Science Signaling

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.

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.

Astrocytes are essential regulators of neuronal function and brain homeostasis. Recent methodological advances have increasingly revealed their active roles through precise manipulation of astrocyte function in the brain. While chemogenetic strategies, such as Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), have been widely used to recapitulate endogenous G-protein-coupled receptors (GPCRs) signaling and selectively modulate astrocyte activity, the molecular mechanisms accompanying DREADD activation remain incompletely understood. Here, we characterized the transcriptional responses of cultured cortical astrocytes and found that DREADD activation induces profound changes in astrocytic gene expression profiles. Activation of Gi-, Gq-, and Gs-coupled DREADDs elicited distinct transcriptomic responses. Transcription factor activity profiling further revealed that each DREADD subtype selectively modulates a distinct set of transcription factors that collectively shape astrocytic transcriptomic responses, thereby indicating subtype-specific transcriptional mechanisms. Importantly, DREADD activation induced persistent changes in the astrocytic transcriptome that remained detectable even three days after stimulation. These transcriptomic alterations were associated with sustained changes in astrocytic calcium responses to extracellular ATP, indicating plastic changes in astrocytic physiological function. Together, these findings provide a molecular framework for interpreting astrocytic DREADD manipulations and reveal a mechanistic basis for functional plasticity and heterogeneity of astrocytes.

Gastric cancer is driven by aberrant kinase signaling that promotes uncontrolled proliferation and malignant progression. Calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) is overexpressed in gastric cancer; however, the global phosphorylation networks downstream of CAMKK2 remain incompletely defined. In this study, we investigated the functional and signaling consequences of CAMKK2 inhibition in gastric cancer cells using an integrated phenotypic and quantitative phosphoproteomics approach. Pharmacological inhibition of CAMKK2 using STO-609 in AGS cells significantly suppressed proliferation, clonogenic growth, migration, and invasion, and induced defects in nuclear morphology indicative of impaired cell cycle progression. Tandem mass tag (TMT) based phosphoproteomic profiling identified over 10,500 phosphopeptides and revealed extensive phosphoproteome remodeling following CAMKK2 inhibition, characterized predominantly by hypophosphorylation of proteins involved in nuclear signaling, RNA processing, and cell cycle regulation. Kinase substrate enrichment and motif analyses demonstrated coordinated attenuation of CDK, MAPK, and mitotic kinase-associated signaling pathways, with convergence on E2F regulated transcriptional programs. Collectively, these findings establish CAMKK2 as a central regulator of kinase signaling networks that sustain proliferative and malignant phenotypes in gastric cancer and highlight CAMKK2 inhibition as a potential therapeutic strategy.

The interleukin-1 receptor type 1 (IL1R1) is a central regulator of inflammatory signaling and functions as a molecular switch, yet it remains unclear how agonists and antagonists that bind the same primary site produce opposite signaling outcomes. Available structural data define inactive and active endpoint conformations but do not explain how antagonist binding dynamically prevents co-receptor recruitment. Here, we combine all-atom molecular dynamics simulations with multiscale flexibility modeling using CABS-flex to systematically compare the intrinsic dynamics of IL1R1 across its unbound, agonist-bound, antagonist-bound, and co-receptor-bound states. Although both agonists and antagonists engage the same conserved interface on the D1/D2 domains, they induce fundamentally different dynamic responses in the receptor. Agonist binding progressively stabilizes interdomain coupling and promotes a stepwise transition toward a signaling-competent conformation. In contrast, antagonist binding selectively increases flexibility of the distal D3 domain, particularly at the co-receptor binding interface, thereby preventing progression along the activation pathway. Importantly, the interaction patterns and dynamic signatures observed in the simulations are consistent with experimentally identified binding determinants, mutational data, and structural features associated with receptor activation and inhibition. These results demonstrate that IL1R1 antagonism is an active, allosteric, dynamics-driven process rather than a simple failure to stabilize an active conformation. Together, this work provides a mechanistic framework that reconciles existing structural and functional observations and highlights receptor dynamics as a key determinant of signaling control in the interleukin-1 system.

Receptor internalization regulates the duration and qualitative output of intracellular signaling. Although generic mechanisms of receptor internalization are well characterized, how these are deployed and regulated in different cell types remains much less understood. Here we show that the p75 neurotrophin receptor (p75NTR), a key regulator of neuron survival and function, internalizes in hippocampal (HCNs)and cerebellar granule (CGNs)neurons with very different kinetics, regulated by distinct mechanisms. Compared to HCNs, p75NTR internalizes at a much slower rate and shows stronger interaction with caveolin in CGNs. In both cell types, p75NTR internalization was enhanced by nerve growth factor (NGF)but reduced by inhibitors of the RhoA and PKC pathways. In line with this, internalization of a p75NTR mutant specifically impaired in RhoA signaling was significantly reduced and insensitive to NGF in both neuron types. Accordingly, this mutant showed a much stronger interaction with caveolin than wild type p75NTR. On the other hand, internalization of a mutant specifically impaired in coupling to the NF-kB signaling pathway was greatly accelerated in CGNs but unaffected in HCNs. These results reveal the crucial role of intracellular signaling in p75NTR internalization and demonstrate that the receptor is differentially wired to the endocytosis machinery in different neuron types, leading to distinct internalization behaviors.

