G protein-coupled receptors (GPCRs) play key roles in neurodevelopment by regulating excitatory and inhibitory neuronal pathways. The G protein subunit Gα is a downstream effector of inhibitory GPCR signaling, and the ge...G protein-coupled receptors (GPCRs) play key roles in neurodevelopment by regulating excitatory and inhibitory neuronal pathways. The G protein subunit Gα is a downstream effector of inhibitory GPCR signaling, and the gene encoding it () is abundantly expressed in the brain. Rare variants are linked to a severe neurodevelopmental disorder called syndrome, which is characterized by intellectual disability, a range of movement issues, and epilepsy. Here, we investigated the effect of five syndrome-associated missense variants on GPCR signaling. Predictions based on genetic biobank data and in silico modeling suggested that these variants were pathogenic. Compared with the wild-type protein, four Gα variants (T48K, T48I, C224Y, and V332E) showed marked increases in dopamine potency at the dopamine D2 receptor (D2R) and increased constitutive G protein activity when expressed in oocytes. By contrast, the Gα G40C variant was unresponsive to D2R activation. All Gα variants displayed reduced GTP-γ-S binding rates and undetectable GTP hydrolysis, except for the T48I variant, which showed more rapid binding and hydrolysis. Thus, four syndrome variants caused a net gain-of-function effect on D2R signaling, and all studied variants disrupted GTP exchange. These biochemical effects may underlie syndrome, and mutations should therefore be considered when screening for rare neurodevelopmental disorders.
Cytosolic calcium ion (Ca) signatures with specific spatiotemporal patterns play crucial roles in plant responses to biotic and abiotic stresses. The perception of microbe- or damage-associated molecular patterns (MAMPs...Cytosolic calcium ion (Ca) signatures with specific spatiotemporal patterns play crucial roles in plant responses to biotic and abiotic stresses. The perception of microbe- or damage-associated molecular patterns (MAMPs or DAMPs, respectively) initiates cytosolic Ca fluxes that are essential for the induction and spread of pattern-triggered immunity, the first line of plant defense against pathogens, at the cellular, organ, and systemic levels. Here, we quantitatively assessed Ca signatures at the single-cell level, as well as the local traveling Ca waves induced by uniform MAMP or DAMP treatment of cotyledons. MAMPs and DAMPs induced distinct local spatiotemporal Ca responses in epidermal pavement cells, with traveling waves of Ca consistently initiated from a randomly distributed subset of cells and spreading in an approximately radial pattern. These local traveling waves propagated at a slow but constant speed of ~1 micrometer per second and spread to a limited number of neighboring cells. In contrast, wound-induced traveling waves of Ca, which are propagated by the diffusion of molecules that activate Ca channels, displayed a diffusion-like decay pattern that moved rapidly away from the wounded cell but with diminishing speed over time and distance. Mathematical modeling supported a Ca-induced Ca release mechanism that recapitulated the constant wave speed induced by MAMPs. These findings contribute to a deeper understanding of plant defense-related Ca signaling mechanisms, as well as how defense responses are spatially restricted in tissues.
Waves of Ca signals rapidly propagate throughout a plant to help integrate responses to stimuli as diverse as wounding, heat stress, and pathogen attack. In this issue of , Zhang . show that bacterial elicitors and physi...Waves of Ca signals rapidly propagate throughout a plant to help integrate responses to stimuli as diverse as wounding, heat stress, and pathogen attack. In this issue of , Zhang . show that bacterial elicitors and physical injury likely use different molecular mechanisms to produce distinct patterns of Ca waves, shedding light on how such a universal signal leads to stimulus-specific responses.
