Signaling Events Research Articles

Soluble amyloid precursor protein-alpha mediates post-ischemic neovascularization after myocardial infarction

Abstract Background Amyloid precursor protein (APP) gives rise to amyloid-β peptides and thus has a key role in the pathogenesis of Alzheimer´s disease. APP is also highly expressed outside the brain including cells of the cardiovascular system, in particular endothelial cells (1). However, the role of APP and its proteolytic fragments in cardiovascular function and disease remains unknown. Purpose The main purpose of this study was to understand the function APP and the close homologue, amyloid precursor-like protein 2 (APLP2) in endothelial cells. Methods To test the potential role of endothelial APP and APLP2 in vivo, we generated inducible endothelium-specific APP/APLP2-double deficient mice (EC-APP/APLP2-KO) by crossing App fl/fl; Aplp2 fl/fl mice with the Cdh5-CreERT2 line. Animals were analyzed after exposure to myocardial infarction (MI). Human umbilical vein endothelial cells were used to study the mechanisms underlying APP/APLP2-mediated endothelial function in vitro. Results APP and APLP2 were found to be upregulated in cardiac endothelial cells after MI both in mice and humans. Loss of APP and APLP2 in endothelial cells had no effect on the basal heart function in mice. But, when subjected to MI, approximately 40 % of the EC-APP/APLP2-KO mice died within 3 weeks after MI in contrast to control animals, where hardly any death occurred. Survival of endothelium-specific APP- or APLP2-single knockouts was indistinguishable from that of control animals after MI. The surviving EC-APP/APLP2-KO mice showed larger infarct scars than the control animals. Magnetic resonance imaging and echocardiography at 3 weeks after MI revealed that EC-APP/APLP2-KO had more severe contractile dysfunction than the control mice (left ventricular ejection fraction 32.8 % vs. 44.9 %, respectively). As the underlying cause of this we identified a strong impairment of post-ischemia neovascularization in the absence of endothelial APP and APLP2. We found that soluble APP-α (APPsα) generated by non-amyloidogenic processing was the predominant form of APP in cardiac endothelial cells after MI. We therefore tested whether the expression of APPsα was able to rescue the observed phenotype of EC-APP/APLP2-KO after MI. Indeed, APPsα knock-in mice that solely express APPsα and no other APP fragments or cell surface APP showed the same phenotype as wild-type animals, indicating that APPsα is sufficient to mimic the function of APP. Under in vitro conditions, we found that APPsα was able to promote tube formation of cultured endothelial cells. APPsα also induced downstream signalling events such as AKT, ERK, and p38 activation which are reminiscent of growth factor induced signaling processes. Conclusions APPsα mediates post-ischemia neovascularization in the heart by exerting growth factor-like activity in endothelial cells, thereby protecting against cardiac injury.

HNCDrugResDb: a platform for deciphering drug resistance in head and neck cancers

Drug resistance poses a significant obstacle to the success of anti-cancer therapy in head and neck cancers (HNCs). We aim to develop a platform for visualizing and analyzing molecular expression alterations associated with HNC drug resistance. Through data mining, we convened differentially expressed molecules and context-specific signaling events involved in drug resistance. The driver genes, interaction networks and transcriptional regulations were explored using bioinformatics approaches. A total of 2364 differentially expressed molecules were identified in 78 distinct drug-resistant cells against 14 anti-cancer drugs, comprising 1131 mRNAs, 746 proteins, 62 lncRNAs, 257 miRNAs, 1 circRNA, and 166 post-translational modifications. Among these, 255 molecules were considerably, the signature driver genes of HNC drug resistance. Further, we also developed a landscape of signaling pathways and their cross-talk with diverse signaling modules involved in drug resistance. Additionally, a publicly-accessible database named “HNCDrugResDb” was designed with browse, query, and pathway explorer options to fetch and enrich molecular alterations and signaling pathways altered in drug resistance. HNCDrugResDb is also enabled with a Drug Resistance Analysis tool as an initial platform to infer the likelihood of resistance based on the expression pattern of driver genes. HNCDrugResDb is anticipated to have substantial implications for future advancements in drug discovery and optimization of personalized medicine approaches.

Phosphoproteomic Analysis of Signaling Pathways in Lymphomas.