Mutations in the adenomatous polyposis coli (APC) gene are a defining feature of colorectal cancer (CRC) and impose metabolic and stress-adaptation requirements that may create exploitable vulnerabilities. Prolyl hydroxylase domain (PHD) inhibitors have been explored as therapeutic agents in CRC, however, their mechanisms of action and off-target effects remain elusive. Serendipitously, we found that Molidustat, a PHD2 inhibitor, induced cell death in APC mutant CRC cells. Ablation of PHD2 alone did not affect cell viability, suggesting an off-target mechanism. Using thermal proteome profiling and chemical proteomics, we identify glutathione S-transferase P1 (GSTP1) as a previously unrecognised off-target of Molidustat and demonstrate direct inhibition of its enzymatic activity. Genetic ablation of PHD2 alone did not phenocopy the cytotoxic effects of Molidustat, whereas combined loss of PHD2 and GSTP1 induced synergistic proteomic changes associated with cell-cycle suppression and apoptotic signalling. Integrated proteomic and metabolomic analyses further revealed energetic and metabolic perturbations specific to simultaneous GSTP1 and PHD2 loss. Consistent with these findings, APC-mutant colonic organoids displayed selective sensitivity to Molidustat that was not reproduced by hydroxylase inhibition alone supporting a synthetic lethal interaction between GSTP1 and PHD2 in APC-mutant contexts. Together, these results identify a functional interaction between GSTP1 and PHD2 in a subset of colorectal cancer and suggest that off-target engagement of GSTP1 contributes to the anti-tumour activity of Molidustat.

Neurogranin (Ng) is a postsynaptic protein highly enriched in forebrain neurons and implicated in synaptic plasticity through its ability to bind calmodulin. However, its impact on neuronal development, network dynamics, and cellular homeostasis remains incompletely understood. In this study, we examined the effects of manipulating Ng expression in primary hippocampal neurons using viral gene delivery, with emphasis on structural, functional, and molecular outcomes. Restoring Ng expression to adult physiological levels enhanced dendritic growth, increased synaptic number, and induced a proximal shift of the axon initial segment, consistent with adaptive responses to increased connectivity. Functionally, Ng markedly increased spontaneous neuronal activity and network synchronization, even under culture conditions that normally show minimal baseline activity. Electrophysiological recordings revealed enhanced burst firing and spike synchrony, indicating strengthened functional coupling rather than increased membrane excitability. Ng-dependent activity required action potential firing and glutamatergic transmission. At the molecular level, Ng increased total calmodulin levels in a binding-dependent manner, reduced overall calcium/calmodulin-dependent protein kinase II abundance while enhancing its relative autophosphorylation, and selectively decreased both total and surface levels of ionotropic glutamate receptors. These changes are consistent with a coordinated homeostatic reorganization of calcium-dependent signaling. Despite robust increases in activity, Ng expression improved neuronal viability, reduced cellular stress markers, and increased expression of the anti-apoptotic protein Bcl-2. Active caspase-3 was selectively elevated without triggering apoptosis, suggesting a non-apoptotic role in activity-dependent structural remodeling. Together, these findings identify Ng as a homeostatic regulator that promotes coordinated network activity, adaptive synaptic remodeling, and neuronal survival.

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.

During arousal and stress, the locus coeruleus (LC) releases noradrenaline (NA) throughout the brain. It remains, however, unclear how LC activity contributes to the cellular response profiles observed during natural arousal. Here, we directly compared natural arousal with selective LC activation in mouse CA1 using physiologically titrated optogenetics, fiber photometry of NA and calcium signals, chronic two-photon imaging, and behavioral monitoring. Natural arousal robustly activated astrocytes, pyramidal cells, and inhibitory interneurons at the population level. In contrast, stimulation of the LC activated only astrocytes and, for higher stimulation intensities, resulted in a slow inhibition of pyramidal cells and interneurons at the population level. A subset of interneurons exhibited a transient activation by LC stimulation and occupied distinct laminar positions in CA1. However, these LC-responsive subpopulations of interneurons did not reliably map onto subpopulations defined by activity patterns during natural arousal. Similarly, single-cell astrocyte responses to LC stimulation only partially aligned with their activity profiles during natural arousal. Together, these findings demonstrate that LC-driven NA release produces cell-specific effects in hippocampal CA1 that are strikingly distinct from, and in some cases opposite to, cellular dynamics during natural arousal. These results challenge the idea that LC activity as a proxy for arousal recapitulates the cellular signatures of arousal in vivo.

