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Small Heat Shock Proteins are considered as the first line of defense when proteostasis fails. Among them, αB-crystallin is expressed in striated muscles in which it interacts with desmin intermediate filaments to stabilize them, maintaining cytoskeleton's integrity and muscular functionalities. Desmin is a key actor for muscle health; its targeting by αB-crystallin is thus crucial, especially in stress conditions. αB-crystallin is phosphorylated and O-GlcNAcylated. Its phosphorylation increases consecutively to various stresses, correlated with its recruitment for cytoskeleton's safeguarding. However, phosphorylation as unique signal for cytoskeleton translocation remains controversial; indeed, O-GlcNAcylation was also proposed to be involved. Thus, there are still some gaps for a deeper comprehension of how αB-crystallin functions are finely regulated by post-translational modifications. Furthermore, desmin also bears both post-translational modifications; while desmin phosphorylation is closely linked to desmin intermediates filaments turnover, it is unclear whereas its O-GlcNAcylation could impact its proper function. In the herein paper, we aim at identifying whether phosphorylation and/or O-GlcNAcylation are involved in αB-crystallin targeting towards cytoskeleton in proteotoxic stress induced by proteasome inhibition in C2C12 myotubes. We demonstrated that proteotoxicity led to αB-crystallin's phosphorylation and O-GlcNAcylation patterns changes, both presenting a dynamic interplay depending on protein subfraction. Importantly, both post-translational modifications showed a spatio-temporal variation correlated with αB-crystallin translocation towards cytoskeleton. In contrast, we did not detect any change of desmin phosphorylation and O-GlcNAcylation. All together, these data strongly support that αB-crystallin phosphorylation/O-GlcNAcylation interplay rather than changes on desmin is a key regulator for its cytoskeleton translocation, preserving it towards stress. [Display omitted] • Small Heat Shock Proteins (sHSPs) are the first line of defense when proteostasis failed in stressful conditions. • The MG132-induced proteotoxicity modifies O-GlcNAcylation/phosphorylation patterns of αB-crystallin. • The MG132-induced proteotoxicity does not modify the O-GlcNAcylation/phosphorylation patterns of desmin. • Phosphorylation/O-GlcNAcylation of αB-crystallin spatio-temporally varies along proteotoxic stress. • Phosphorylation/O-GlcNAcylation interplay is a key regulator of αB-crystallin cytoskeleton translocation. [ABSTRACT FROM AUTHOR]

Saxidomus purpurata extract (SPE) is a highly consumable seafood worldwide with known health-related benefits. However, there are no reports of its' anti-obesity effect. This study explores the potential of SPE for anti-obesity effects by modulating adipogenesis and lipolysis. SPE reduced intracellular lipid and triglyceride accumulation while increasing free glycerol release in adipocytes. SPE inhibited lipogenesis protein expressions and increased the phosphorylation of hormone-sensitive lipase and Adenosine monophosphate-activated protein kinase (AMPK) to promote lipolysis. In addition, SPE suppressed adipogenesis by downregulating protein expression of key adipogenic markers, peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), and sterol regulatory element-binding protein 1 (SREBP1) via Wnt/β-catenin signaling. SPE augmented the heme oxygenase-1 (HO-1) expression. Thus, pharmacological intervention with Zinc protoporphyrin (ZnPP-HO-1 antagonist) was employed to validate the HO-1 role. The presence of ZnPP increased the lipid accumulation and reduced the free glycerol release. At the molecular level, adipogenic transcription factors (PPARγ, C/EBPα, and SREBP1) expressions were restored in the presence of ZnPP. GC-MS analysis revealed that SPE was comprised of several fatty acids, contributing to its anti-obesity activity. SPE is an effective nutraceutical that can be used to reduce the progression of obesity. HO-1 expression during adipogenesis might be the mechanism of action for the anti-obesity effect of SPE. [ABSTRACT FROM AUTHOR]