Cellular fate is regulated by intricate signal transduction mediated by posttranslational protein modifications like phosphorylation to transmit information. As other cancer types, lymphomas frequently show dysregulation of signaling pathways that contribute to malignant transformation and tumor progression. For example, in diffuse large B-cell lymphoma the B-cell antigen receptor was identified as an oncogenic driver mediating cellular growth and survival signals. Thus, the elucidation of these complex signaling networks is crucial to gain insight into the mechanisms underlying tumorigenesis and to identify target proteins for innovative therapeutic approaches.Here, we describe a mass spectrometry-based phosphoproteomic approach for the global analysis of intracellular signaling events and their dynamics. The workflow combines phosphopeptide enrichment and fractionation with liquid chromatography-coupled mass spectrometry for the amino acid site-specific identification and quantification of thousands of phosphorylation events. Such global signaling analyses have great potential for the elucidation of oncogenic pathomechanisms, diagnostic biomarkers, and drug targets.

The Influence of Cholesterol on Membrane Targeted Bioactive Peptides: Modulating Peptide Activity Through Changes in Bilayer Biophysical Properties.

Cholesterol is a biological molecule that is essential for cellular life. It has unique features in terms of molecular structure and function, and plays an important role in determining the structure and properties of cell membranes. One of the most recognized functions of cholesterol is its ability to increase the level of lipid packing and rigidity of biological membranes while maintaining high levels of lateral mobility of the bulk lipids, which is necessary to sustain biochemical signaling events. There is increased interest in designing bioactive peptides that can act as effective antimicrobial agents without causing harm to human cells. For this reason, it becomes relevant to understand how cholesterol can affect the interaction between bioactive peptides and lipid membranes, in particular by modulating the peptides' ability to penetrate and disrupt the membranes through these changes in membrane rigidity. Here we discuss cholesterol and its role in modulating lipid bilayer properties and discuss recent evidence showing how cholesterol modulates bioactive peptides to different degrees.

Farm exposure is associated with human breast milk immune profile and microbiome.

Prenatal and early life farm exposure, and breastfeeding, are associated with protection from allergic diseases. We hypothesize that farm exposure influences the human breast milk microbiome and immune proteins. The immune protein profiles and microbial communities of 152 human breast milk samples were compared among three maternal farm exposure groups (traditional agrarian, farm, and non-farm) in rural Wisconsin to identify signatures associated with farm status and atopic disease. We found significant differences between farm groups for 23 immune proteins (p-adj<0.05), microbiome diversity (p=2.2E-05), and microbiome richness (p=8.0e-06). Traditional agrarian human breast milk had the highest immune protein levels and microbiome diversity and richness, followed by farm and non-farm human breast milk. Furthermore, Gram-positive bacterial species correlated with IL-23 mediated signaling events (p-adj<1.0E-05). These data suggest that increased farm exposures promotes human breast milk that is more microbially-diverse and rich in immune-associated proteins, ultimately influencing immune development in the infant.

Mapping Nanoscale-To-Single-Cell Phosphoproteomic Landscape by Chip-DIA.

Protein phosphorylation plays a crucial role in regulating disease phenotypes and serves as a key target for drug development. Mapping nanoscale-to-single-cell samples can unravel the heterogeneity of cellular signaling events. However, it remains a formidable analytical challenge due to the low detectability, abundance, and stoichiometry of phosphorylation sites. Here, we present a Chip-DIA strategy,integratinga microfluidic-based phosphoproteomic chip (iPhosChip) with data-independent acquisition mass spectrometry (DIA-MS) for ultrasensitive nanoscale-to-single-cell phosphoproteomic profiling. The iPhosChip operates as an all-in-one station that accommodates both quantifiable cell capture/imaging and the entire phosphoproteomic workflow in a highly streamlined and multiplexed manner. Coupled with a sample size-comparable library-based DIA-MS strategy, Chip-DIA achieved ultra-high sensitivity, detecting 1076±158 to 15869±1898 phosphopeptides from 10±0 to 1013±4 cells, and revealed the first single-cell phosphoproteomic landscape comprising druggable sites and basal phosphorylation-mediated networks in lung cancer. Notably, the sensitivity and coverage enabled the illumination of heterogeneous cytoskeleton remodeling and cytokeratin signatures in patient-derived cells resistant to third-generation EGFR therapy, stratifying mixed-lineage adenocarcinoma-squamous cell carcinoma subtypes, and identifying alternative targeted therapy for late-stage patients. With flexibility in module design and functionalization, Chip-DIA can be adapted to other PTM-omics to explore dysregulated PTM landscapes, thereby guiding therapeutic strategies toward precision oncology.

