Antigen receptor diversity depends on the assembly of variable (V), diverse (D), and joining (J) exons in genes encoding immunoglobulins (Igs) and T cell receptors (TCRs). During V(D)J recombination, DNA double-strand br...Antigen receptor diversity depends on the assembly of variable (V), diverse (D), and joining (J) exons in genes encoding immunoglobulins (Igs) and T cell receptors (TCRs). During V(D)J recombination, DNA double-strand breaks (DSBs) introduced by the RAG1/2 nuclease complex are repaired by the process of nonhomologous end-joining (NHEJ). We hypothesized that functional redundancies between NHEJ and the chromatin DSB response, which depends on the kinase ATM, potentially masked the activity of additional factors that regulate V(D)J recombination. We performed targeted CRISPR-Cas9 knockout screens for genes implicated in V(D)J recombination in pro-B cells that were either untreated or treated with an ATM inhibitor. We found that loss of the RNA/DNA helicase senataxin (SETX) impaired V(D)J recombination and led to the formation of aberrant hybrid joints between coding ends and signal ends, both in vitro and in mice. The loss of SETX in a background deficient in the NHEJ factor XLF or in which ATM was inhibited led to substantial impairment of V(D)J recombination and to the presence of unsealed coding ends. SETX limited aberrant activation-induced cytidine deaminase (AID)-induced DNA end-joining between -containing alleles during the process of class-switch recombination. Together, our findings reveal a previously uncharacterized role for SETX in promoting recombination fidelity during antigen receptor gene diversification.
Osteochondromas characterize the rare pediatric disorder hereditary multiple osteochondromas (HMO). The tumors originate from the growth plate perichondrium along skeletal elements, appear first as ectopic cartilage, and...Osteochondromas characterize the rare pediatric disorder hereditary multiple osteochondromas (HMO). The tumors originate from the growth plate perichondrium along skeletal elements, appear first as ectopic cartilage, and then grow unidirectionally, colliding with and damaging surrounding structures. HMO is caused by mutations that affect the heparan sulfate (HS) synthases EXT1 or EXT2, leading to HS deficiency and aberrant activity of HS-binding growth factors. We investigated the signaling pathways and mechanisms underlying tumor growth in HMO using mice with conditional deficiency in the growth plate and perichondrium. Developing tumors displayed active Hedgehog (Hh) signaling within their cartilaginous moiety and the presence of parathyroid hormone-related protein (PTHrP) at their distal edge, generating an ectopic Hh-PTHrP axis orthogonal to the one directing normal bone lengthening at the adjacent growth plate. In mutants, loss of the Hh signaling effector Smoothened (Smo) reduced tumor growth, whereas heterozygous loss of the Smo inhibitor Patched1 (Ptch1) increased tumor growth. Two HS-binding growth factors that promote normal cartilage growth in the growth plate, BMP2 and activin A, did not exert their normal prochondrogenic activity when Hh signaling was blocked, demonstrating that Hh signaling is essential for chondrogenesis. Together, our findings show that osteochondromas usurp a physiological signaling mechanism to guide and propel their directional outgrowth, enabling them to damage surrounding tissues, and suggest potential targets for therapeutic intervention.
Ryanodine receptor 1 (RYR1) is the sarcoplasmic reticulum (SR) Ca release channel required for both skeletal muscle contraction and Ca leak. Mutations in cause malignant hyperthermia susceptibility (MHS) and enhanced se...Ryanodine receptor 1 (RYR1) is the sarcoplasmic reticulum (SR) Ca release channel required for both skeletal muscle contraction and Ca leak. Mutations in cause malignant hyperthermia susceptibility (MHS) and enhanced sensitivity to heat stroke (ESHS), which can result in death due to excessive skeletal muscle thermogenesis upon exposure to volatile anesthetics or heat. Here, we investigated the molecular and physiological functions of phosphorylation of RYR1 at Ser by the kinase striated muscle preferentially expressed protein (SPEG). Muscle from SPEG-deficient mice expressing RYR1 with a Ser→Asp (S2902D) point mutation to mimic phosphorylation by SPEG showed decreased SR Ca sparks. Muscle from mice homozygous for the S2902D point mutation had reduced SR Ca transients and small changes in force generation but overall mild phenotypic changes. YS mice, which are heterozygous for a Tyr→Ser point mutation in RYR1, show increased Ca leak and are a model of MHS and ESHS. Crossing YS mice with S2902D mice led to decreased SR Ca leak and desensitized the mice to both volatile anesthetics and heat. Thus, SPEG inhibits SR Ca leak in skeletal muscle by phosphorylating Ser on RYR1, and mutation of Ser to Asp to mimic this phosphorylation event rescues YS mice from heat-induced death.