Chimeric antigen receptor (CAR) T cells have transformed cancer immunotherapy, yet their truncated or suboptimal intracellular signaling can limit therapeutic efficacy. To enhance proximal signaling of a CD19-targeted CAR, we systematically inserted short Lck-recruiting motifs derived from Lck-adaptor proteins into the CAR intracellular tail. Six candidate sequences from four adaptor molecules (SH2D2A, SKAP1, LAT, LIME), with a sequence from CD3{varepsilon}, known to affect CAR functionality, as a positive control, were tested for expression and functional impact. Three CAR constructs (containing SH2D2A, LAT and LIME1 sequences respectively) displayed reduced surface expression, but only SH2D2A elicited a pronounced rewiring of CAR T cell phenotype following co-culture with CD19+ tumor lines. SH2D2A CAR T cells showed increased CD27 and CD56 expression and reduced expression of effector-associated mediators including granzyme B, IL-2, TNF, and IFN{gamma}. Through systematic mutagenesis and comparative phenotyping of SH2D2A CAR variants, we identified SH2D2A tyrosine 290 (Tyr290) as the critical residue mediating both the altered signaling phenotype and the low surface expression. Additionally, mutation of Tyr254 in the LIME1 CAR restored surface expression in Jurkat T cells, indicating insert- and context-dependent effects on receptor surface expression. Collectively, these results demonstrate that short, intrinsically disordered adaptor-derived sequences -- and single tyrosine residues within them -- can profoundly reprogram CAR signaling and expression.

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.

V-domain Ig Suppressor of T cell activation (VISTA) has emerged as a critical target for anti-cancer immunotherapy. VISTA inhibits T cell activity, suppressing the anti-cancer immune responses. Here, we use atomistic molecular dynamics simulations to investigate the conformational behavior of membrane-bound human VISTA using both a glycan-free truncated model and a glycosylated, full-length model. Our simulations show that the extracellular and transmembrane domains remain structurally stable; notably, the unusually long CC' loop is stabilized by extensive hydrogen-bonding interactions. Principal component analysis and conformational clustering reveal a dominant rotational motion of the extracellular domain relative to the membrane, giving rise to two recurrent conformational states: an "Up" state in which the CC' loop is solvent-exposed and accessible for ligand engagement, and a "Down" state in which the loop transiently associates with the lipid bilayer. These transitions are allosterically coupled to proline-mediated bending of the transmembrane helix. We propose that this membrane-coupled Up/Down conformational switching regulates CC' loop accessibility, favoring cis interactions with cognate protein partners on the same cell surface, consistent with recent experimental observations, while permitting context-dependent trans interactions. Together, our findings reveal a membrane-coupled conformational mechanism regulating VISTA immune checkpoint function.

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

Polymorphonuclear neutrophils (PMNs) serve as frontline defenders against injury and infection, eliminating pathogens and initiating mucosal tissue repair. However, excessive PMN transepithelial migration (TEpM) contributes to chronic mucosal inflammatory disorders, including inflammatory bowel disease. PMN pro-inflammatory and pro-repair functions are regulated by incompletely defined signaling cascades involving kinases and phosphatases. Here, we determined how the protein tyrosine phosphatase CD45/PTPRC regulates PMN trafficking and effector functions in the gut. Pharmacologic inhibition of CD45 significantly reduced PMN colonic TEpM in vitro and in vivo and decreased intestinal PMN trafficking was observed in transgenic mice with PMN-specific deletion of CD45 (MRP8-Cre;Cd45fl/fl). Beyond limiting TEpM, CD45 depletion impaired key antimicrobial functions, including degranulation and phagocytosis, indicating broader effects on PMN effector activity. Importantly, recovery from dextran sodium sulfate (DSS)-induced colitis and biopsy-induced colonic wounding was delayed in MRP8-Cre;Cd45fl/fl mice, linking altered PMN function to defective mucosal healing. Mechanistically, CD45 depletion reduced surface expression of the {beta}2 integrin CD11b/CD18 and inactivated the Src family kinase member Lyn. Together, data highlight a novel CD45-CD11b-Lyn signaling axis that regulates PMN trafficking and effector functions in the intestine and identify CD45 as a promising target for modulating PMN function to promote mucosal tissue repair.

KRAS is mutated in over 90% of pancreatic ductal adenocarcinomas (PDAC), where hotspot alterations in codons 12, 13, and 61 drive tumor initiation and progression. Although distinct biochemical properties have been described for individual KRAS mutants, whether they generate unique allele-specific signaling programs in PDAC cells remains unresolved. Here, we systematically interrogated the molecular consequences of seven common KRAS mutant variants in reconstituted isogenic, KRAS-deficient PDAC cell lines by integrated transcriptomic, proteomic, and phosphoproteomic profiling. We found that baseline cellular state, rather than allele identity, was the predominant driver of molecular variation. Comparisons with established KRAS reference signatures revealed significant but moderate overlap at the mRNA level and less so at the proteome level. Pathway analyses highlighted interferon response and mitochondrial translation as recurrently altered across alleles, while phosphoproteomic data confirmed robust ERK1/2 activity and suppression of DYRK kinase substrates by mutant KRAS expression. Importantly, no robust allele-specific molecular programs were identified. Together, our study establishes a comprehensive multi-omics resource for KRAS signaling in PDAC and demonstrates that cellular context exerts a stronger influence than allele identity in shaping molecular profiles, with implications for interpreting putative allele-specific signaling dependencies and therapeutic vulnerabilities.