Science Signaling

Protein S-palmitoylation is a reversible lipid modification that regulates protein trafficking, membrane association, and synaptic signaling, yet its role in learning-induced hippocampal plasticity remains incompletely understood. Here, we investigated how spatial learning remodels the hippocampal palmitoylome in rats trained in a Morris water maze using short-term training (1 session, 15 trials; test at 1 h, STT) or long-term training (4 sessions over 4 days; 4 trials/session; test at 24 h, LTT). Palmitoylated proteins were profiled using acyl-biotin exchange followed by tandem mass tag labeling and LC-MS/MS. In total, 5,260 proteins were identified, including 763 palmitoylated species. Spatial learning induced robust and time-dependent remodeling of protein S-palmitoylation, with pronounced differences between STT and LTT. Comparison of trained and yoked controls revealed 186 differentially palmitoylated proteins (DPPs) in STT and 62 in LTT, indicating stronger early molecular reorganization. Notably, yoked animals also displayed substantial palmitoylation changes versus cage controls, indicating that locomotor activity and mild stress independently reshape the hippocampal palmitoylome. DPPs were broadly distributed across cellular compartments, with enrichment of synaptic proteins at both stages. STT preferentially engaged functional enrichment in synaptic vesicle cycling, GTPase signaling, cytoskeletal remodeling, mitochondrial metabolism, and secretory pathways, whereas LTT was associated with protein translation, synaptic membrane organization, and structural plasticity, consistent with consolidation processes. Protein-protein interaction and KEGG analyses supported a transition from widespread early network remodeling toward more selective regulation of synaptic and translational machinery. Site-specific analysis further identified previously unreported palmitoylation sites in rat hippocampal proteins. Together, these data demonstrate that spatial learning dynamically reshapes the hippocampal palmitoylome in a temporally structured manner, suggesting a key role for S-palmitoylation in coordinating metabolic and synaptic adaptations underlying memory formation.

G protein-coupled receptors rely on dynamic conformational changes to coordinate G protein activation and recruitment of regulatory transducers such as G protein-coupled receptor kinases and {beta}-arrestins. The chemotactic receptor GPR183 has been implicated in a context-dependent role in hematological malignancies. Here, we investigated the impact of A338V mutation located within the C-terminal tail of GPR183. This mutation is associated with acute myeloid leukaemia. Using bioluminescence resonance energy transfer-based assays in HEK293A cells, we assessed receptor-proximal signaling events. The A338V variant displayed preserved agonist potency and comparable agonist-induced Gi activation relative to wild type, although constitutive activity towards Gi was modestly reduced. In contrast, recruitment of GRK2 and {beta}-arrestin2 was consistently impaired across multiple assay configurations. These differences were not attributable to altered receptor abundance, as the C-tail untagged mutant exhibited increased plasma membrane expression despite reduced regulatory transducer engagement. While intramolecular conformational biosensor measurements revealed subtle differences in global receptor conformation between WT and A338V, extensive molecular dynamics simulations supported the altered conformational sampling of the C-terminal tail in the A338V variant. Together, these data support a model in which the A338V substitution selectively alters C-terminal structural dynamics, impairing GRK2 and {beta}-arrestin2 recruitment while preserving G protein activation.

Many intracellular pathogens stimulate host cell stress by directly or indirectly causing an imbalance in host nutrients; depletion of amino acid pools in particular can act as a danger signal to infected cells. Using a restrictive host model of Salmonella enterica serovar Typhi (S. Typhi) infection, we identify early induction the integrated stress response (ISR) by viable bacteria, but not heat-killed bacteria. Genetic deletion of the amino acid sensing ISR kinase GCN2 (also known as EIF2AK4) prevented early ISR activation during S. Typhi infection, and murine macrophages lacking GCN2 show impaired bacterial clearance and decreased cytokine output. Supplementation of wildtype C57BL/6 murine macrophages with only the non-essential amino acid asparagine was sufficient to suppress S. Typhi-induced ISR activation and deletion of S. Typhi ansB, encoding an asparaginase, prevented ISR activation during infection. Pharmacological inhibition of mammalian target of rapamycin (mTOR), the other major amino acid sensing pathway in eukaryotic cells, prevented GCN2 activation and ISR induction in murine macrophages, indicating an upstream role for mTOR in signaling to GCN2. These findings suggest a role for the ISR in macrophage innate immune responses to S. Typhi infection and highlight a potential difference in nutrient-dependent signaling between the S. Typhi-susceptible human host and the restrictive murine host centered around asparagine, mTOR, and GCN2.

Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system that eliminate virus-infected and transformed cells through the formation of a specialized immune synapse. Effective target cell killing requires coordinated plasma membrane remodeling and dynamic reorganization of the actin and microtubule cytoskeletons, enabling centrosome polarization and directed secretion of lytic granules. The scaffold protein CIP4 has emerged as an important regulator of cytoskeletal coordination in NK cells, yet how its subcellular localization is controlled during NK cell activation is unknown. CIP4 contains a unique protein kinase A (PKA) phosphorylation site (threonine 225, T225) within its F-BAR domain, a domain that mediates interactions with microtubules and the plasma membrane. We hypothesized that localized PKA signaling controls CIP4 redistribution during immune synapse assembly. To test this hypothesis, we analyzed CIP4 localization and phosphorylation in NK cells engaged with sensitive target cells using biochemical and imaging approaches. We show that NK-target cell interaction enhances PKA activity and promotes phosphorylation of CIP4, coinciding with its delocalization from microtubules and accumulation at the immune synapse. Importantly, this relocalization process requires the PKA-anchoring protein AKAP350, which positions PKA and CIP4 within the same protein complex, thereby facilitating CIP4 phosphorylation. Consistently, pharmacological inhibition of PKA prevented CIP4 delocalization from microtubules and reduced its accumulation at the immune synapse. The non-phosphorylatable CIP4 mutant T225A displayed increased association with microtubules compared with a phosphomimetic mutant, identifying phosphorylation at T225 as a key determinant of CIP4 spatial regulation. Together, these findings identify a signaling mechanism that links compartmentalized PKA activity to the spatial control of CIP4 during immune synapse formation, providing new insight into the molecular mechanisms governing immune synapse maturation.

Crosstalk across two major receptor families involved in signal transduction, namely receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs), have been observed at different levels of their signaling cascades. Using newly developed enhanced bystander bioluminescence resonance energy transfer (ebBRET)-based biosensors that monitor the recruitment of SH2 domains to activated RTKs, we assessed the ability of GPCRs to modulate cellular localization of SH2 domains. Receptor-mediated activation of either Gq/11 or G12/13 but not Gs or Gi/o (e.g., thromboxane A2 receptor, TP, and type-2 protease activated receptor, PAR2) resulted in the plasma membrane (PM) dissociation of SH2 domains derived from RTKs effectors such as GRB2, STAT5 and PLC{gamma}1. The role of Gq/11, G12/13, Rho and downstream kinases in the subcellular SH2 domain redistribution was further confirmed using both pharmacological and genetic approaches. BRET imaging and spectrometric analyses showed that the dissociation of SH2 domains from the PM was accompanied by their accumulation in the nucleus and a reduction in RTK signaling activity, as determined using a STAT5 transcriptional assay. The effect of Gq/11 and G12/13 activation on STAT5 transcriptional activity was observed both in engineered systems and in HeLa cells endogenously expressing all the components of the regulatory mechanism. The Gq/11 / G12/13-mediated redistribution of SH2 domain-containing proteins represents an undescribed mechanism through which GPCRs regulate RTKs activity. Significance StatementThis study reveals a novel crosstalk mechanism between G protein coupled receptors and receptor tyrosine kinases showing that Gq/11 and G12/13 activation triggers Rho-dependent translocation of SH2-containing effector proteins, such as GRB2, PLC{gamma}1 and STAT5. This process causes compartmentalization inside the nucleus and thus reduces their availability at the plasma membrane, leading to attenuated RTK responses.