The voltage-gated potassium channel Kv1.3 contributes to action potential conduction in sensory neurons and to sustained increases in cytoplasmic Ca that activate immune cells. Here, we found that two distinct endocytosi...The voltage-gated potassium channel Kv1.3 contributes to action potential conduction in sensory neurons and to sustained increases in cytoplasmic Ca that activate immune cells. Here, we found that two distinct endocytosis-inducing stimuli acted through the same residues in Kv1.3 to control surface abundance and activity of the channel. Upon stimulation of the growth factor receptor EGFR, which stimulates Tyr-directed kinases and is important in neuronal differentiation, or of the Ser/Thr kinase PKC, which participates in the down-regulation of inflammatory responses, Kv1.3 underwent ubiquitination-dependent endocytosis that routed channel proteins to lysosomes for degradation. We mapped two lysine clusters in the N and C termini of Kv1.3, both of which became ubiquitinated upon activation of either Tyr or Ser/Thr kinases and whose combined mutation had an additive effect in reducing ubiquitination and endocytosis. Manipulations that prevented the ubiquitination or decreased the endocytosis of Kv1.3 resulted in increased Kv1.3 abundance at the immunological synapse and activity in primary human T cells. Prolonged channel accumulation at this location would be expected to increase Kv1.3-dependent leukocyte activation and lead to chronic inflammatory pathologies. Thus, ubiquitination fine-tunes cell biology by inducing the endocytosis and turnover of Kv1.3 in response to biological stimuli and insults.
Of the thousands of genes and substrates identified in KRAS-mutant signaling networks in pancreatic ductal adenocarcinoma (PDAC), more than 200 are transcription factors, implying extensive and complex transcriptional re...Of the thousands of genes and substrates identified in KRAS-mutant signaling networks in pancreatic ductal adenocarcinoma (PDAC), more than 200 are transcription factors, implying extensive and complex transcriptional regulation. However, we observed that genetic suppression of the transcription factor MYC alone was sufficient to phenocopy the effect of KRAS suppression in signaling, growth, and metabolic processes in PDAC cells. We determined the gene transcription changes caused by acute suppression of MYC function in -mutant PDAC cell lines and performed dependency map and pathway analyses on the affected gene sets. The expression of 1685 genes was increased upon suppression of MYC, and this gene set may comprise the bulk of the MYC-regulated genes essential for PDAC growth. In contrast, the 1325 genes whose expression was inhibited may comprise a compensatory response to oncogenic stress, mediated in part by the GTPase RHO. MYC-dependent transcriptional activity was largely ERK dependent, and almost one-third of ERK-regulated genes were also regulated by MYC in PDAC cells. Furthermore, chemical proteomic profiling revealed MYC-regulated protein kinases that can be targeted therapeutically. Together, these data provide a molecular portrait of MYC-dependent signaling that encompasses potentially exploitable mechanisms for treating PDAC.
Mitochondrial stress drives brown fat whitening through a pathway involving reduced nuclear stiffness.Mitochondrial stress drives brown fat whitening through a pathway involving reduced nuclear stiffness.
