Changes In Protein Phosphorylation Research Articles

In High Glucose Exposed Endothelial Cells BLNK and BTK are Novel Components of the Nuclear Factor-kappa B Pathway

Peripheral artery disease (PAD) is atherosclerotic occlusion of vessels outside the heart and most commonly affects the lower extremities. Diabetes worsens PAD outcomes, but the mechanisms involved are poorly understood. A key component of perfusion recovery in experimental PAD is induction of NFκB signaling in ischemic endothelial cells (EC). We have previously shown that prolonged exposure of ECs to high glucose before simulated ischemic exposure resulted in impairment of the canonical NFκB pathway through activation of protein kinase C β (PKCβ). However, the signaling pathways upstream of PKCβ involved in this high glucose induced desensitization of NFκB signaling are not known. We used arrays of antibodies to approximately 100 proteins known to participate in the NFκB pathway to identify the changes in their phosphorylation states in human umbilical vein endothelial cells (HUVEC). Cells grown for three days either in culture medium with normal glucose (NG) or high glucose (HG) were subjected to ischemia for 24 hours (NGI and HGI, respectively). Cell lysates from NG, HG, NGI and HGI groups were then incubated with the array of antibodies printed on glass slides and fluorescent signals were digitally recorded and normalized. The change in protein phosphorylation was calculated by dividing the intensity of the phosphorylated spots by the signal intensity of the corresponding non-phosphorylated spots for each protein. Differential expression between samples, were calculated by dividing the phosphorylation ratio of the NGI and HGI with that of the NG and HG controls, respectively. A combination of pathway analyses using bioinformatics tools on 65 modulated phosphorylation sites in HGI (represented by 35 genes) and analysis of differential phosphorylation between NGI and HGI samples suggested involvement of B cell linker/adapter protein (BLNK)/Bruton’s tyrosine kinase (BTK). BTK plays a key role in B cell antigen receptor (BCR)-coupled signaling by associating with the scaffold protein BLNK. However, neither of these proteins has been known to be expressed in ECs or play a role in EC function. In this study, we confirmed BLNK and BTK protein and transcript expression in ECs. Inhibition of BTK by a selective inhibitor terreic acid decreased PKCβ-S661 phosphorylation and restored NFκB activation through IκBα in ECs suggesting a critical role of BLNK/BTK in the EC. Thus, we have identified BLNK/BTK as potentially new components of the NFκB pathway in ECs that may be a therapeutic target to improve PAD outcomes in diabetes. National Heart, Lung, and Blood Institute (R01 HL130399) to AOD. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Yiqi Kaimi prescription regulates protein phosphorylation to promote intestinal motility in slow transit constipation

Ethnopharmacological relevanceThe clinical efficacy of the Yiqi Kaimi prescription has been confirmed in slow transit constipation. However, the effects and biological mechanism of Yiqi Kaimi prescription are still unclear. Aims of the studyTo identify the effects of Yiqi Kaimi prescription on intestinal motility; To reveal the potential key targets and pathways of Yiqi Kaimi prescription for the treatment of slow transit constipation. Materials and methodsThe effects of Yiqi Kaimi prescription on slow transit constipation were investigated in a mouse model. The terminal ink propulsion experiment and fecal indocyanine green imaging was used to measure the intestinal transit time. Protein phosphorylation changes in colon tissues treated with Yiqi Kaimi prescription were detected using a Phospho Explorer antibody microarray. Bioinformatic analyses were performed using the Database for Annotation Visualization and Integrated Discovery (DAVID) and the Search Tool for the Retrieval of Interacting Genes (STRING). Western blot analysis and immunohistochemistry confirmed the observed changes in phosphorylation. Results: Yiqi Kaimi prescription significantly increased the intestinal transit rate (P < 0.05 vs. model) and reduced the time to first discharge of feces containing fecal indocyanine green imaging in mice (P < 0.05 vs. model). The administration of Yiqi Kaimi prescription induced phosphorylation changes in 41 proteins, with 9 upregulated proteins and 32 downregulated proteins. Functional classification of the phosphorylated proteins with DAVID revealed that the critical biological processes included tyrosine protein kinases, positive regulation of calcium-mediated signaling and response to muscle stretch. The phosphorylation of the spleen tyrosine kinase (SYK) at Tyr348 increased 2.19-fold, which was the most significant change. The phosphorylation level of the transcription factor p65 (RELA) at Thr505 was decreased 0.57-fold. SYK was a hub protein in the protein-protein interaction network and SYK and RELA formed the core of the secondary subnetwork. The key protein phosphorylation after treatment with Yiqi Kaimi prescription were verified by Western blot analysis and immunohistochemistry. ConclusionYiqi Kaimi prescription significantly enhanced intestinal motility. This effect was attributed to alterations in the phosphorylation levels of various target proteins. The observed changes in protein phosphorylation, including SYK and RELA, may serve as crucial factors in the treatment of slow transit constipation.