Post-traumatic stress disorder (PTSD) involves complex neuroimmune-synaptic crosstalk in the hippocampus. Here we show that interleukin-18 (IL-18), an extensively studied pro-inflammatory cytokine, serves a protective role against traumatic fear memory through a microglia-to-neuron signaling axis. Traumatic stress induces sustained upregulation of IL-18 in the hippocampus. Exogenous IL-18 administration attenuates fear memory, whereas blockade of IL-18 signaling exacerbates it. Mechanistically, microglial-derived IL-18 acts on neuronal IL-18R1 to restore stress-impaired synaptic plasticity and reduce perineuronal net density, thereby facilitating structural synaptic remodeling. In addition, IL-18 modulates the synaptic organization of fear memory-encoding engram cells within hippocampal ensembles. Together, these findings redefine IL-18 as a homeostatic regulator of post-trauma hippocampal synaptic function and identify the hippocampal IL-18 pathway as a potential therapeutic target for PTSD.

Corticotroph cells convert hypothalamic inputs into adrenocorticotrophic hormone secretion via intracellular calcium (Ca2+) signalling, but how they integrate corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP) across physiological concentration ranges remains unclear. Here, we quantified intracellular Ca2+ responses in isolated rat corticotrophs to CRH and AVP, applied alone and in combination, to characterise response magnitude, temporal dynamics, and cell recruitment. Both secretagogues increased Ca2+ signalling in a concentration-dependent manner, but with distinct effects: AVP generally evoked larger responses, faster response onset, and greater cell recruitment than CRH when applied alone. Under co-stimulation, increasing CRH concentration increased the proportion of cells classified as synergistic without altering positive synergy values, suggesting CRH-dependent control of interaction likelihood rather than interaction strength. Marked cell-to-cell heterogeneity was observed across all conditions, consistent with corticotroph subpopulations differing in activation thresholds. Together, these findings show that AVP drives broad corticotroph recruitment, whereas CRH modulates how corticotrophs integrate convergent inputs, defining complementary roles in shaping pituitary output.

Desensitization and internalization of most G protein-coupled receptors (GPCRs) depend on phosphorylation by GPCR kinases (GRKs), promoting {beta}-arrestin recruitment. Atypical chemokine receptors (ACKRs), including ACKR3, are structurally related to classical chemokine receptors but do not activate heterotrimeric G proteins. ACKR3 signaling and trafficking have been proposed to depend on GRK5-mediated phosphorylation and {beta}-arrestin interaction. However, the respective roles of {beta}-arrestins, GRKs, and receptor phosphorylation in chemokine scavenging and in constitutive or ligand-induced trafficking remain debated. Using bioluminescence resonance energy transfer (BRET)-based biosensors and immunofluorescence imaging with fluorescently labeled receptors and chemokines, we examined ACKR3 interaction with {beta}-arrestin1/2 and assessed chemokine scavenging and receptor trafficking in {beta}-arrestin-deficient ({Delta}{beta}arr1/2) cells. We also evaluated the contribution of GRK-mediated phosphorylation. {beta}-arrestins supported agonist-independent receptor internalization but were dispensable for chemokine-induced internalization and chemokine scavenging. In contrast, GRKs were required for ligand-promoted endocytosis, with either GRK2/3 or GRK5/6 being sufficient. Mutation of ACKR3 phosphorylation sites impaired {beta}-arrestin recruitment but did not completely block internalization and scavenging, whereas complete C-terminal truncation abolished both processes. Consistently, kinase-dead GRK2 rescued ACKR3 endocytosis in {Delta}GRK2/3/5/6 cells, indicating a scaffolding role partially independent of kinase activity. Moreover, G{beta}{gamma} was not required for GRK2-mediated ACKR3 endocytosis, as a PH-domain-deleted GRK2 mutant restored internalization in {Delta}GRK2/3/5/6 cells, and G{beta}{gamma} sequestration by {beta}ARKct-CAAX did not inhibit this process consistent with the notion that ACKR3 does not promote G protein activation. Thus, ligand-promoted ACKR3 internalization and chemokine scavenging occur independently of {beta}-arrestins but requires GRKs. One-sentence summaryGRKs are essential for ACKR3 endocytosis and chemokine scavenging, whereas {beta}-arrestins and receptor phosphorylation are dispensable.