Candidate Molecular Biomarkers of Traumatic Brain Injury: A Systematic Review.

Traumatic brain injury (TBI) is one of the leading causes of mortality and disability among young and middle-aged individuals. Adequate and timely diagnosis of primary brain injuries, as well as the prompt prevention and treatment of secondary injury mechanisms, significantly determine the potential for reducing mortality and severe disabling consequences. Therefore, it is crucial to have objective markers that indicate the severity of the injury. A number of molecular factors-proteins and metabolites-detected in the blood immediately after trauma and associated with the development and severity of TBI can serve in this role. TBI is a heterogeneous condition with respect to its etiology, clinical form, and genesis, being accompanied by brain cell damage and disruption of blood-brain barrier permeability. Two oppositely directed flows of substances and signals are observed: one is the flow of metabolites, proteins, and nucleic acids from damaged brain cells into the bloodstream through the damaged blood-brain barrier; the other is the infiltration of immune cells (neutrophils and macrophages) and serological proteins. Both flows aggravate brain tissue damage after TBI. Therefore, it is extremely important to study the key signaling events that regulate these flows and repair the damaged tissues, as well as to enhance the effectiveness of treatments for patients after TBI.

8053 A Ligand-Based Differentiation Protocol for Human Induced Pluripotent Stem Cells Recapitulates Granulosa Cell Development and Induces Steroidogenic Pathway Genes

Abstract Disclosure: H. Kubo: None. M.M. Laronda: None. Sex determination is a developmental process that defines the gonad as a testis or ovary, and influences sex hormone production. While key signaling events are well conserved across mammals, there can be differences between species. Therefore, there is a need to develop a human model to fully understand this critical process in humans. We have developed a ligand-based differentiation protocol for human induced pluripotent stem cells (hiPSCs) to generate granulosa-like cells (GLCs). Additionally, hiPSC-derived GLCs could become the foundation for a cell-based hormone replacement therapy that recapitulates cyclicity, physiologic levels of hormones, and production of peptide hormones that are not included in currently available hormone replacement therapies. We aim to gain further understanding of the key factors and culture conditions necessary for functional GLCs that produce ovarian hormones, which includes maintained expression of FOXL2. We used our monolayer differentiation protocol that includes WNT-agonist CHIR99021 treatment for 2 days, followed by a combination treatment of BMP4, FST, and DKK1-inhibitor Gallocyanine for 3 days. We have tested our protocol on three different hiPSC lines: IISH2i-BM9s, STiPS-A3, and STiPS-A6. Expression of pluripotency marker POU5F1 is reduced during differentiation, while expression of the intermediate mesoderm markers PAX2, LHX1, and WT1 and bipotential gonad marker GATA4 peak at day 2. Peak expression of the bipotential gonad marker NR5A1 occurred at day 2 in the STiPS-A6 cells, but at day 5 in IISH2i-BM9 and STiPS-A3 cells. Interestingly, FOXL2 expression peaks at day 2 in all three lines and drops by day 5. In the IISH2i-BM9s, FOXL2 protein peaks at day 2 by Jess Western while there is the highest percentage of FOXL2-positive cells at day 5 by immunofluorescence. In murine granulosa cells, FOXL2 maintenance depends on NR5A2. NR5A2 expression drops in the GLCs, which suggests that NR5A2 loss may be preventing FOXL2 maintenance. Additionally, as FOXL2 is required for expression of aromatase and HSD17B1, we are using this model to interrogate NR5A2 upstream of FOXL2 and control hormone production. CYP19A1 and HSD17B1 along with AMH are induced during this differentiation protocol, suggesting these GLCs can be steroidogenic and produce peptide hormones. Additionally, scRNA-seq analysis of the GLCs is underway to identify types of granulosa cells produced by this protocol. Future directions include optimizing GLC culture conditions to generate hormones and to test if NR5A2 inhibitors alter FOXL2 expression. Put together, the data suggests that cells differentiated with our protocol show promise as a human model of ovarian development and a foundation for a personalized cell-based hormone replacement therapy. This work is supported by the NIH/NICHD R01HD104683 (MML), HuBMAP U01HD110336 (MML), and Gesualdo Family Research Scholar (MML). Presentation: 6/2/2024

Towards New Anti-inflammatory Agents: Design, Synthesis and Evaluation of Molecules Targeting XIAP-BIR2.