Blocking the cell cycle is a promising avenue for cancer therapy, with cyclin-dependent kinase 2 (CDK2) emerging as a key target. However, in multiple cell types, the activities of CDK4 and CDK6 (CDK4/6) compensate for C...Blocking the cell cycle is a promising avenue for cancer therapy, with cyclin-dependent kinase 2 (CDK2) emerging as a key target. However, in multiple cell types, the activities of CDK4 and CDK6 (CDK4/6) compensate for CDK2 inhibition and sustain tumor cell proliferation, enabling CDK2 reactivation. Thus, we hypothesized that sensitivity to CDK2 inhibition is linked to the absence of this CDK4/6-mediated compensatory mechanism. We found that cyclin E1-driven ovarian cancers often coexpressed the tumor suppressor p16, which inhibited CDK4/6 signaling. Single-cell time-lapse imaging showed that high abundance of p16 conferred increased sensitivity to CDK2 inhibitors, whereas depletion of p16 rendered cells more resistant to CDK2 inhibition through CDK4/6-dependent compensation. Concordantly, acquired resistance to CDK2 inhibitors correlated with reduced p16 and increased cyclin D1 protein abundance. Multiplexed immunofluorescence of 225 ovarian tumors from patients revealed that 18% of the tumors had high cyclin E1 and p16 expression. Thus, p16 may be a useful biomarker for identifying patients most likely to benefit from CDK2 inhibitors.
Stem cells in hair follicles can be activated after skin injury by fatty acids released from adipocytes.Stem cells in hair follicles can be activated after skin injury by fatty acids released from adipocytes.
Osimertinib is the mainstay of therapy for patients with non-small cell lung cancer (NSCLC) driven by the receptor tyrosine kinase (RTK) EGFR. In most patients, however, therapeutic pressure promotes RTK-dependent mechan...Osimertinib is the mainstay of therapy for patients with non-small cell lung cancer (NSCLC) driven by the receptor tyrosine kinase (RTK) EGFR. In most patients, however, therapeutic pressure promotes RTK-dependent mechanisms that support tumor cell survival and the emergence of osimertinib resistance. Here, we found that inhibiting the proximal RTK signaling intermediate SOS1 promoted continued osimertinib efficacy in sensitive cells and restored sensitivity in cells with acquired resistance. In three-dimensional spheroid cultures of naïve NSCLC cells, SOS1 inhibition enhanced osimertinib potency by limiting the reactivation of RTK-dependent adaptive Ras effectors. SOS1 inhibition resensitized drug-tolerant persister cells to osimertinib, and knockout or inhibition of SOS1 reduced the frequency of tumor-initiating cells to curb spheroid growth in situ and tumorigenesis in vivo. SOS1 inhibition further limited the development of acquired osimertinib resistance and resensitized resistant cells to osimertinib. In mice, tumors regressed nearly completely when treated with either osimertinib or a combination of osimertinib and a SOS1 inhibitor, with the combination providing a slightly greater effect. However, only the combination delayed tumor regrowth after treatment removal. Our data provide a mechanistic rationale for the clinical investigation of combining SOS1 inhibitors with osimertinib to achieve more durable responses and suppress resistance in NSCLC.
Protein domains are conserved structural and functional units that serve as the building blocks of proteins. Through evolutionary expansion, domain families are represented by multiple members in diverse configurations w...Protein domains are conserved structural and functional units that serve as the building blocks of proteins. Through evolutionary expansion, domain families are represented by multiple members in diverse configurations with other domains, evolving new specificities for their interacting partners. Here, we developed a structure-based interface analysis to map domain interfaces from experimental and predicted structures, including interfaces with macromolecules and intraprotein interfaces. We hypothesized that contact mapping of domains could yield insights into domain selectivity and the conservation of domain-domain interfaces across proteins and identify conserved posttranslational modifications (PTMs) relative to interaction interfaces, enabling the inference of specific effects as a result of PTMs or mutations. We designed this modular approach as an open-source Python package called Comprehensive Domain Interface Analysis of Contacts (CoDIAC) and applied it to the family of human Src homology 2 (SH2) domains, a modular unit central to phosphotyrosine-mediated signaling. CoDIAC revealed coordinated regulation of SH2 domain binding interfaces by tyrosine and serine/threonine phosphorylation and acetylation that may underlie binding selectivity. These findings suggest that multiple signaling systems can regulate protein activity and SH2 domain interactions in a coordinated manner. Applying CoDIAC to the study of other protein domains should provide insights into their binding interfaces and molecular interactions.