Novel (nua) Kinase 1 (NUAK1) encodes a serine-threonine protein kinase, mutations in which are associated with autism spectrum disorder. Direct phosphorylation targets of NUAK1 have been elusive hindering mechanistic understanding of its role in brain development. Here, we characterize autism-associated NUAK1 variants and show their differential impact on catalytic activity and subcellular distribution. We engineered ATP-analog sensitive NUAK1 and utilized its specificity towards bulky analogs to identify over 30 hitherto unknown direct phosphorylation targets of NUAK1. We demonstrate that Pleckstrin Homology and Sec7-domain containing protein 3 (PSD3) is a bona fide phosphorylation target of NUAK1. A guanine exchange factor (GEF) for ARF6 GTPase, PSD3 is phosphorylated by NUAK1 at S476. Expression of phosphodeficient PSD3 leads to aberrant activation of ARF6 and generation of PI(4,5)P2 that accumulates in intracellular vesicles. In neurons, phosphomutant PSD3 leads to enhanced spine maturation in an ARF6 dependent fashion. This study reveals direct neuronal substrates of an autism risk gene NUAK1, and delineates a mechanism by which PSD3 phosphorylation regulates ARF6 activation and spine maturation.

Macrophages are abundant in the colorectal tumour microenvironment and can alter drug response. Using mouse Apc/Kras/Trp53 (AKP) colorectal cancer organoids, we found that macrophages and/or macrophage-conditioned medium reduced sensitivity to the MEK inhibitor trametinib and the pan-RAS inhibitor RMC-6236. In contrast, macrophage-conditioned medium had little effect on regorafenib and increased sensitivity to dabrafenib, suggesting that resistance depends on the inhibitory profile of each drug. Secretome profiling identified PDGF-BB and GDF-15 as candidate mediators. Adding both ligands to organoid medium reproduced much of the conditioned-medium effect, whereas either ligand alone was insufficient. Inhibition of PDGFR or RET partially reduced drug resistance, suggesting that PDGF-BB and GDF-15 likely act through canonical signalling by that additional macrophage-derived signals also contribute. Kinome profiling pointed to increased tyrosine kinase signalling during trametinib treatment, with SRC family kinases emerging as a key downstream node. Consistent with this, SRC inhibition reduced the difference between control and conditioned-medium responses. The multi-kinase inhibitor masitinib--which targets several kinases along this resistance network--strongly restored sensitivity to trametinib and RMC-6236. Together, these data define a macrophage-driven resistance network in KRAS-mutant colorectal cancer organoids and support combined inhibition of RAS-pathway and tyrosine kinase signalling.

Progesterone (P4) plays key roles in reproductive and metabolic function and signals through two receptor classes: classical nuclear receptors that regulate gene transcription and membrane progesterone receptors (mPR) that mediate rapid, non-genomic signaling. Whether mPR signaling influences systemic glucose homeostasis remains unclear. Here, we investigated whether mPR activation regulates glucose homeostasis and insulin sensitivity. Using the selective mPR agonist OD02-0, we show that mPR activation enhances glucose uptake in skeletal muscle and hepatocytes, associated with AMP-activated protein kinase (AMPK) activation. In HepG2 cells, mPR activation induces metabolic reprogramming characterized by reduced mitochondrial respiration and increased glycolytic flux. Pharmacological inhibition of AMPK suppresses this effect, indicating that these responses require AMPK activity. In diet-induced obese mice, chronic mPR activation reduces fasting glucose and insulin levels, improves glucose tolerance, and restores glucose-stimulated insulin secretion without detectable toxicity. Integrated proteomic and phosphoproteomic analyses in mouse liver reveal modulation of AMPK signaling and inhibition of mTORC1. Transcriptomic changes were limited, supporting a predominantly non-genomic mode of action. Together, these findings identify mPR signaling as a regulator of glucose homeostasis that engages central energy-sensing pathways to improve metabolic control in obesity.