Comprehensive multi-omics analysis reveals WEE1 as a synergistic lethal target with hyperthermia through CDK1 super-activation

Hyperthermic intraperitoneal chemotherapy’s role in ovarian cancer remains controversial, hindered by limited understanding of hyperthermia-induced tumor cellular changes. This limits developing potent combinatory strategies anchored in hyperthermic intraperitoneal therapy (HIPET). Here, we perform a comprehensive multi-omics study on ovarian cancer cells under hyperthermia, unveiling a distinct molecular panorama, primarily characterized by rapid protein phosphorylation changes. Based on the phospho-signature, we pinpoint CDK1 kinase is hyperactivated during hyperthermia, influencing the global signaling landscape. We observe dynamic, reversible CDK1 activity, causing replication arrest and early mitotic entry post-hyperthermia. Subsequent drug screening shows WEE1 inhibition synergistically destroys cancer cells with hyperthermia. An in-house developed miniaturized device confirms hyperthermia and WEE1 inhibitor combination significantly reduces tumors in vivo. These findings offer additional insights into HIPET, detailing molecular mechanisms of hyperthermia and identifying precise drug combinations for targeted treatment. This research propels the concept of precise hyperthermic intraperitoneal therapy, highlighting its potential against ovarian cancer.

Quantitative phosphoproteomics explain cryopreservation-induced reductions in ram sperm motility

Sperm cryopreservation decreases motility, probably due to changes in protein phosphorylation. Our objective was to use quantitative phosphoproteomics for systematic comparative analyses of fresh versus frozen-thawed sperm to identify factors causing cryo-injury. Ejaculates were collected (artificial vagina) from six Dorper rams, pooled, extended, and frozen over liquid nitrogen. Overall, 915, 3382, and 6875 phosphorylated proteins, phosphorylated peptides, and phosphorylation sites, respectively, were identified. At least two modified sites were present in 57.94% of the 6875 phosphosites identified, of which AKAP4 protein contained up to 331 modified sites. There were 732 phosphorylated peptides significantly up-regulated and 909 significantly down-regulated in frozen-thawed versus fresh sperm. Moreover, the conserved motif [RxxS] was significantly down-regulated in frozen-thawed sperm. Phosphorylation of sperm-specific proteins, e.g., AKAP3/4, CABYR, FSIP2, GSK3A/B, GPI, and ODF1/2 make them potential biomarkers to assess the quality of frozen-thawed ram sperm. Furthermore, these differentially phosphorylated proteins and modification sites were implicated in cryopreservation-induced changes in sperm energy production, fiber sheath composition, and various biological processes. We concluded that abnormal protein phosphorylation modifications are key regulators of reduced sperm motility. These novel findings implicated specific protein phosphorylation modifications in sperm cryo-injury. SignificanceThis study used phosphorylated TMT quantitative proteomics to explore regulation of epigenetic modifications in frozen-thawed ram sperm. This experiment demonstrated that ram sperm freezing affects phosphorylation site modifications of proteins, especially those related to functions such as sperm motility and energy production. Furthermore, it is important to link functions of phosphorylated proteins with changes in sperm quality after freezing and thawing, and to clarify intrinsic reasons for sperm quality changes, which is of great importance for elucidating mechanisms of sperm freezing damage. Based on these protein markers and combined with cryoprotectant design theory, it provides a theoretical basis and data reference to study sperm cryoprotectants.

Rapid auxin signaling: An ancient and conserved response in plants

&lt;p&gt;Rapid auxin responses in plants are crucial in initiating cellular changes. These responses are involved in processes such as plasma membrane depolarization, cytoplasmic streaming, apoplastic pH changes, calcium influx, etc. Recent studies illustrated how auxin triggers rapid changes in protein phosphorylation in different species through both the ABP-TMK auxin perception at the cell surface and a conserved RAF-like kinase-based mechanism. These works uncovered an ancient system for rapid responses to the auxin signaling molecule, shedding light on its profound impact on various cellular pathways and functions.&lt;/p&gt;

Surviving winter on the Qinghai-Xizang Plateau: Extensive reversible protein phosphorylation plays a dominant role in regulating hypometabolism in hibernating Nanorana parkeri.