Alamir H, Wong CCW, Alsubaiti A
… +12 more, Edmunds GL, Alismail M, Huynh L, Shi Y, Lewis PA, Grant T, Alsulaimani S, Boyd J, Holland CJ, Morgan DJ, Gallimore AM, Wülfing C
TIM3 is a coregulatory receptor that is highly abundant on multiple immune cell types, including T cells in response to prolonged exposure to antigen, and it marks functionally suppressed cytotoxic T lymphocytes (CTLs) i...TIM3 is a coregulatory receptor that is highly abundant on multiple immune cell types, including T cells in response to prolonged exposure to antigen, and it marks functionally suppressed cytotoxic T lymphocytes (CTLs) in the tumor microenvironment. TIM3 exhibits inhibitory function in vivo but paradoxically has costimulatory T cell signaling capability in vitro. Here, we found that TIM3 directly inhibited the function of murine and human CTLs in direct interaction with target tumor cell spheroids. TIM3 regulated the ability of suppressed CTLs to polarize their actin cytoskeleton as a required step in cytolysis. Whereas the expression of the proposed TIM3 ligands CEACAM1 and galectin 9 in trans on target tumor cells enhanced TIM3 function, expression of CEACAM1 in cis on CTLs had the opposite effect. TIM3 functioned as an inhibitory receptor on spheroid-suppressed CTLs but not on active CTLs in a two-dimensional tissue culture model. Together, these data suggest that TIM3 enhances T cell function, serving as either a coinhibitory or costimulatory receptor depending on the functional context of the T cell on which it is expressed.
Metabolic reprogramming controls protective and pathogenic T helper 17 (T17) cell responses. When naïve T cells are differentiated into T17 cells in vitro, the presence of the cytokine activin A promotes their maturation...Metabolic reprogramming controls protective and pathogenic T helper 17 (T17) cell responses. When naïve T cells are differentiated into T17 cells in vitro, the presence of the cytokine activin A promotes their maturation into a nonpathogenic state. Here, we found that nonpathogenic T17 cells induced by activin A displayed reduced aerobic glycolysis and increased oxidative phosphorylation (OXPHOS). In response to activin A, signaling through the adenosine A receptor (AR) and AMP-activated protein kinase (AMPK) enhanced OXPHOS and reprogrammed pathogenic T17 cells toward nonpathogenic states that did not induce central nervous system autoimmunity in a mouse model of multiple sclerosis. In pathogenic T17 cells, the transcriptional coactivator p300/CBP-associated factor (PCAF) increased acetylation at histone 3 Lys (H3K9ac) of genes involved in aerobic glycolysis and T17 pathogenic programs. In contrast, in nonpathogenic activin A-treated T17 cells, AMPK signaling suppressed PCAF-mediated H3K9ac modification of genes involved in aerobic metabolism and enhanced H3K9ac modification of genes involved in OXPHOS and nonpathogenic T17 programs. Together, our findings uncover AR-AMPK signaling as a central metabolic checkpoint that suppresses T17 cell pathogenicity.
Mutated RAS proteins activate downstream effector pathways (RAF-MEK-ERK and PI3K-AKT) to drive oncogenic transformation and progression. Because RAS family members differentially engage these pathways, combined inhibitio...Mutated RAS proteins activate downstream effector pathways (RAF-MEK-ERK and PI3K-AKT) to drive oncogenic transformation and progression. Because RAS family members differentially engage these pathways, combined inhibition of both pathways is required to effectively treat -mutated cancers. Here, we found that this was due to signaling contributed by wild-type RAS family members that activated an effector pathway that was poorly engaged by the mutant RAS family member. Wild-type KRAS and NRAS promoted RAF-MEK-ERK signaling in cells expressing mutant HRAS, whereas wild-type HRAS and NRAS promoted PI3K-AKT signaling in cells expressing mutant KRAS. Combining inhibitors targeting the poorly engaged RAS effector pathways with inhibitors targeting the mutant RAS resulted in synergistic cytotoxicity in a manner that depended on wild-type RAS expression. The farnesyltransferase inhibitor tipifarnib blocked mutant HRAS-PI3K signaling and synergized with MEK inhibitors in -mutated cells, whereas KRAS inhibitors blocked mutant KRAS-MEK signaling and synergized with PI3K inhibitors in -mutated cells. Synergy was abolished in MEFs lacking all RAS proteins and in cancer cell lines in which nonmutated RAS family members were deleted. Our data highlight the critical role of wild-type RAS family members in supporting mutant RAS signaling and its importance as a therapeutic cotarget in -mutated cancers.