Klebsiella pneumoniae is an opportunistic bacterial pathogen associated with high morbidity and mortality, exacerbated by the rapid emergence of resistance to last-resort antibiotics, such as carbapenems. Adaptation to nutrient limitation, particularly fluctuations in metal availability, is critical for bacterial survival and virulence, yet the regulatory mechanisms coordinating these responses remain incompletely understood. Protein phosphorylation represents a key post-translational modification governing bacterial physiology and offers a promising avenue for identifying novel antimicrobial targets. Here, we applied mass spectrometry-based phosphoproteomics to define nutrient-responsive signaling networks in K. pneumoniae under varying iron and zinc conditions. This analysis identified iron-dependent phosphorylation of TolQ, a conserved inner membrane component of the Tol-Pal system that maintains cell envelope integrity. Structural modeling predicted that phosphorylation modulates TolQ-TolR conformation, suggesting a mechanism by which iron availability regulates Tol-Pal function. Functional characterization demonstrated that deletion of tolQ results in reduced bacterial viability, increased susceptibility to host immune clearance, and heightened sensitivity to antibiotic treatment. To further explore the therapeutic potential of this pathway, we integrated high-throughput compound screening with computational modeling and identified small molecules that phenocopy {Delta}tolQ. Collectively, these findings reveal a previously unrecognized link between iron availability and phosphoregulation of the Tol-Pal system and establish TolQ as a critical mediator of bacterial survival. This work highlights phosphoproteomics as a powerful strategy to uncover regulatory vulnerabilities and identify targets for antimicrobial development in drug-resistant pathogens.

The multiple endocrine neoplasia type 1 (MEN1) gene encodes Menin, a nuclear scaffold protein and tumor suppressor that regulates transcription. It is frequently mutated in endocrine neoplasia. MEN1-gastrinomas are aggressive neuroendocrine tumors (NETs) that arise predominantly in the submucosal Brunners glands of the duodenum, an organelle rich in extracellular growth factors. Many duodenal NETs retain wild-type MEN1 allele and nuclear Menin, suggesting post-translational inactivation of its tumor-suppressor function. The Menin C-terminal domain (CTD) contains a conserved phosphorylation site at Ser487 within the first of three nuclear localization signals (NLS1-3). We hypothesized that extracellular signaling regulates Menin by phosphorylating the CTD at Ser487 blocking its nuclear localization. Using CTD deletion mapping, site-directed mutagenesis, and kinase activation in gastric cell lines, we show that loss of NLS1-3 reduces Menins nuclear localization, stability, and repression of GASTRIN. Cell stimulation by epiregulin, forskolin, or phorbol ester induced Menin Ser487 phosphorylation and its nuclear translocation, relieving repression of GASTRIN. The phospho-mimetic S487D mutant remained cytoplasmic and phenocopied CTD deletion of NLS1-3 sustaining de-repression of GASTRIN. These findings showed that Ser487 phosphorylation restricts nuclear accumulation of Menin and functionally links extracellular signaling to post-translational modification of Menin that ultimately contributes to transcriptional derepression and neuroendocrine tumorigenesis. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=127 HEIGHT=200 SRC="FIGDIR/small/717082v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1f96df4org.highwire.dtl.DTLVardef@a1db4borg.highwire.dtl.DTLVardef@4435f9org.highwire.dtl.DTLVardef@3373b3_HPS_FORMAT_FIGEXP M_FIG C_FIG