Changes in protein abundance and reversible protein phosphorylation (RPP) play important roles in regulating hypometabolism but have never been documented in overwintering frogs at high altitudes. To test the hypothesis that protein abundance and phosphorylation change in response to winter hibernation, we conducted a comprehensive and quantitative proteomic and phosphoproteomic analysis of the liver of the Xizang plateau frog, Nanorana parkeri, living on the Qinghai-Xizang (Tibet) Plateau (QTP). In total, 5 170 proteins and 5 695 phosphorylation sites in 1 938 proteins were quantified. Based on proteomic analysis, 674 differentially expressed proteins (438 up-regulated, 236 down-regulated) were screened in hibernating N. parkeri versus summer individuals. Functional enrichment analysis revealed that higher expressed proteins in winter were significantly enriched in immune-related signaling pathways, whereas lower expressed proteins were mainly involved in metabolic processes. A total of 4 251 modified sites (4 147 up-regulated, 104 down-regulated) belonging to 1 638 phosphoproteins (1 555 up-regulated, 83 down-regulated) were significantly changed in the liver. During hibernation, RPP regulated a diverse array of proteins involved in multiple functions, including metabolic enzymatic activity, ion transport, protein turnover, signal transduction, and alternative splicing. These changes contribute to enhancing protection, suppressing energy-consuming processes, and inducing metabolic depression. Moreover, the activities of phosphofructokinase, glutamate dehydrogenase, and ATPase were all significantly lower in winter compared to summer. In conclusion, our results support the hypothesis and demonstrate the importance of RPP as a regulatory mechanism when animals transition into a hypometabolic state.

Gallic acid attenuates metastatic potential of human colorectal cancer cells through the miR-1247-3p-modulated integrin/FAK axis.

Colorectal cancer (CRC) exhibits highly metastatic potential even in the early stages of tumor progression. Gallic acid (GA), a common phenolic compound in plants, is known to possess potent antioxidant and anticancer activities, thereby inducing cell death or cell cycle arrest. However, whether GA reduces the invasiveness of CRC cells without inducing cell death remains unclear. Herein, we aimed to investigate the antimetastatic activity of low-dose GA on CRC cells and determine its underlying mechanism. Cell viability and tumorigenicity were analyzed by MTS, cell adhesion, and colony formation assay. Invasiveness was demonstrated using migration and invasion assays. Changes in protein phosphorylation and expression were assessed by Western blot. The involvement of microRNAs was validated by microarray analysis and anti-miR antagonist. Our findings showed that lower dose of GA (≤100 μM) did not affect cell viability but reduced the capabilities of colony formation, cell adhesion, and invasiveness in CRC cells. Cellularly, GA downregulated the cellular level of integrin αV/β3, talin-1, and tensin and diminished the phosphorylated FAK, paxillin, Src, and AKT in DLD-1 cells. Microarray results revealed that GA increased miR-1247-3p expression, and pretreatment of anti-miR antagonist against miR-1247-3p restored the GA-reduced integrin αV/β3 and the GA-inhibited paxillin activation in DLD-1 cells. Consistently, the in vivo xenograft model showed that GA administration inhibited tumor growth and liver metastasis derived from DLD-1 cells. Collectively, our findings indicated that GA inhibited the metastatic capabilities of CRC cells, which may result from the suppression of integrin/FAK axis mediated by miR1247-3p.

Targeting Covalent and Non-Covalent Btki-Resistant CLL Using the Dual Irreversible/Reversible 4 th Generation BTK Inhibitor LP-168