The X-chromosome-linked inhibitor of apoptosis protein (XIAP) plays a crucial role in controlling cell survival across multiple regulated cell death pathways and coordinating a range of inflammatory signalling events. The discovery of selective inhibitors for XIAP-BIR2, able to disrupt the direct physical interaction between XIAP and RIPK2, offer promising therapeutic options for NOD2-mediated diseases like Crohn's disease, sarcoidosis, and Blau syndrome. The objective of this study was to design, synthesize, and evaluate small synthetic molecules with binding selectivity to XIAP-BIR2 domain. To achieve this, we applied an interdisciplinary drug design approach and firstly we have synthesized an initial fragment library to achieve a first XIAP inhibition activity. Then using a growing strategy, larger compounds were synthesized and one of them presents a good selectivity for XIAP-BIR2 versus XIAP-BIR3 domain, compound 20c. The ability of compound 20c to block the NOD1/2 pathway was confirmed in cell models. These data show that we have synthesized molecules capable of blocking NOD1/2 signalling pathways in cellulo, and ultimately leading to new anti-inflammatory compounds.

Soma-to-germline BMP signal is essential for Drosophila spermiogenesis

In the Drosophila testis, developing germ cells are encapsulated by somatic support cells throughout development. Soma-germline interactions are essential for successful spermiogenesis. However, it is still not fully understood what signaling events take place between the soma and the germline. In this study, we found that a Bone Morphogenetic Protein (BMP) ligand, Glass bottom boat (Gbb), secreted from somatic cyst cells (CCs), signals to differentiating germ cells to maintain proper spermiogenesis. Knockdown of Gbb in CCs or the type I BMP receptor Saxophone (Sax) in germ cells leads to a defect in sperm head bundling and decreased fertility. Our Transmission Electron Microscopy (TEM) analyses revealed that the mutant germ cells have aberrant morphology of mitochondria throughout the stages of spermiogenesis and exhibit a defect in nebenkern formation. Elongating spermatids show uncoupled nuclei and elongating mitochondrial derivatives, suggesting that improper mitochondrial development may cause sperm bundling defects. Taken together, we propose a new role of soma-derived BMP signaling, which is essential for spermiogenesis.

Intracellular lipid droplets are exploited by Junín virus in a nucleoprotein-dependent process.

Lipid droplets (LDs) are organelles involved in lipid storage, maintenance of energy homeostasis, protein sequestration, signaling events and inter-organelle interactions. Recently, LDs have been shown to favor the replication of members from different viral families, such as the Flaviviridae and Coronaviridae. In this work, we show that LDs are essential organelles for members of the Arenaviridae family. A virus-driven reduction of LD number was observed in cultures infected with Junín mammarenavirus (JUNV), caused in part by action of the viral nucleoprotein. Notably, we identified a new pool of nucleoprotein and viral RNA that localizes in the vicinity of LDs, suggesting that LDs play a role during the viral replication cycle. Regarding the mechanism behind LD exhaustion, we found evidence that lipophagy is involved in LD degradation with the resulting fatty acids being substrates of fatty acid β-oxidation, which fuels viral multiplication. This work highlights the importance of LDs during the replication cycle of JUNV, contributing to the knowledge of the metabolic changes these mammarenaviruses cause in their hosts.

The RNA receptor RIG-I binding synthetic oligodeoxynucleotide promotes pneumonia survival.