Migration of leukocytes in the context of immune homeostasis or inflammatory diseases is regulated by activation of chemokine receptors by chemokine ligands. To elucidate how these interactions give rise to cell migration, we mapped the chemokine-stimulated signal transduction network in monocytic THP-1 cells. Global phosphoproteomics revealed 630 time-resolved changes in phosphorylated proteins downstream of the chemokine receptor CCR2. We used the "PHONEMeS" network modeling algorithm to generate the most parsimonious signal transduction network consistent with the observed protein phosphorylation data. The CCR2 signaling network is highly divergent, acting via multiple branches to regulate proteins required for cell migration. We validated this model using kinase inhibitors targeting different branches of the network and successfully blocked chemokine-stimulated cell migration. Thus, chemotaxis is an emergent property resulting from an integrated cellular response to divergent signaling pathways. This paradigm suggests that physiological regulation or pharmacological blockade of chemokine-driven inflammation could potentially be achieved by inhibiting any of the divergent pathways within the network.

Protein tyrosine kinases (PTKs) regulate cellular biochemistry by phosphorylating tyrosine residues that alter protein function; their substrate preferences define the topology of signaling cascades. Previous studies of PTKs have mapped their average preferences for amino acids surrounding phosphorylation sites, but their ability to discriminate between highly similar substrate sequences (i.e., their sensitivity to minor changes in sequence within different regions of a substrate, and the sequence-dependent nature of this sensitivity) remains poorly understood. Here, we use a genetically encoded biosensor for PTK activity to examine the influence of local sequence context on substrate specificity. Across five well-studied PTKs, we identified amino acid substitutions within consensus substrates that could enhance selectivity for one PTK over others, confer sensitivity to substrate length, or improve PTK compatibility beyond the consensus. These effects were not predicted by classical specificity maps or advanced molecular modeling tools. Using a dual-selection screen that incorporates decoy substrates, we found additional sequence-diverse substrates with unexpectedly orthogonal PTK compatibilities. Our findings show how context-specific sequence features alter PTK substrate specificity far beyond what might be expected from classical consensus models and establish an experimental framework for defining the limits of substrate overlap between closely related kinases.

Tricomplex inhibitors (TCIs) are a novel class of direct Ras inhibitors that target the GTP-bound Ras(on) state trough recruitment of Cyclophilin A. Daraxonrasib (RMC-6236) is a pan-Ras TCI that was recently shown to restore GTPase activity of G12-mutant Ras proteins. Structural analysis of a pan-Ras TCI bound to K-Ras(GDP- AlF3) reveals a transition-state arrangement of Tyr32 and Gln61 that closely resembles endogenous GTPase- GAP complexes. This includes a closed Switch-I conformation engaging the cis-GTPase machinery in a manner analogous to non-arginine-finger GAPs such as RanGAP. These observations position pan-Ras TCIs as pharmacologic GAP mimetics. The GTPase-promoting activity of daraxonrasib suggests synergy with Switch-II pocket K-Ras inhibitors, including the approved GDP-state selective K-Ras G12C inhibitor adagrasib (MRTX-849), whose engagement of K-Ras(GTP) is kinetically constrained by slow endogenous hydrolysis of the mutant GTPase. We demonstrate that daraxonrasib sensitizes K-Ras(GTP) to adagrasib labeling in both recombinant protein and cellular context. In K-Ras G12C and G12D mutant cell lines, combinations of daraxonrasib with adagrasib or HRS-4642 (MRTX-1133 analog) yield more rapid K-Ras engagement, rapid p-ERK suppression, and significant Loewe synergy scores in viability assays. These findings establish GAP mimetics as rational and potent combination partners for SW-II inhibitors. The synergistic combination has potential to deepen and prolong pathway suppression while enabling dose reductions that may mitigate on-target toxicity and resistance.