Background: Treatment of chronic lymphocytic leukemia (CLL) has been transformed with targeted therapies including inhibitors of Bruton's tyrosine kinase (BTKi). Currently three covalent BTK inhibitors (cBTKi) are approved for CLL, but most patients eventually relapse, commonly through acquisition of the C481S BTK mutation (Woyach et al. 2014). Pirtobrutinib and nemtabrutinib are non-covalent BTK inhibitors (ncBTKi) developed to target and inhibit C481S mutant BTK. However novel secondary site mutations in BTK, namely T474I and L528W, have been found to confer resistance to both cBTKi and ncBTKi (Wang et al. 2022). Regardless of these mutations, BCR signaling remains intact, suggesting that inhibition of BTK maintains its therapeutic importance. LP-168 is a novel ultra-selective 4 th generation BTKi with an active warhead capable of covalent interaction with WT BTK or non-covalent binding when a BTK C481 mutation is present. Methods: To determine the target selectivity of LP-168 we screened the compound against 468 kinases in the scanMAX Kinase Assay. For validation of covalent binding, dialysis against WT BTK was performed. Primary CLL B cells were isolated by negative selection and treated with LP-168 for all experiments. BCR signaling alterations were assessed via immunoblot to observe changes in target protein phosphorylation. Changes in CLL migration towards CXCL12 or CXCL13 were interrogated by transwell migration. Cytotoxicity towards primary CLL cells was measured via Annexin V/PI flow cytometry with and without HS-5 stromal cell support. Experiments using TMD8 cells were performed following CRISPR modification to insert WT, C481S, or T474I BTK. In vivo survival studies were performed using both the Eμ-TCL1 and Eμ-MTCP1 mouse engraftment models. Results: LP-168 demonstrated selectivity towards BTK with roughly 700-fold selectivity for BTK vs its next off-target. In an enzymatic assay, LP-168 displayed nanomolar potency towards both WT BTK (IC 50=0.11 nM) and C481S BTK (IC 50=1.0 nM). Following 2-hour drugging, LP-168 inhibited BCR signaling in primary CLL B cells at increasing concentrations, demonstrated by reduced phosphorylation of BTK (92%, p&amp;lt;0.0001, n=8) and its immediate downstream target PLCγ2 (41%, p=0.0013, n=8). Migration capacity of primary CLL cells towards CXCL12 and CXCL14 was also found to decrease after 2-hours of treatment with LP-168 (52%, p&amp;lt;0.001, n=10 and 51%, p&amp;lt;0.001, n=10, respectively). After 48 hours of exposure, LP-168 was able to induce cytotoxicity of primary CLL cells alone and in co-culture with HS-5 stromal cells in a dose-dependent manner, with similar or improved efficacy to other BTKi. LP-168 decreased CLL cell production of CCL3 and CCL4 chemokines (CCL3: 93%, p&amp;lt;0.001, n=8; CCL4: 93%, p&amp;lt;0.001, n=8). LP-168 also demonstrated modest cytotoxicity towards TMD8 cells harboring either C481S BTK (24%, p=0.0008 , n=3) or T474I BTK (13%, p=0.0690, n=3), as well as decreased production of CCL3 (C481S: 95%, p&amp;lt;0.0001, n=3; T474I: 99%, p&amp;lt;0.0001, n=3) and CCL4 (C481S: 97%, p&amp;lt;0.0001, n=3; T474I: 98%, p&amp;lt;0.0001, n=3) (Figure 1A). Further, we observed inhibition of downstream BCR signaling in primary CLL B cells harboring C481S via immunoblotting and cytokine production. Finally, to evaluate the in vivo efficacy of LP-168 we tested this compound in both the Eµ-TCL1 and Eµ-MTCP1 models. We treated mice daily via oral gavage and found that 50 mg/kg of LP-168 significantly improved survival in the Eµ-TCL1 model when compared to vehicle (median 51 vs 44 days; p=0.0018) or ibrutinib at 50 mg/kg (median 51 vs 45 days; p=0.0098) and the Eµ-MTCP1 model when compared with vehicle (median 122 vs 62 days; p&amp;lt;0.0001) or ibrutinib at 50 mg/kg (median 122 vs 96 days; p=0.0162) (Figure 1B). Conclusions: Collectively, our data demonstrate that LP-168 is a potent and selective inhibitor of BTK with activity against CLL, even in the presence of mutations that mediate resistance to cBTKi and ncBTKi. These data support the continued preclinical and clinical investigation of LP-168, which is currently being studied in the phase 1 setting of CLL (NCT04775745) and NHL (NCT04993690).

The PKC-β Inhibitor MS-553 Displays Preclinical Efficacy in BTK Inhibitor Resistant Chronic Lymphocytic Leukemia