Cinat D, van der Wal R, Baanstra M
… +8 more, Soto-Gamez A, Maturi R, Jellema-de Bruin AL, Brouwer U, van Goethem MJ, van Vugt MATM, Barazzuol L, Coppes RP
The goal of radiotherapy in cancer treatment is to maximize DNA damage in tumors while minimizing harm to surrounding healthy tissues, especially to stem and progenitor cells essential for tissue regeneration and organ f...The goal of radiotherapy in cancer treatment is to maximize DNA damage in tumors while minimizing harm to surrounding healthy tissues, especially to stem and progenitor cells essential for tissue regeneration and organ function. Here, we investigated the molecular responses to photon and proton irradiation, two key modalities in head and neck cancer treatment. Multiomics and in vitro analyses revealed that both photon and proton irradiation of mouse salivary gland organoids induced similar early responses, including DNA damage, micronuclei formation, increased amounts of the cytosolic DNA sensor cGAS, and type I interferon (IFN-I) signaling. In addition, both types of radiation induced comparable increases in the release of mitochondrial DNA (mtDNA) into the cytoplasm and stimulated the production of ZBP1, a cytosolic nucleic acid sensor involved in mtDNA recognition. However, proton irradiation resulted in a more pronounced loss of heterochromatin regulators and derepression of transposable elements at later times after irradiation, which was accompanied by increased accumulation of intracellular double-stranded RNA (dsRNA) and an enhanced RIG-I-mediated IFN-I response. Genetic and pharmacological modulation demonstrated its critical role for IFN-I signaling in enhancing salivary gland stem and progenitor cell activity after irradiation in vitro and in vivo. Our findings reveal more pronounced molecular changes after proton irradiation as compared with photon irradiation and uncover a proregenerative role of IFN-I signaling in the salivary gland, suggesting this pathway as a promising therapeutic target to mitigate radiation-induced side effects.
Innate immune signaling is important for tissue regeneration. In this issue of , Cinat . report that a cell-intrinsic type I interferon (IFN-I) response drives salivary gland organoid regeneration after radiation and tha...Innate immune signaling is important for tissue regeneration. In this issue of , Cinat . report that a cell-intrinsic type I interferon (IFN-I) response drives salivary gland organoid regeneration after radiation and that proton irradiation induces a stronger regenerative response than photon irradiation because it amplifies the IFN-I response.
Chimeric antigen receptor (CAR) T cells have demonstrated unprecedented success in treating relapsed or refractory blood cancers. Previous studies of the mechanisms underlying the interactions and responses of CAR T cell...Chimeric antigen receptor (CAR) T cells have demonstrated unprecedented success in treating relapsed or refractory blood cancers. Previous studies of the mechanisms underlying the interactions and responses of CAR T cells and their targets have largely ignored the responses of tumors to CAR ligation. We compared the signaling of a second-generation, ligand-based CAR built from colony-stimulating factor 1 (CSF1) to target the CSF1 receptor (CSF1R) on target cells with a conventional, single-chain variable fragment-based CAR against the B cell antigen CD19. Using SILAC coculture with phosphotyrosine enrichment and LC-MS/MS analysis, we showed that ligation of CSF1R-expressing THP-1 cells with CSF1R-CAR T cells stimulated CSF1R-like signaling in the THP-1 cells. In contrast, no target cell signaling response was observed after the ligation of CD19-CAR T cells with target Raji cells. Using small-molecule inhibitors of the tyrosine kinase Lck, actin polymerization, and CSF1R, we found that CAR-induced CSF1R signaling in THP-1 cells depended exclusively on the kinase activity of CSF1R with no participation from T cell activation. Consistently, CSF1R-CAR T cells promoted THP-1 cell proliferation at low effector-to-target ratios but prevented THP-1 cell proliferation at high effector-to-target ratios. Our data provide evidence for CAR-induced signaling in target cells, an unintended consequence of CARs that may have implications for the choice of CAR antigen for optimal clinical efficacy.