Pneumonia is a worldwide threat to public health, demanding novel preventative and therapeutic strategies. The lung epithelium is a critical environmental interface that functions as a physical barrier to pathogen invasion while also actively sensing and responding to pathogens. We have reported that stimulating lung epithelial cells with a combination therapeutic consisting of a diacylated lipopeptide and a synthetic CpG oligodeoxynucleotide (ODN) induces synergistic pneumonia protection against a wide range of pathogens. We report here that mice deficient in Toll-like receptor 9 (TLR9), the previously described receptor for ODN, still displayed partial ODN-induced protection. This prompted us to seek an alternate ODN receptor, and we discovered by mass spectroscopy that the RNA sensor RIG-I could also bind DNA-like ODN. ODN binding by RIG-I resulted in MAVS-dependent pneumonia-protective signaling events. While RIG-I is essential to native defenses against viral infections, we report that therapeutic RIG-I activation with ODN promoted pathogen killing and host survival following both viral and bacterial challenges. These data indicate that maximal ODN-induced pneumonia protection requires activation of both TLR9/MyD88 and RIG-I/MAVS signaling pathways. These findings not only identify what we believe to be a novel pattern recognition receptor for DNA-like molecules, but reveal a potential therapeutic strategy to protect susceptible individuals against lethal pneumonias during periods of peak vulnerability.

Chronic stimulation desensitizes β2-adrenergic receptor responses in natural killer cells.

Adrenergic receptors (ARs) are preferentially expressed by innate lymphocytes such as natural killer (NK) cells. Here, we study the effect of epinephrine-mediated stimulation of the β2-adrenergic receptor (β2AR) on the function of human NK cells. Epinephrine stimulation inhibited early NK cell signaling events and blocked the function of the integrin LFA-1. This reduced the adhesion of NK cells to ICAM-1, explaining how NK cells are mobilized into the peripheral blood upon epinephrine release during acute stress or exercise. Additionally, epinephrine stimulation transiently reduced NK cell degranulation, serial killing, and cytokine production and affected metabolic changes upon NK cell activation via the cAMP-protein kinase A (PKA) pathway. Repeated exposure to β2AR agonists resulted in the desensitization of the β2AR via a PKA feedback loop-initiated G-protein switch. Therefore, acute epinephrine stimulation of chronically β2AR stimulated NK cells no longer resulted in inhibited signaling and reduced LFA-1 activity. Sustained stimulation by long-acting β2-agonists (LABA) not only inhibited NK cell functions but also resulted in desensitization of the β2AR. However, peripheral NK cells from LABA-treated asthma patients still reacted unchanged to epinephrine stimulation, demonstrating that local LABA administration does not result in detectable systemic effects on NK cells.

The natural flavonoid pinocembrin shows antithrombotic activity and suppresses septic thrombosis

Sepsis, an extreme host response to systemic infection, remains one of the leading causes of mortality worldwide. Platelets, which are integral to both thrombosis and inflammation, play a crucial role in the pathophysiology of sepsis. Excessive platelet activation and aggregation significantly increase the risk of thrombosis, thereby elevating mortality in septic patients. However, the etiology and treatment of this condition have not been comprehensively studied. This study identifies pinocembrin, a natural flavonoid compound derived from propolis, as a potential therapeutic agent for mitigating platelet activation and treating sepsis. In vivo, pinocembrin effectively inhibited FeCl3-induced carotid arterial occlusive thrombus formation and collagen/epinephrine-induced pulmonary thromboembolism in mouse models. In vitro, pinocembrin treatment suppressed multiple facets of platelet activation, including aggregation, secretion, and αIIbβ3-mediated signaling events. Mechanistically, pinocembrin repressed platelet functions by inhibiting Src/Syk/PLCγ2/MAPK signaling pathway. Using cecal ligation and puncture (CLP) mouse model to simulate human sepsis, pinocembrin reduced inflammatory cytokine release and septic thrombosis, thereby improving the survival rate of septic mice. Lipopolysaccharide (LPS)-induced model further substantiated these results. Overall, the inhibition of platelet activity by pinocembrin demonstrates significant therapeutic potential for managing life-threatening septic thrombosis.

Type I IFN Induces TCR-dependent and -independent Antimicrobial Responses in γδ Intraepithelial Lymphocytes.