Background: Treatment of chronic lymphocytic leukemia (CLL) has been revolutionized by usage of targeted therapies against the B-cell receptor (BCR) signaling cascade, specifically Bruton's Tyrosine Kinase (BTK). However, resistance to BTK inhibition occurs through acquisition of various mutations in BTK or its immediate downstream signaling partner, PLCγ2. Clinical outcomes for these patients are poor, emphasizing the need for alternative therapeutics. In the presence of these mutations, BCR signaling remains intact, suggesting that targeting molecules downstream of BTK may be effective. Protein kinase C-β (PKCβ) is a downstream component of the BCR pathway that has been shown to be over-expressed in CLL. Moreover, PKCβ is essential to the development of CLL in the Eμ-TCL1 mouse model and its expression in stromal cells is required for the survival of leukemic B-cells (Holler et al. 2009). This suggests that signaling through PKCβ, in both CLL cells and cells in the microenvironment, is essential to promote leukemogenesis. MS-553 is a potent, ATP competitive, reversible inhibitor of multiple PKC isoforms including PKCβ, with the potential to overcome BTKi mediated resistance. Methods: Primary CLL cells were isolated by negative selection and treated with up to 5 μM MS-553. Cell viability was assessed via Annexin V/PI with and without HS-5 stromal cell co-culture. BCR and canonical WNT signaling changes were evaluated by change in target protein phosphorylation or total protein by immunoblot following drugging. Changes in downstream NFκB and WNT signaling were interrogated by RT-qPCR. Using genetically modified TMD8 cells that express only C481S or T474I mutated BTK, we tested MS-553's effect on cell-viability and BCR signaling. In vivo survival studies were performed using the Eμ-MTCP1 (Walker et al. 2021) adoptive transfer mouse model, an aggressive model of CLL, with subsequent randomization into vehicle or MS-553 treatment arms. Results: Following 72 hours of drugging, 5 μM MS-553 displayed modest cytotoxicity against primary CLL cells both alone (50%, p&amp;lt;0.0001, n=8) and in the presence of HS-5 stromal cells (25%, p=0.0008, n=8). In primary CLL cells harboring the C481S BTK mutation, we observed retained cytotoxicity following 72 hours of drugging (29%, p=0.00029, n=6). At 24 hours, 5 μM MS-553 inhibited downstream BCR and WNT signaling in primary CLL cells, demonstrated by reduced phosphorylation of PKC-β (43%, p=0.0024, n=15) and several of its downstream targets including pGSK3β (45%, p&amp;lt;0.0001, n=15), pIκBα (66%, p&amp;lt;0.0001, n=15), and pERK (17%, p=0.0301, n=15), and reduced total protein of β-Catenin (82%, p&amp;lt;0.0001, n=9), Cyclin D2 (71%, p=0.0011, n=9), and cMyc (85%, p&amp;lt;0.0001, n=9) compared to vehicle treated, stimulated samples. In both treatment naïve and C481S patient samples, mRNA expression of NFκB and WNT pathway targets BCL-2, OCT-2, BFL-1, MYC, CD40, Cyclin D1, and Cyclin D2 were all decreased after 48 hours of treatment with 1 or 5 μM MS-553. MS-553 also showed efficacy in the presence of BTK mutations: viability in TMD8 cells was reduced following 72-hour drugging with MS-553 regardless of BTK mutational status (WT: 22%, p=0.0006, n=3; C481S BTK: 18%, p=0.0032, n=3; T474I BTK: 19%, p=0.0015, n=3). Inflammatory cytokine production is inhibited by MS-553 through its ability to decrease CCL3 and CCL4 cytokine expression in WT and mutant BTK-harboring TMD8 cells (p&amp;lt;0.0001, n=3) (Figure 1A). To validate inhibition of BCR signaling in the C481S BTK setting, we treated primary CLL cells harboring this mutation and observed a reduction in both CCL3 and CCL4 production (61%, p=0.001229, n=5). As proof of concept, we examined MS-553's efficacy in vivo in the Eμ-MTCP1 mouse engraftment model, and found a significant reduction in disease progression and increase in survival (median 117 days) when compared to vehicle control (median 56 days, p=0.0003) (Figure 1B). Conclusion: Our data demonstrate the efficacy of the PKCβ inhibitor MS-553 in preclinical models of CLL including that with BTK mutations conferring BTKi resistance. These findings support continued preclinical and clinical investigation of MS-553 in CLL, and the phase 1 trial of this agent is currently underway (NCT03492125) (Blachly et al. 2022).

Doxorubicin-induced modulation of TGF-β signaling cascade in mouse fibroblasts: insights into cardiotoxicity mechanisms

Doxorubicin (DOX)-induced cardiotoxicity has been widely observed, yet the specific impact on cardiac fibroblasts is not fully understood. Additionally, the modulation of the transforming growth factor beta (TGF-β) signaling pathway by DOX remains to be fully elucidated. This study investigated DOX’s ability to modulate the expression of genes and proteins involved in the TGF-β signaling cascade in mouse fibroblasts from two sources by assessing the impact of DOX treatment on TGF-β inducible expression of pivotal genes and proteins within fibroblasts. Mouse embryonic fibroblasts (NIH3T3) and mouse primary cardiac fibroblasts (CFs) were treated with DOX in the presence of TGF-β1 to assess changes in protein levels by western blot and changes in mRNA levels by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Our results revealed a dose-dependent reduction in cellular communication network factor 2 (CCN2) protein levels upon DOX treatment in both NIH3T3 and CFs, suggesting an antifibrotic activity by DOX in these fibroblasts. However, DOX only inhibited the TGF-β1 induced expression of COL1 in NIH3T3 cells but not in CFs. In addition, we observed that DOX treatment reduced the expression of BMP1 in NIH3T3 but not primary cardiac fibroblasts. No significant changes in SMAD2 protein expression and phosphorylation in either cells were observed after DOX treatment. Finally, DOX inhibited the expression of Atf4 gene and increased the expression of Cdkn1a, Id1, Id2, Runx1, Tgfb1, Inhba, Thbs1, Bmp1, and Stat1 genes in NIH3T3 cells but not CFs, indicating the potential for cell-specific responses to DOX and its modulation of the TGF-β signaling pathway.

Inhibition of HSP90 distinctively modulates the global phosphoproteome of Leishmania mexicana developmental stages.