Intracellular pH dynamics regulate many cell biological processes. We developed a computational pipeline to identify pH-sensitive proteins and the molecular mechanisms that regulate their pH-dependent activity. By applyi...Intracellular pH dynamics regulate many cell biological processes. We developed a computational pipeline to identify pH-sensitive proteins and the molecular mechanisms that regulate their pH-dependent activity. By applying the pipeline to the phosphatase SHP2, which regulates signaling pathways that control pH-dependent cellular processes, we found that SHP2 phosphatase activity was sensitive to pH in vitro and in cells and that mutation of His and Glu abolished SHP2 pH-sensitive activity. We also found that the activity of the kinase SRC was pH dependent and that mutations in a network of ionizable amino acids abolished pH-sensitive activity. Furthermore, we found that SRC kinase activity was pH sensitive even in the presence of the growth factor EGF, which stimulates SRC activity in a phosphorylation-dependent manner, or with a phosphomimetic substitution (Y527E) that promotes SRC autoinhibition. These data suggest that pH-sensitive regulation functions in concert with established phosphorylation-dependent mechanisms to regulate SRC kinase activity. Constant pH molecular dynamics simulations performed on both SHP2 and SRC supported allosteric regulation mediated by pH-dependent binding of inhibitory SH2 domains to the respective catalytic domain in each protein. We also identified evolutionarily conserved putative pH-sensing networks in other SH2 domain-containing signaling proteins. Together, our computational, biophysical, and cellular analyses reveal a role for intracellular pH dynamics in allosterically regulating the activities of modular SH2 signaling proteins to control cell biology.
Citrullination enhances the tumor-suppressor function of p53 by redirecting its gene target selectivity.Citrullination enhances the tumor-suppressor function of p53 by redirecting its gene target selectivity.
The penetrant D463H mutation in , which encodes the kinase PKCα, is a biomarker and driver of chordoid glioma, a type of brain cancer. Here, we found that heterozygous knock-in expression of the D463H mutant in mice elic...The penetrant D463H mutation in , which encodes the kinase PKCα, is a biomarker and driver of chordoid glioma, a type of brain cancer. Here, we found that heterozygous knock-in expression of the D463H mutant in mice elicited the development of chondrosarcomas. The mutant protein kinase was catalytically inactive, but no such oncogenic phenotype was observed for the related inactivating mutation D463N, indicating that the lack of activity per se was not the cause of the oncogenicity of the D463H mutant. In cultured glioma cells, the behavior of the D463H mutant closely mirrored that of wild-type PKCα and retained ATP binding, unlike the related D463N mutant. Mechanistically, PKCα D463H displayed quantitative alterations in its interactome compared with that of the wild-type kinase, with enhanced association with epigenetic regulators. This change in the interactome aligned with transcriptomic changes that resembled an increased PKCα-induced expression program, with enhanced gene signatures mediated by BRD4, MYC, and TGF-β. D463H expression reduced the sensitivity of cells to the BET inhibitors JQ1 and AZD5153, indicating the functional importance of these pathways. The findings indicate that D463H is a dominant gain-of-function oncogenic mutant that operates through a noncatalytic allosteric mechanism.
The glycocalyx is a proteoglycan-rich layer present on the surface of all mammalian cells and is particularly prevalent on endothelial cells lining the vasculature. The glycocalyx is thought to affect leukocyte migration...The glycocalyx is a proteoglycan-rich layer present on the surface of all mammalian cells and is particularly prevalent on endothelial cells lining the vasculature. The glycocalyx is thought to affect leukocyte migration by masking adhesion molecules and reducing leukocyte adhesion to the endothelium. Leukocyte recruitment is a key driver of inflammatory diseases, including psoriasis. Here, we found that leukocytes had the glycocalyx component heparan sulfate on their cell surface and that it was lost in response to psoriasis-like skin inflammation. In contrast, endothelial heparan sulfate was not affected. Treatment with a heparan sulfate mimetic during psoriasis-like skin inflammation in mice protected heparan sulfate from cleavage by myeloid cell-derived heparanase and resulted in reduced leukocyte accumulation in the skin. However, clinical signs of inflammation were increased because of the reduced numbers of T regulatory cells that were recruited. These findings refine our understanding of immune cell recruitment by revealing the presence and function of a heparan sulfate glycocalyx on immune cells and highlight the complex effects of heparanase inhibitors on the immune response in this context.