Intraepithelial lymphocytes (IELs) expressing the TCRγδ survey the intestinal epithelium to limit the invasion of microbial pathogens. The production of type I IFN is a central component of an antiviral immune response, yet how these proinflammatory cytokines contribute to γδ IEL effector function remains unclear. Based on the unique activation status of IELs and their ability to bridge innate and adaptive immunity, we investigated the extent to which type I IFN signaling modulates γδ IEL function. Using an ex vivo culture model, we find that type I IFN alone is unable to drive IFN-γ production, yet low-level TCR activation synergizes with type I IFN to induce IFN-γ production in murine γδ IELs. Further investigation into the underlying molecular mechanisms of costimulation revealed that TCRγδ-mediated activation of NFAT and JNK is required for type I IFN to promote IFN-γ expression in a STAT4-dependent manner. Whereas type I IFN rapidly upregulates antiviral gene expression independent of a basal TCRγδ signal, neither tonic TCR triggering nor the presence of a TCR agonist was sufficient to elicit type I IFN-induced IFN-γ production invivo. However, bypassing proximal TCR signaling events synergized with IFNAR/STAT4 activation to induce γδ IEL IFN-γ production. These findings indicate that γδ IELs contribute to host defense in response to type I IFN by mounting a rapid antimicrobial response independent of TCRγδ signaling, and may produce IFN-γ in a TCR-dependent manner under permissive conditions.

Improving Compton camera imaging of multi-energy radioactive sources by using machine learning algorithms for event selection

Event selection and background reduction for Compton camera imaging of multi-energy radioactive sources has been performed by employing neural networks. A Compton camera prototype with detectors made of LaBr3 crystals coupled to silicon photomultiplier arrays was used to acquire experimental data from a circular array of 22Na sources. The prototype and two arrays of 22Na sources were simulated with GATE v8.2 Monte Carlo code, to obtain data for neural network training. Neural network models were trained on simulated data for event classification. The optimum models were found by using Weights & Biases platform tools. The trained models were used to classify simulated and real data for selecting signal events and rejecting background prior to image reconstruction. The models performed well on simulated data. The image obtained with experimental data showed an improvement with respect to event selection with energy cuts. The method is promising for Compton camera imaging of multi-energy radioactive sources.

PAIReD jet: A multi-pronged resonance tagging strategy across all Lorentz boosts

We propose a new approach of jet-based event reconstruction that aims to optimally exploit correlations between the products of a hadronic multi-pronged decay across all Lorentz boost regimes. The new approach utilizes clustered small-radius jets as seeds to define unconventional jets, referred to as PAIReD jets. The constituents of these jets are subsequently used as inputs to machine learning-based algorithms to identify the flavor content of the jet. We demonstrate that this approach achieves higher efficiencies in the reconstruction of signal events containing heavy-flavor jets compared to other event reconstruction strategies at all Lorentz boost regimes. Classifiers trained on PAIReD jets also have significantly better background rejections compared to those based on traditional event reconstruction approaches using small-radius jets at low Lorentz boost regimes. The combined effect of a higher signal reconstruction efficiency and better classification performance results in a two to four times stronger rejection of light-flavor jets compared to conventional strategies at low Lorentz-boosts, and rejection rates similar to classifiers based on large-radius multi-pronged jets at high Lorentz-boost regimes.

Glucoraphanin and sulforaphane mitigate TNFα-induced Caco-2 monolayers permeabilization and inflammation

Intestinal permeabilization is central to the pathophysiology of chronic gut inflammation. This study investigated the efficacy of glucoraphanin (GR), prevalent in cruciferous vegetables, particularly broccoli, and its derivative sulforaphane (SF), in inhibiting tumor necrosis factor alpha (TNFα)-induced Caco-2 cell monolayers inflammation and permeabilization through the regulation of redox-sensitive events. TNFα binding to its receptor led to a rapid increase in oxidant production and subsequent elevation in the mRNA levels of NOX1, NOX4, and Duox2. GR and SF dose-dependently mitigated both these short- and long-term alterations in redox homeostasis. Downstream, GR and SF inhibited the activation of the redox-sensitive signaling cascades NF-κB (p65 and IKK) and MAPK ERK1/2, which contribute to inflammation and barrier permeabilization. GR (1 μM) and SF (0.5–1 μM) prevented TNFα-induced monolayer permeabilization and the associated reduction in the levels of the tight junction (TJ) proteins occludin and ZO-1. Both GR and SF also mitigated TNFα-induced increased mRNA levels of the myosin light chain kinase, which promotes TJ opening. Molecular docking suggests that although GR is mostly not absorbed, it could interact with extracellular and membrane sites in NOX1. Inhibition of NOX1 activity by GR would mitigate TNFα receptor downstream signaling and associated events. These findings support the concept that not only SF, but also GR, could exert systemic health benefits by protecting the intestinal barrier against inflammation-induced permeabilization, in part by regulating redox-sensitive pathways. GR has heretofore not been viewed as a biologically active molecule, but rather, the benign precursor of highly active SF. The consumption of GR and/or SF-rich vegetables or supplements in the diet may offer a means to mitigate the detrimental consequences of intestinal permeabilization, not only in disease states but also in conditions characterized by chronic inflammation of dietary and lifestyle origin.