In the unicellular parasites Leishmania spp., the etiological agents of leishmaniasis, a complex infectious disease that affects 98 countries in 5 continents, chemical inhibition of HSP90 protein leads to differentiation from promastigote to amastigote stage. Recent studies indicate potential role for protein phosphorylation in the life cycle control of Leishmania. Also, recent studies suggest a fundamentally important role of RNA-binding proteins (RBPs) in regulating the downstream effects of the HSP90 inhibition in Leishmania. Phosphorylation-dephosphorylation dynamics of RBPs in higher eukaryotes serves as an important on/off switch to regulate RNA processing and decay in response to extracellular signals and cell cycle check points. In the current study, using a combination of highly sensitive TMT labeling-based quantitative proteomic MS and robust phosphoproteome enrichment, we show for the first time that HSP90 inhibition distinctively modulates global protein phosphorylation landscapes in the different life cycle stages of Leishmania, shedding light into a crucial role of the posttranslational modification in the differentiation of the parasite under HSP90 inhibition stress. We measured changes in phosphorylation of many RBPs and signaling proteins including protein kinases upon HSP90 inhibition in the therapeutically relevant amastigote stage. This work provides insights into the importance of HSP90-mediated protein cross-talks and regulation of phosphorylation in Leishmania, thus significantly expanding our knowledge of the posttranslational modification in Leishmania biology.

Proteomics, Transcriptomics, and Phosphoproteomics Reveal the Mechanism of Talaroconvolutin-A Suppressing Bladder Cancer via Blocking Cell Cycle and Triggering Ferroptosis

Talaroconvolutin-A (TalaA) is a compound from the endophytic fungus T. convolutispora of the Chinese herbal medicine Panax notoginseng. Whether TalaA exerts anticancer activity in bladder cancer remains unknown. Using CCK8 assay, EdU staining, crystal violet staining, flow cytometry, living/dead cell staining, and Western blotting, we studied the anticancer activity of TalaA in vitro. Moreover, we performed xenograft tumor implantation. The antitumor effects were evaluated through H&E and immunohistochemistry staining. Proteomics was conducted to detect changes in the protein profile; transcriptomics was performed to detect changes in mRNA abundance; phosphoproteomics was used to detect changes in protein phosphorylation. TalaA inhibited tumor cell proliferation, DNA replication, and colony formation in a dose-dependent manner in bladder cancer cells. The IC50 values of TalaA on SW780 and UM-UC-3 cells were 5.7 and 8.2 μM, respectively. TalaA (6.0 mg/kg) significantly repressed the growth of xenografted tumors and did not affect the body weight nor cause obvious hepatorenal toxicity. TalaA arrested the cell cycle by downregulating cyclinA2, cyclinB1, and AURKB and upregulating p21/CIP. TalaA also elevated intracellular reactive oxygen species and upregulated transferrin and heme oxygenase 1 to induce ferroptosis. Moreover, TalaA was able to bind to MAPKs (MAPK1, MAPK8, and MAPK14) to inhibit the phosphorylation of ∗SP∗ motif of transcription regulators. This study revealed that TalaA inhibited bladder cancer by arresting cell cycle to suppress proliferation and triggering ferroptosis to cause cell death. Conclusively, TalaA would be a potential candidate for treating bladder cancer by targeting MAPKs, suppressing the cell cycle, and inducing ferroptosis.

Phosphoproteomic insight into the changes in structural proteins of muscle architecture associated with texture softening of grass carp (Ctenopharyngodon idella) fillets during chilling storage.

Texture is an important sensory attribute of fish affected by modifications of structural proteins in muscle architecture. To investigate the changes in protein phosphorylation during texture softening of fish, the proteins of grass carp muscle after chilling storage of 0day and 6days were compared by phosphoproteomics, and their association with texture was analyzed. Totally 1026 unique phosphopeptides on 656 phosphoproteins were identified as differential. They were mainly classified as intracellular myofibril and cytoskeleton, and extracellular matrix, of which the molecular function and biological process were binding into supramolecular assembly and myofilament contraction. The concomitant dephosphorylation of kinases and assembly regulators indicated dephosphorylation and disassembly tendency of sarcomeric architecture. Correlation analysis defined the relation between texture and dephosphorylation of myosin light chain, actin, collagen and cytoskeleton. This study revealed that protein phosphorylation may affect the texture of fish muscle through regulating sarcomeric assembly of structural proteins in muscle architecture.