Fallone L, Pouxvielh K, Arbez L
… +17 more, Rousseaux N, Picq L, Drouillard A, Mathieu AL, Nombel A, Benezech S, Bourdonnay E, Degouve S, Machy P, Mortier E, Bouscasse E, Chaoui K, Malissen B, Gonzalez de Peredo A, Roncagalli R, Walzer T, Marçais A
The multiprotein complex mTORC1 is essential for the increase in protein synthesis and bioenergetic metabolism that supports the proliferation of many cell types, including natural killer (NK) cells, which are important...The multiprotein complex mTORC1 is essential for the increase in protein synthesis and bioenergetic metabolism that supports the proliferation of many cell types, including natural killer (NK) cells, which are important innate effectors of the antitumoral response. Here, we investigated the mechanisms of mTORC1 activation in NK cells by interleukin-15 (IL-15) and IL-18, which promote NK cell function and are components of a cytokine cocktail used to preactivate NK cells for cancer immunotherapy. Through genetic and pharmacological approaches, we showed that IL-15 activated mTORC1 through the PI3K/Akt/ERK pathway, whereas IL-18 signaled through the p38 effectors MK2 and MK3 in both murine and human primary NK cells. Both pathways synergized to promote NK cell proliferation and effector functions in an mTORC1-dependent manner. Moreover, both pathways operated independently of the inhibitor TSC and the activator Rheb, revealing a noncanonical mode of mTORC1 activation by cytokines. Treating mice with IL-15 and IL-18 in combination led to increased NK cell numbers and improved antitumoral activity, suggesting that this cytokine combination could be exploited to enhance NK cell potential in therapeutic settings.
Mabry SJ, Cao X, Zhu Y
… +14 more, Rowe C, Patel S, González-Arancibia C, Romanazzi T, Saleeby DP, Elam A, Lee HT, Turkmen S, Lauzon SN, Hernandez CE, Sun H, Wu H, Carter AM, Galli A
Amphetamines are psychostimulants that are commonly used to treat neuropsychiatric disorders and are prone to misuse. The pathogenesis of amphetamine use disorder (AUD) is associated with dysbiosis (an imbalance in the b...Amphetamines are psychostimulants that are commonly used to treat neuropsychiatric disorders and are prone to misuse. The pathogenesis of amphetamine use disorder (AUD) is associated with dysbiosis (an imbalance in the body's microbiome) and bacterially produced short-chain fatty acids (SCFAs), which are implicated in the gut-brain axis. Amphetamine exposure in both rats and humans increases the amount of intestinal , which releases SFCAs. Here, we found that colonization of gnotobiotic with or supplementing the flies' diet with the SCFA butyrate enhanced the psychomotor and reward properties of amphetamine. Butyrate inhibits histone deacetylases (HDACs), and knockdown of HDAC1 recapitulated the effects induced by or butyrate. The enhancement in amphetamine-induced behaviors was mediated by an increase in the amount of released dopamine that resulted from amphetamine-induced reversal of dopamine transporter (DAT) function, termed nonvesicular dopamine release (NVDR). The magnitude of amphetamine-induced NVDR was partially mediated by an increase in DAT abundance stimulated at a transcriptional level, and the administration of or butyrate enhanced NVDR by increasing DAT expression. The findings indicate that supports AUD through epigenetic regulation of dopamine signaling and identify potential targets for AUD treatment.