Ubiquitylomics: An Emerging Approach for Profiling Protein Ubiquitylation in Skeletal Muscle.

Skeletal muscle is a highly adaptable tissue, finely tuned by various physiological and pathological factors. Whilst the pivotal role of skeletal muscle in overall health is widely acknowledged, unravelling the underlying molecular mechanisms poses ongoing challenges. Protein ubiquitylation, a crucial post-translational modification, is involved in regulating most biological processes. This widespread impact is achieved through a diverse set of enzymes capable of generating structurally and functionally distinct ubiquitin modifications on proteins. The complexity of protein ubiquitylation has presented significant challenges in not only identifying ubiquitylated proteins but also characterising their functional significance. Mass spectrometry enables in-depth analysis of proteins and their post-translational modification status, offering a powerful tool for studying protein ubiquitylation and its biological diversity: an approach termed ubiquitylomics. Ubiquitylomics has been employed to tackle different perspectives of ubiquitylation, including but not limited to global quantification of substrates and ubiquitin linkages, ubiquitin site recognition and crosstalk with other post-translational modifications. As the field of mass spectrometry continues to evolve, the usage of ubiquitylomics has unravelled novel insights into the regulatory mechanisms of protein ubiquitylation governing biology. However, ubiquitylomics research has predominantly been conducted in cellular models, limiting our understanding of ubiquitin signalling events driving skeletal muscle biology. By integrating the intricate landscape of protein ubiquitylation with dynamic shifts in muscle physiology, ubiquitylomics promises to not only deepen our understanding of skeletal muscle biology but also lay the foundation for developing transformative muscle-related therapeutics. This review aims to articulate how ubiquitylomics can be utilised by researchers to address different aspects of ubiquitylation signalling in skeletal muscle. We explore methods used in ubiquitylomics experiments, highlight relevant literature employing ubiquitylomics in the context of skeletal muscle and outline considerations for experimental design.

PIP5K-Ras bistability initiates plasma membrane symmetry breaking to regulate cell polarity and migration.

Symmetry breaking, polarity establishment, and spontaneous cell protrusion formation are fundamental but poorly explained cell behaviors. Here, we demonstrate that a biochemical network, where the mutually inhibitory localization of PIP5K and Ras activities plays a central role, governs these processes. First, in resting cells devoid of cytoskeletal activity, PIP5K is uniformly elevated on the plasma membrane, while Ras activity remains minimal. Symmetry is broken by spontaneous local displacements of PIP5K, coupled with simultaneous activations of Ras and downstream signaling events, including PI3K activation. Second, knockout of PIP5K dramatically increases both the incidence and size of Ras-PI3K activation patches, accompanied by branched F-actin assembly. This leads to enhanced cortical wave formation, increased protrusive activity, and a shift in migration mode. Third, high inducible overexpression of PIP5K virtually eliminates Ras-PI3K signaling, cytoskeletal activity, and cell migration, while acute recruitment of cytosolic PIP5K to the membrane induces contraction and blebs in cancer cells. These arrested phenotypes are reversed by reducing myosin II activity, indicating myosin's involvement in the PIP5K-Ras-centered regulatory network. Remarkably, low inducible overexpression of PIP5K unexpectedly facilitates polarity establishment, highlighting PIP5K as a highly sensitive master regulator of these processes. Simulations of a computational model combining an excitable system, cytoskeletal loops, and dynamic partitioning of PIP5K recreates the experimental observations. Taken together, our results reveal that a bistable, mutually exclusive localization of PIP5K and active Ras on the plasma membrane triggers the initial symmetry breaking. Coupled actomyosin reduction and increased actin polymerization lead to intermittently extended protrusions and, with feedback from the cytoskeleton, self-organizing, complementary gradients of PIP5K versus Ras steepen, raising the threshold of the networks at the rear and lowering it at the front to generate polarity for cell migration.