Actuated tissue engineered muscle grafts restore functional mobility after volumetric muscle loss

Damage that affects large volumes of skeletal muscle tissue can severely impact health, mobility, and quality-of-life. Efforts to restore muscle function by implanting tissue engineered muscle grafts at the site of damage have demonstrated limited restoration of force production. Various forms of mechanical and biochemical stimulation have been shown to have a potentially beneficial impact on graft maturation, vascularization, and innervation. However, these approaches yield unpredictable and incomplete recovery of functional mobility. Here we show that targeted actuation of implanted grafts, via non-invasive transcutaneous light stimulation of optogenetic engineered muscle, restores motor function to levels similar to healthy mice 2 weeks post-injury. Furthermore, we conduct phosphoproteomic analysis of actuated engineered muscle in vivo and in vitro to show that repeated muscle contraction alters signaling pathways that play key roles in skeletal muscle contractility, adaptation to injury, neurite growth, neuromuscular synapse formation, angiogenesis, and cytoskeletal remodeling. Our study uncovers changes in phosphorylation of several proteins previously unreported in the context of muscle contraction, revealing promising mechanisms for leveraging actuated muscle grafts to restore mobility after volumetric muscle loss.

Abstract 131: Phosphoproteomics Analysis Reveals That Angiotensin-(1-5) Inhibits MTOR Signalling Pathway In Endothelial Cells

Introduction: Our group demonstrated that Angiotensin-(1-5) [Ang-(1-5)] is a biologically active component of the protective arm of the Renin-Angiotensin System (RAS), displaying high efficacy towards the AT 2 R. However, signaling mechanisms for Ang-(1-5) in vitro are still unknown. Objective: To characterize Ang-(1-5) signaling signature in human aortic endothelial cells. Methods: Human aortic endothelial cells (HAEC) were stimulated with Ang-(1-5) (1μM) for 1, 3, 5 or 20 minutes. Proteins were isolated, digested and labeled with TMTpro TM 16-plex. Phosphorylation changes of proteins in response to Ang-(1-5) treatment were analyzed by mass spectrometry. Raw data were processed in Proteome Discoverer. General bioinformatic analysis was performed in R Studios and functional analysis was performed with DAVID and Cytoscape. Validation of phosphoproteomics data was performed by western blot in HAEC treated with Ang-(1-5) (1μM) for 1, 3, 5 or 20 minutes. Results: Treatment of HAEC with Ang-(1-5) significantly modified the phosphorylation status of 831 proteins at 1799 residues, majority of these being dephosphorylated (1079). Functional bioinformatic analysis revealed a cluster of proteins involved in cell cycle and cell division regulation. Upon further analysis, we observed that Ang-(1-5) led to dephosphorylation of residues of CLIP-170 (Ser 195 , Ser 200 , Ser 204 ), 4EBP1 (Ser 65 ), S6K1 (Ser 447 ) and ribosomal S6 protein (Ser 244 , Ser 247 ), suggesting an inhibitory effect on mTOR activity. Western blot analysis confirmed the inhibitory effect of Ang-(1-5) on mTOR signaling, as a significant dephosphorylation of Ser 65 -4EBP1 [% vs. CT = 69.9%] and Thr 389 -S6K1 [% vs. CT = 43.7%] was observed at all timepoints tested. These effects were AT 2 R-mediated, since PD123319 prevented dephosphorylation of Ser 65 -4EBP1 and Thr 389 -S6K1 driven by Ang-(1-5). Conclusion: This study provides an in-depth analysis of Ang-(1-5) signaling patterns in HAEC. The overall similarity of Ang-(1-5) and C21 intracellular signaling further supports that Ang-(1-5) is a hormone of the protective RAS. Since inhibition of mTOR in endothelial cells is known as an anti-senescence mechanism, further investigations are needed to confirm an anti-senescence effect of Ang-(1-5).

Advancing therapeutics using antibody-induced dimerization of receptor tyrosine phosphatases.

Receptor protein tyrosine phosphatases (RPTPs) are involved in a broad list of cellular, developmental, and physiological functions. Altering their expression leads to significant changes in protein phosphorylation linked to a growing list of human diseases, including cancers and neurological disorders. In this issue of Genes & Development, Qian and colleagues (pp. 743-759) present the identification of a monoclonal antibody targeting PTPRD extracellular domain-inducing dimerization and inhibition of the phosphatase activities, causing the proteolysis of dimeric PTPRD by a mechanism involving intracellular degradation pathways. Their study supports the potential of modulating PTPRD via its extracellular domains. This opens a new framework in the clinical manipulation of PTPRD and its closely related family members.

Pan-cancer analysis of post-translational modifications reveals shared patterns of protein regulation

Post-translational modifications (PTMs) play key roles in regulating cell signaling and physiology in both normal and cancer cells. Advances in mass spectrometry enable high-throughput, accurate, and sensitive measurement of PTM levels to better understand their role, prevalence, and crosstalk. Here, we analyze the largest collection of proteogenomics data from 1,110 patients with PTM profiles across 11 cancer types (10 from the National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium [CPTAC]). Our study reveals pan-cancer patterns of changes in protein acetylation and phosphorylation involved in hallmark cancer processes. These patterns revealed subsets of tumors, from different cancer types, including those with dysregulated DNA repair driven by phosphorylation, altered metabolic regulation associated with immune response driven by acetylation, affected kinase specificity by crosstalk between acetylation and phosphorylation, and modified histone regulation. Overall, this resource highlights the rich biology governed by PTMs and exposes potential new therapeutic avenues.