Replication of HIV-1 requires the coordinated action of host and viral transcription factors, most critically the viral transactivator Tat and the host nuclear factor κB (NF-κB). This activity is disrupted in infected ce...Replication of HIV-1 requires the coordinated action of host and viral transcription factors, most critically the viral transactivator Tat and the host nuclear factor κB (NF-κB). This activity is disrupted in infected cells that are cultured with extracellular vesicles (EVs) present in human semen, suggesting that they contain factors that could inform the development of new therapeutics. Here, we explored the contents of semen-derived EVs (SEVs) from uninfected donors and individuals with HIV-1 and identified host proteins that interacted with HIV Tat and the NF-κB subunit p65. Integrative network and pathway enrichment analyses of these complexes revealed associations with an array of biological functions regulating gene expression. Several proteins in SEVs bound to both Tat and NF-κB p65: the scaffolding and cell signaling regulatory protein AKAP9, the G protein signaling regulator ARHGEF28, the epigenetic reader BRD2, the small nuclear RNA processor INTS1, and the transcription elongation inhibitor NELFB. When complexed with p65, NELFB also interacted with HEXIM1, another transcription elongation inhibitor, suggesting that SEVs may inhibit HIV-1 propagation through multiple networks of transcriptional activation and repression. Exploring these data and the underlying mechanisms may inform the development of more effective or more durable therapeutics against HIV.
Opioids relieve pain by activating μ-opioid receptors (MORs), which inhibit communication between pain-sensing neurons (nociceptors) and the spinal cord. However, prolonged opioid use can paradoxically lead to increased...Opioids relieve pain by activating μ-opioid receptors (MORs), which inhibit communication between pain-sensing neurons (nociceptors) and the spinal cord. However, prolonged opioid use can paradoxically lead to increased pain sensitivity (hyperalgesia) and reduced analgesic efficacy (tolerance), partly because of the activation of NMDA-type glutamate receptors (NMDARs) at the central terminals of primary sensory neurons in the spinal cord. Here, we identified a critical role for the G protein Gα in this paradox. Pharmacological inhibition of Gα in rats reversed morphine-induced increases in NMDAR phosphorylation, synaptic trafficking, and activity at sensory neuron terminals and reduced morphine-induced excitatory nociceptive input to spinal dorsal horn neurons. Morphine enhanced Gα coupling specifically to metabotropic glutamate receptor 5 (mGluR5) dimers in the spinal cord. Furthermore, targeted knockdown of Gα in dorsal root ganglion neurons in mice normalized NMDAR-related changes and prevented NMDAR-mediated synaptic potentiation triggered by MOR activation. In addition, either pharmacological or genetic disruption of Gα signaling enhanced morphine's analgesic effects while reducing hyperalgesia and tolerance. These findings reveal that Gα signaling contributes to opioid-induced NMDAR hyperactivity at nociceptor central terminals by promoting MOR-mGluR5 cross-talk. Targeting this pathway may improve the safety and efficacy of opioid-based pain management.
In -mutant melanoma cells treated with inhibitors of the kinases BRAF and MEK, a subset of cells rapidly and nongenetically adapts to escape drug-induced quiescence and reenters the cell cycle. Here, we investigated the...In -mutant melanoma cells treated with inhibitors of the kinases BRAF and MEK, a subset of cells rapidly and nongenetically adapts to escape drug-induced quiescence and reenters the cell cycle. Here, we investigated the mechanisms enabling this drug escape by computationally reconstructing single-cell lineages from time-lapse imaging data, linking dynamic signaling pathways to distinct cell-cycle fate outcomes. We found that reactivation of the MEK substrate ERK was necessary but not sufficient to drive escape; rather, the activity of the protein complex mTORC1 was also required to promote cell growth and protein synthesis in drug-treated cells destined for cell-cycle reentry. ERK and mTORC1 signaling converged to increase the abundance of cyclin D1 protein, a critical bottleneck for cell-cycle commitment under drug pressure. In cells in which endogenous cyclin D1 was fluorescently tagged using CRISPR, the subset that escaped drug treatment exhibited marked accumulation of cyclin D1 at least 15 hours before cell-cycle reentry, enabling early prediction of future drug escape. Cyclin D1 thus represents both an early biomarker and potential therapeutic target for suppressing drug escape in melanoma. We observed a similar mTORC1-driven mechanism underlying escape in lung cancer cells, but not colon cancer cells, highlighting partial generalizability across cancer types.