Savitsky M, Larasati YA, Solis GP
… +10 more, Koval A, Camacho A, Núñez N, Patat O, Wallach E, Galosi S, Novelli M, Martinelli S, Leuzzi V, Katanaev VL
The α subunits of heterotrimeric guanine nucleotide-binding proteins (G proteins) in G protein-coupled receptor (GPCR) pathways operate as gate switches by cycling through inactive GDP-bound and active GTP-bound states....The α subunits of heterotrimeric guanine nucleotide-binding proteins (G proteins) in G protein-coupled receptor (GPCR) pathways operate as gate switches by cycling through inactive GDP-bound and active GTP-bound states. Three flexible regions (switches I to III) of Gα subunits undergo the most substantial conformational rearrangements and determine interactions with receptors, signaling regulators, and effectors. Here, we describe three patients with severe pediatric encephalopathy harboring c.723+1G>A or c.723+2T>A variants in the splicing donor site of intron 6 of , the gene encoding the major neuronal G protein α subunit Gα. These substitutions destroyed the conserved GU sequence of the pre-mRNA and rendered the donor site unrecognizable, prompting cryptic splice site engagement and production of the dominant pathogenic Gα[V234_T241del] variant, which lacked switch III (amino acid residues 232 to 243 in Gα). Structural, biochemical, and cellular analyses characterized V234_T241del as a strong neomorphic variant that was severely deficient in guanine nucleotide handling and cellular interactions and sensitive to zinc salts, an emerging targeted treatment for a encephalopathy subgroup. Our studies provide pathological and molecular insights into the function and effect of removal of an entire switch region in 1 of the 16 human G protein α subunits that underlie multiple physiological processes and diverse pathologies.
Sensory neurons promote bone homeostasis and repair by providing osteopontin to skeletal stem cells.Sensory neurons promote bone homeostasis and repair by providing osteopontin to skeletal stem cells.
Metabolic dysfunction-associated steatotic liver disease (MASLD) is linked to a shift in the composition of the gut microbiome. Here, we found that depletion of the gut microbiome in adult zebrafish led to the developmen...Metabolic dysfunction-associated steatotic liver disease (MASLD) is linked to a shift in the composition of the gut microbiome. Here, we found that depletion of the gut microbiome in adult zebrafish led to the development of steatotic liver disease in animals on standard diets. Using metabolomics and isotope tracing, we found that dietary glucose was converted to sorbitol by host intestinal cells. Although bacteria degraded sorbitol in control animals, sorbitol was transferred to the livers of fish when the gut microbiome had been depleted. Within the liver, sorbitol was converted into fructose 1-phosphate, which subsequently activated glucokinase and increased glycolytic flux, leading to increased hepatic glycogen and fat content. Inhibition of sorbitol production in microbiome-depleted animals was sufficient to prevent the development of steatotic liver, and colonizing the intestines of microbiome-depleted fish with sorbitol-degrading bacterial strains rescued the steatotic liver phenotype. Conversely, exogenous administration of high concentrations of sorbitol phenocopied gut microbiota depletion and induced hepatic steatosis. Together, these findings show that sorbitol-degrading bacteria in the gut protect against steatotic liver disease and suggest that excessive intake of dietary sorbitol may pose a risk for the development of MASLD.