Nutritional stress-induced regulation of microtubule organization and mRNP transport by HDAC1 controlled α-tubulin acetylation

In response to nutritional stress, microtubules in cells of the Drosophila female germline are depleted from the cytoplasm and accumulate cortically. This triggers aggregation of mRNPs into large processing bodies (P-bodies) and oogenesis arrest. Here, we show that hyperacetylation of α-tubulin at lysine 40 (K40) alters microtubule dynamics and P-body formation. We found that depletion of histone deacetylase 1 (HDAC1) by RNAi phenocopies the nutritional stress response, causing α-tubulin hyperacetylation and accumulation of maternally deposited mRNPs in P-bodies. Through in vitro and in vivo studies, we identify HDAC1 as a direct regulator of α-tubulin K40 acetylation status. In well-fed flies, HDAC1 maintains low levels of α-tubulin acetylation, enabling the microtubule dynamics required for mRNP transport. Using quantitative phosphoproteomics we identify nutritional stress-induced changes in protein phosphorylation that act upstream of α-tubulin acetylation, including phosphorylation of HDAC1 at S391, which reduces its ability to deacetylate α-tubulin. These results reveal that Drosophila HDAC1 senses and relays the nutritional status, which regulates germline development through modulation of cytoskeleton dynamics.

Phosphoproteomic analysis highlights phosphorylation of primary metabolism-related proteins in response to postharvest cold storage and subsequent shelf-life in tomato fruit

Tomato fruit are susceptible to postharvest chilling injuries when stored at low temperatures, resulting in quality deterioration and economic losses worldwide. Protein phosphorylation is an important post-translational modification that has been proven to be involved in plant cold tolerance. However, the changes of protein phosphorylation underlying cold stress responses in tomatoes remain poorly understood. In this study, microscopic analyses showed that endomembranes and intracellular organelles were broken down after cold storage, and the breakdown became more pronounced when the fruit was restored to room temperature. Primary metabolomic analysis revealed that large amounts of lipids, amino acids, sugars, and organic acids were altered following cold treatment. Additionally, we performed comparative phosphoproteomics and determined the overall phosphoprotein profile of tomato fruit after postharvest cold storage and subsequent shelf life. A total of 10260 phosphopeptides corresponding to 9640 phosphosites in 3740 phosphoproteins were identified. Differentially phosphorylated proteins were mainly involved in primary metabolism, such as glycerolipid metabolism, glycolysis/gluconeogenesis, and the tricarboxylic acid cycle. Correspondingly, the phosphorylation of large amounts of important primary metabolic enzymes, such as fatty acid desaturase, sucrose synthase, and alcohol dehydrogenase, was upregulated after cold treatment. The present study revealed the involvement of phosphorylation of primary metabolism-related proteins in the response to postharvest cold storage in tomato fruit.

Isoflurane Rapidly Modifies Synaptic and Cytoskeletal Phosphoproteomes of the Supraoptic Nucleus of the Hypothalamus and the Cortex

Introduction: Despite the widespread use of general anaesthetics, the mechanisms mediating their effects are still not understood. Although suppressed in most parts of the brain, neuronal activity, as measured by FOS activation, is increased in the hypothalamic supraoptic nucleus (SON) by numerous general anaesthetics, and evidence points to this brain region being involved in the induction of general anaesthesia (GA) and natural sleep. Posttranslational modifications of proteins, including changes in phosphorylation, enable fast modulation of protein function which could be underlying the rapid effects of GA. In order to identify potential phosphorylation events in the brain-mediating GA effects, we have explored the phosphoproteome responses in the rat SON and compared these to cingulate cortex (CC) which displays no FOS activation in response to general anaesthetics. Methods: Adult Sprague-Dawley rats were treated with isoflurane for 15 min. Proteins from the CC and SON were extracted and processed for nano-LC mass spectrometry (LC-MS/MS). Phosphoproteomic determinations were performed by LC-MS/MS. Results: We found many changes in the phosphoproteomes of both the CC and SON in response to 15 min of isoflurane exposure. Pathway analysis indicated that proteins undergoing phosphorylation adaptations are involved in cytoskeleton remodelling and synaptic signalling events. Importantly, changes in protein phosphorylation appeared to be brain region specific suggesting that differential phosphorylation adaptations might underlie the different neuronal activity responses to GA between the CC and SON. Conclusion: In summary, these data suggest that rapid posttranslational modifications in proteins involved in cytoskeleton remodelling and synaptic signalling events might mediate the central mechanisms mediating GA.