The current working model of the innate immune protein calprotectin (CP) focuses on its metal-sequestering activity, which contributes to host defense against infection. Recently, CP was reported to enhance the survival of Staphylococcus aureus in coculture with Pseudomonas aeruginosa in a metal-independent manner. This prior work indicated that the CP protein scaffold, even in the absence of its metal-binding sites, possesses activities that impact interspecies dynamics between these bacterial pathogens. In this study, we employ {Delta}{Delta}, a CP variant lacking both functional metal-binding sites, to assess the responses of each pathogen to the CP protein scaffold in monoculture and coculture. Using dual-species transcriptomics, we report that {Delta}{Delta} treatment induced gene expression changes indicative of cell envelope modifications for both P. aeruginosa and S. aureus during coculture. The presence of the CP protein scaffold also attenuated the production of the quorum sensing molecule C4-homoserine lactone and the anti-staphylococcal alkylquinolone (AQ) metabolite 2-heptyl-4-hydroxyquinoline N-oxide. Cocultures with S. aureus and P. aeruginosa mutants defective in AQ biosynthesis demonstrated that AQ production was required for {Delta}{Delta} to impact expression of membrane remodeling genes in both species during coculture. Furthermore, we showed that in the absence of AQ production, the effect of CP on S. aureus in coculture resembled that of Fe depletion. Collectively, our findings demonstrate that the functional versatility of CP extends beyond multi-metal sequestration and that its intertwined metal-dependent and -independent activities have important consequences for bacterial physiology and polymicrobial interactions. IMPORTANCERecent studies of the innate immune protein calprotectin (CP), which is known for its metal-sequestering ability and contributions to nutritional immunity, have uncovered that the protein also exerts metal-independent activities on bacterial pathogens. In this work, we investigate the metal-independent effects of CP on the interspecies dynamics of Pseudomonas aeruginosa and Staphylococcus aureus, two high-priority pathogens that co-colonize various polymicrobial infection sites. We report that the ability of the CP protein scaffold to attenuate the anti-staphylococcal activity of P. aeruginosa results from perturbed quorum sensing and reduced production of alkylquinolone (AQ) metabolites. We further show that pseudomonal AQs contribute to cell envelope remodeling responses exhibited by both pathogens in the presence of the CP protein scaffold. These results afford an updated working model wherein both canonical metal-dependent and noncanonical metal-independent activities of CP elicit physiological changes in both pathogens, resulting in perturbed coculture dynamics.

Necroptosis is a lytic form of programmed cell death that requires activation of the RIPK1/3- MLKL complex and results in plasma membrane permeabilization. Although the protein components governing necroptosis are well defined, the lipid determinants of this process remain poorly understood. Here, we combined lipidomics, pharmacological perturbations of sphingolipid metabolism and functional assays to identify sphingolipid pathways that contribute to necroptotic cell death. Using a panel of small molecule inhibitors, we found that inhibition of acid sphingomyelinase (ASMase) with ARC39 restored cell viability and membrane integrity during necroptosis without altering canonical necroptotic signaling. Lipidomic analysis revealed that ARC39 treatment prevented ceramide accumulation in necroptosis, linking reduced ceramide levels to decreased membrane permeability. Interestingly, ARC39 treatment did not reduce total cellular levels of phosphorylated MLKL (pMLKL) nor its initial membrane association, suggesting that the observed decrease in membrane permeability arises downstream of MLKL activation. Instead, our findings support a model in which the reduction of ceramide levels impairs productive membrane insertion and pore formation by pMLKL. Consistent with this interpretation, genetic knockdown of ASMase similarly resulted in increased cell viability, decreased membrane permeabilization, and decreased ceramide levels during necroptosis, further linking ceramide homeostasis to necroptotic membrane damage. Together, these results indicate that ASMase-derived ceramides are important for efficient MLKL-mediated membrane permeabilization in necroptosis.

