-G expressed by fetal trophoblast cells at the maternal-fetal interface in early pregnancy stimulated tr...Most of the lymphocytes in the human pregnant uterus are natural killer (NK) cells. Here, we showed that soluble HLA-G expressed by fetal trophoblast cells at the maternal-fetal interface in early pregnancy stimulated transcription of mostly the same genes in primary NK cells as an agonistic antibody to the receptor KIR2DL4. An exception was the transcription of type I interferon (IFN-I)-stimulated genes (ISGs), which were selectively induced by HLA-G through a noncanonical pathway. This ISG response required the transcription factor IRF7 and the kinase JAK1. The carboxyl-terminal portion of the KIR2DL4 cytoplasmic tail includes a sequence analogous to conserved JAK1 binding sites in IFN receptors and was required for JAK1 binding to KIR2DL4. Phosphorylation of IRF7 and the JAK substrate STAT2, which link IFN-I stimulation to ISG transcription, was detected in the nuclei of HLA-G-stimulated NK cells. Single-cell RNA sequencing (scRNA-seq) showed that HLA-G induced a broader transcriptional response in CD56 NK cells than in CD56 NK cells and that ISG expression was similar in both NK cell subsets. Comparison of our data with scRNA-seq data from the early maternal-fetal interface revealed that HLA-G induced the transcription of genes that distinguish decidual NK cells from maternal blood NK cells. Thus, ISG transcription, which is inducible by HLA-G, is detected in early pregnancy and may underlie intrinsic antipathogen resistance of NK cells in the maternal decidua.
Olbei M, Hautefort I, Thomas JP
… +12 more, Csabai L, Bohar B, Koigi SS, Ibraheim H, Saifuddin A, Coman D, Højmose Kromann E, Neves JF, Papp D, Powell N, Modos D, Korcsmaros T
Ulcerative colitis (UC) is a chronic inflammatory disorder of the gastrointestinal tract that is characterized by dysregulated cytokine signaling. Treatment outcomes for patients with UC remain suboptimal despite the adv...Ulcerative colitis (UC) is a chronic inflammatory disorder of the gastrointestinal tract that is characterized by dysregulated cytokine signaling. Treatment outcomes for patients with UC remain suboptimal despite the advent of cytokine-targeting therapies, necessitating a better understanding of the interconnected cytokine signaling networks that are perturbed in patients with UC. To address this, we undertook systems immunology modeling of single-cell transcriptomics data from colonic biopsies of treatment-naive and treatment-exposed patients with UC to build complex cytokine signaling networks underpinned by putative cytokine-cytokine interactions. The generated cytokine networks effectively captured known, physiologically relevant cytokine-cytokine interactions, which we validated in vitro in colonic epithelial organoids derived from patients with UC and with organoids cocultured with innate lymphoid cells. These networks revealed several previously unrecognized aspects of UC pathogenesis. These included the identification of a cytokine subnetwork that is unique to treatment-naive patients with UC, of cytokines with altered interaction patterns across UC disease states (including IL-22, TL1A, IL-23A, and OSM), and of cytokine-cytokine interactions that were mediated by specific members of the Janus-associated kinase (JAK) family. In particular, our network analysis positioned TL1A as an important upstream regulator of TNF and IL-23A (both of which are targeted by approved UC drugs) and suggested it as a potential therapeutic target. Together, these findings open several avenues for guiding future cytokine-targeting therapeutic approaches in UC, and the presented methodology can be readily applied to gain similar insights into other immune-mediated inflammatory diseases.
Pain associated with oral cancer is debilitating. Opioids are the gold standard for cancer pain management, but tolerance and side effects limit their use. Epidermal growth factor receptor (EGFR) signaling is commonly am...Pain associated with oral cancer is debilitating. Opioids are the gold standard for cancer pain management, but tolerance and side effects limit their use. Epidermal growth factor receptor (EGFR) signaling is commonly amplified in oral tumors. Here, we found that EGFR activation contributed to both oral cancer pain and opioid tolerance by sensitizing trigeminal ganglion (TG) cells, the main sensory neurons innervating the face and mouth. EGFR ligands were secreted by oral squamous cell carcinoma (OSCC) cells and by peripheral glial cells cocultured with OSCC cells. In human OSCC and an orthotopic mouse model, EGFR was abundant in tumor-innervating TG nerves. Oral cancer pain and opioid tolerance in the mice were increased by EGFR ligands and reduced by EGFR inhibitors. In mice, the abundance of glutamate-type NMDA receptors (NMDARs) was also increased in both the TG and the brainstem. Upon activation by ligands or OSCC cell supernatant, EGFR phosphorylated the NMDAR subunit GluN2B, which increased electrical currents and sensitized pre- and postsynaptic NMDARs in the brainstem. This sensitization was also seen in the brainstems of mice receiving chronic morphine treatment and was mitigated by EGFR blockade. These findings suggest that EGFR-targeted cancer therapeutics may be repurposed to manage cancer pain and reduce opioid tolerance in patients with OSCC.