The transcriptional coactivator YAP1 regulates numerous biological processes, including organ size control and tissue homeostasis. Although its hyperactivity promotes tumor development and progression, YAP1 itself is not...The transcriptional coactivator YAP1 regulates numerous biological processes, including organ size control and tissue homeostasis. Although its hyperactivity promotes tumor development and progression, YAP1 itself is not yet druggable. Here, we found that the poly(ADP-ribose) polymerase PARP1 promoted the transcriptional activity of YAP1-TEAD4 complexes that mediate breast cancer cell stemness, metastatic behavior, and evasion of antitumor immunity. This PARP1-mediated mechanism was independent of its role in the DNA damage response. Specifically, PARP1 directly interacted with and promoted the formation of the YAP1-TEAD4 complex by PARylating TEAD4 at a conserved Arg-Lys sequence. This PARP1-enhanced YAP1-TEAD4 binding attenuated the interaction between YAP1 and the E3 ubiquitin ligase CRL4, thus preventing its ubiquitylation and degradation. Furthermore, the abundance of PARP1 protein correlated with that of YAP1 and the immune checkpoint protein PD-L1 in breast cancer tissues and cell lines. In a mouse model of triple-negative breast cancer, pharmacological inhibition of PARP1 enhanced the ability of antibody blockade of PD-L1 to increase cytolytic and tumor-suppressive T cell infiltration and reduce tumor growth. The findings reveal a mechanism that promotes YAP1-TEAD4 transcriptional activity and immune escape in breast cancer cells and is targetable with clinically approved therapies.
Stem cells reside in niches that determine how they respond to injury to expand in number, migrate to the injury site, and differentiate into cells to regenerate lost tissue. Suture stem cells (SuSCs) are important for h...Stem cells reside in niches that determine how they respond to injury to expand in number, migrate to the injury site, and differentiate into cells to regenerate lost tissue. Suture stem cells (SuSCs) are important for homeostasis and regeneration of cranial bone and can be used as a model to understand stem cell regulation for bone regeneration at a distance. Using a mouse cranial bone injury model, we identified the chemokine Cxcl12 and the Hedgehog family ligands Shh and Ihh as injury-induced niche factors that coordinately promoted the proliferation, directional migration, and osteoblastic differentiation of the Gli1 subset of SuSCs from the sagittal suture. Cxcl12 was constitutively produced in the SuSC niche, induced at the injury site, and activated its cognate receptor Cxcr4 on Gli1 SuSCs to stimulate Gli1 SuSC proliferation and migration to the injury site. Cxcl12-Cxcr4 signaling also induced the production of Shh and Ihh, which promoted Gli1 SuSC proliferation and osteoblastic differentiation. Furthermore, expressing loss- or gain-of-function mutant forms of the G protein Gα, which cause inherited diseases characterized by cranial bone defects, led to aberrant Cxcl12, Shh, and Ihh signaling during regeneration and resulted in cranial bone phenotypes similar to those in human patients. Our results indicate that the injury-induced niche factors Cxcl12, Shh, and Ihh orchestrate SuSC activation and migration to promote injury repair and suggest that disrupting this system impairs regeneration and contributes to human disease.
Ligands of the WNT family induce formation of the WNT receptor signalosome and promote stabilization of the transcriptional coactivator β-catenin. The homologous transmembrane E3 ubiquitin ligases ZNRF3 and RNF43 inhibit...Ligands of the WNT family induce formation of the WNT receptor signalosome and promote stabilization of the transcriptional coactivator β-catenin. The homologous transmembrane E3 ubiquitin ligases ZNRF3 and RNF43 inhibit WNT-dependent stabilization of β-catenin by stimulating the degradation of the WNT receptor FZD, whereas the secreted R-spondin proteins promote the stabilization of FZD by inducing the degradation of ZNRF3 and RNF43. Here, we report that the R-spondin-induced stabilization of β-catenin in HEK293 cells was not mimicked by FZD overexpression, highlighting a gap in our understanding of this important regulatory mechanism. Contrary to the conventional view that ZNRF3 constitutively mediates the ubiquitylation and degradation of FZD, we found that ZNRF3-induced FZD degradation depended on endogenous WNT and that ZNRF3 selectively degraded WNT-engaged FZD. WNT enhanced the association between FZD and the intracellular adaptor protein DVL, and DVL subsequently recruited ZNRF3 to FZD to promote FZD degradation. Our data suggest that WNT signaling actively restricts itself through ZNRF3-dependent degradation of WNT-engaged FZD and that R-spondin enhances WNT signaling by prolonging the action of the WNT-engaged FZD complex, rather than by simply increasing the abundance of FZD on the cell surface.