Reversible inactivation of protein tyrosine phosphatases by reactive oxygen species (ROS) is essential to the phosphorylation of growth factor receptors. An important outcome of the inactivation of protein tyrosine phosphatase 1B (PTP1B) by ROS involves the conformational change of its phosphotyrosine binding loop which adopts a solvent exposed position in its oxidized form. We previously demonstrated that 14-3-3{zeta} binds to the phosphotyrosine binding loop of the oxidized form of PTP1B. Using a rational approach, we developed a unique protein-protein interaction (PPI) inhibitor peptide derived from the phosphotyrosine binding loop of PTP1B designed to disrupt the interaction between PTP1B and the 14-3-3{zeta}-complex. Exploiting this cell-permeable peptide, we showed decreased association between PTP1B and the 14-3-3{zeta}-complex in cells treated with epidermal growth factor (EGF). We also demonstrated that preventing the association of this 14-3-3{zeta}-complex to PTP1B deterred oxidation and inactivation of PTP1B following EGF receptor (EGFR) activation and generation of ROS. Treating cells with our PPI inhibitor decreased EGFR phosphorylation on PTP1B-specific sites. Furthermore, treating EGFR-driven epidermal cancer cells with our PPI inhibitor also significantly inhibited colony formation and cell viability, consitent with increased activation of PTP1B. These data highlight the ability of PTP1B to downregulate critical signaling pathways in cancer when activated using peptide drugs such as our protein-protein interaction inhibitor. We anticipate that preventing or destabilizing the reversible oxidation of other members of the protein tyrosine phosphatase superfamily using PPI inhibitors may offer a foundation for a broad therapeutic approach to rectify dysregulated signaling pathways in vivo. Significance StatementLimited understanding of redox mechanisms regulating PTP catalytic activity is a major knowledge gap that has hampered our efforts to develop activation strategies. In its reversibly oxidized and inactivated form, conformational changes of PTP1B influence its association with regulatory proteins. We demonstrate that designing a cell-permeable peptide based on a loop of PTP1B that becomes exposed during oxidation can block its interaction with the 14-3-3{zeta}-multiprotein complex and activate the phosphatase. Moreover, activating PTP1B using our protein-protein interaction inhibitor peptide decreases the phosphorylation of its substrate EGFR and decreases the effectiveness of cancer cells to form colonies. This study provides important insights into the therapeutic potential of protein-protein interaction inhibitors that regulate the redox cycle of PTPs to reestablish physiological signaling.

The uterine endometrium is capable of scarless regeneration under coordinated estrogen and progesterone signaling across the menstrual cycle. Obesity suppresses progesterone production, leading to chronic estrogen exposure and increased endometrial hyperplasia (EH) risk. To define how obesity alters endometrial cell states, endometrial tissues from control and EH-predisposed mice fed either a control diet or a high-fat diet (HFD) were analyzed by single-cell RNA sequencing and tissue phenotyping. HFD reprogrammed endometrial stroma towards an inflammatory, pro-fibrotic state, reducing progesterone receptor-network-associated Aldh1a2+ fibroblasts and expanding estrogen receptor-network-associated Gsn fibroblasts. HFD further impaired macrophage recruitment and promoted hyperplastic epithelial signatures, consistent with increased disease severity in an EH mouse model. Stromal deletion of Estrogen Receptor established stromal estrogen signaling as a driver of HFD-induced extracellular matrix (ECM) accumulation. Collectively, these findings identify HFD-driven fibroblast reprogramming as a central mechanism linking estrogen dominance to stromal fibrosis, defective immune clearance, and heightened EH susceptibility. We propose that, in response to progesterone, fibroblast-mediated ECM remodeling is vital to normal endometrial homeostasis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=161 SRC="FIGDIR/small/713224v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@125d0f7org.highwire.dtl.DTLVardef@1ba1714org.highwire.dtl.DTLVardef@41314borg.highwire.dtl.DTLVardef@b4585_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO HFD-induced estrogen dominance disrupts endometrial fibroblast homeostasis to predispose the endometrium to diseaseThis study demonstrates that HFD drives estrogen-dependent reprogramming of stromal fibroblasts, characterized by inflammation, stromal ECM accumulation and fibrosis, and a post-ovulatory shift from PGR-network-associated Aldh1a2+ Fibroblasts toward increasing ER-network-associated Gsn+ Fibroblasts. These fibroblast changes are accompanied by a reduction in endometrial macrophages and a transcriptomic shift of HFD epithelium toward hyperplastic epithelium seen in a mouse model of EH. Figure made with BioRender. C_FIG