Bacterial small proteins (≤50 amino acids) are an emerging class of regulators that modulate the activity of signaling networks that enable bacterial adaptation to stress. The genome encodes at least 150 small proteins,...Bacterial small proteins (≤50 amino acids) are an emerging class of regulators that modulate the activity of signaling networks that enable bacterial adaptation to stress. The genome encodes at least 150 small proteins, most of which are functionally uncharacterized. We identified and characterized 17 small proteins induced in . during magnesium (Mg) starvation using ribosome profiling, RNA sequencing, and transcriptional reporter assays. Several of these were transcriptionally activated by the PhoQ-PhoP two-component signaling system, which is crucial for Mg homeostasis. Deletion or overexpression of some of these small proteins led to growth defects and changes in cell size under low-Mg conditions, indicating physiological roles in stress adaptation. The small transmembrane protein YoaI, which was transcriptionally induced by the phosphate-responsive PhoR-PhoB signaling pathway, increased in abundance under Mg limitation independently of transcription or PhoQ-PhoP signaling. YoaI activated a third signaling system, EnvZ-OmpR, which mediates responses to osmotic stress. Overall, this study establishes an initial framework for understanding how small proteins contribute to bacterial stress adaptation by facilitating cross-talk between different signaling systems. Our results suggest that these proteins play broader roles in coordinating stress responses, reflecting the interconnected nature of cellular stress networks rather than strictly compartmentalized pathways responding to specific stressors.
Estrogen protects against kidney injury through both genomic and nongenomic mechanisms.Estrogen protects against kidney injury through both genomic and nongenomic mechanisms.
Noradrenaline released from sympathetic neurons accelerates cancer metastasis by activating β-adrenergic receptors (β-adrenoceptors) on tumor cells to promote invasion. We previously showed that the β-adrenoceptor promot...Noradrenaline released from sympathetic neurons accelerates cancer metastasis by activating β-adrenergic receptors (β-adrenoceptors) on tumor cells to promote invasion. We previously showed that the β-adrenoceptor promotes invasive behavior in a metastatic triple-negative breast cancer (TNBC) cell line by activating a cAMP- and calcium-mediated feedforward loop. Here, we found this mechanism in most TNBC lines that have an active β-adrenoceptor. Integrated analysis of transcriptomic datasets revealed , which encodes a developmental homeobox transcription factor, as the most discriminating gene separating cell lines with the feedforward loop and those without it. The high expression of did not correlate with transcriptional changes in integral proteins associated with cAMP or calcium signaling, and immunostaining showed cytosolic localization of Hox-C12, suggesting that it played a nontranscriptional role. Knocking out prevented β-adrenoceptor-mediated calcium signaling and invasion in cultured TNBC cells. In basal breast cancers, expression in tumors negatively correlated with overall and disease-free survival in patients. These findings identify a key mediator, Hox-C12, in the coordination of invasion driven by cAMP and calcium signaling in β-adrenoceptor-responsive TNBC cells.
Three amylin receptors (AMYRs) mediate the metabolic actions of the peptide hormone amylin and are drug targets for diabetes and obesity. AMYR, AMYR, and AMYR are heterodimers consisting of the calcitonin receptor (CTR),...Three amylin receptors (AMYRs) mediate the metabolic actions of the peptide hormone amylin and are drug targets for diabetes and obesity. AMYR, AMYR, and AMYR are heterodimers consisting of the calcitonin receptor (CTR), a G protein-coupled receptor, paired with a RAMP1, RAMP2, or RAMP3 accessory subunit, respectively, which increases amylin potency. Here, we found that the AMYRs had distinct basal subunit equilibria that were modulated by peptide agonists and determined the extent of cAMP signaling downstream of receptor activation. By developing a biochemical assay that resolves the AMYR heterodimers and free subunits, we found that the AMYR and AMYR subunit distributions favored free CTR and RAMPs and that rat amylin promoted association of the constituent subunits of AMYR and AMYR. The agonist αCGRP also induced AMYR subunit association. A stronger interaction between the CTR and the RAMP3 transmembrane domains yielded a more stable AMYR, and human and salmon calcitonin agonists promoted AMYR dissociation. Similar changes in subunit association and dissociation were observed in live-cell membranes, and G protein coupling and cAMP signaling assays showed how these changes altered signaling. Our findings have implications for AMYR biology and drug development and reveal regulation of heteromeric GPCR signaling through subunit interaction dynamics.