Background: Huashi Baidu Formula (HBF) is a clinical formula known for its efficacy against coronavirus disease 2019 (COVID-19). HBF may reduce the number of patients with abnormal serum creatinine while improving respiratory symptoms, suggesting that this formula may have potential for treating acute kidney injury (AKI). However, the protective effect of HBF on AKI has not been definitively confirmed, and the mechanism remains unclear. Therefore, the present study explored the renoprotective effects and molecular mechanisms of HBF and screened for its active ingredients to identify new potential applications of renoprotection by HBF. Methods: The present study first assessed the protective effects of HBF on AKI in a DOX-induced mouse model. Then, RNA-seq and bioinformatics analyses were used to explore the related pathological processes and potential molecular mechanisms, which were subsequently validated using qRT-PCR and Western blotting. Furthermore, candidate compounds with potential binding affinity to two pivotal targets, sphingosine kinase 1 (SphK1) and plasminogen activator inhibitor-1 (PAI-1), were screened from the 29 constituents present in the blood using Microscale Thermophoresis (MST). Finally, to identify the active ingredients, the candidate components were re-screened using the SphK1 kinase activity detection system or the uPA/PAI-1 substrate colorimetric assay system. Results: In the DOX-induced AKI mouse model, therapeutic administration of HBF significantly reduced the levels of CRE, BUN, TNF-α, IL-1β, IL-6, and UA in plasma and the levels of MDA, T-CHO, and TG in kidney tissue. Additionally, the levels of TP and Alb in plasma and SOD and CAT in the kidney tissue were significantly increased. Histopathological assessment revealed that HBF reduced tubular vacuolation, renal interstitial inflammatory cell infiltration, tubular atrophy, and positive staining of renal interstitial collagen. RNA-seq and bioinformatics analyses showed that oxidative stress, the immune-inflammatory response, and extracellular matrix (ECM) formation could be the pathological processes that HBF targets to exerts its renoprotective effects. Furthermore, HBF regulated the APJ/SPHK1/NF-κB and APJ/PAI-1/TGFβ signaling axes and reduced the phosphorylation levels of NF-κB p65 and SMAD2 and the expression of cytokines and the ECM downstream of the axis. Finally, six SphK1 inhibitors (paeoniflorin, astragalin, emodin, glycyrrhisoflavone, quercetin, and liquiritigenin) and three PAI-1 inhibitors (glycyrrhisoflavone, licochalcone B, and isoliquiritigenin) were identified as potentially active ingredients in HBF. Conclusion: In brief, our investigation underscores the renoprotective effect of HBF in a DOX-induced AKI model mice, elucidating its mechanisms through distinct pathological processes and identifying key bioactive compounds. These findings offer new insights for broadening the clinical applications of HBF and unravelling its molecular mode of action. [ABSTRACT FROM AUTHOR]

Objective: Autoinflammation and phospholipase C (PLC) γ2–associated antibody deficiency and immune dysregulation (APLAID) syndrome is an autoinflammatory disease caused by gain‐of‐function variants in PLCG2. This study investigates the pathogenic mechanism of a novel variant of PLCG2 in a patient with APLAID syndrome. Methods: Whole‐exome sequencing and Sanger sequencing were used to identify the pathogenic variant in the patient. Single‐cell RNA sequencing, immunoblotting, luciferase assay, inositol monophosphate enzyme‐linked immunosorbent assay, calcium flux assay, quantitative PCR, and immunoprecipitation were used to define inflammatory signatures and evaluate the effects of the PLCG2 variant on protein functionality and immune signaling. Results: We identified a novel de novo variant, PLCG2 p.D993Y, in a patient with colitis, pansinusitis, skin rash, edema, recurrent respiratory infections, B‐cell deficiencies, and hypogammaglobulinemia. The single‐cell transcriptome revealed exacerbated inflammatory responses in the patient's peripheral blood mononuclear cells. Expression of the D993Y variant in HEK293T, COS‐7, and PLCG2 knock‐out THP‐1 cell lines showed heightened PLCγ2 phosphorylation; elevated inositol‐1,4,5‐trisphosphate production and intracellular Ca2+ release; and activation of the MAPK, NF‐κB, and NFAT signaling pathways compared with control‐transfected cells. In vitro experiments indicated that the D993Y variant altered amino acid properties, disrupting the interaction between the catalytic and autoinhibitory domains of PLCγ2, resulting in PLCγ2 autoactivation. Conclusion: Our findings demonstrated that the PLCG2 D993Y variant is a gain‐of‐function mutation via impairing its autoinhibition, activating multiple inflammatory signaling pathways, thus leading to APLAID syndrome. This study further broadens the molecular underpinnings and phenotypic spectrum of PLCγ2‐related disorders. [ABSTRACT FROM AUTHOR]

Secreted protein Reelin is implicated in neuropsychiatric disorders and its supplementation ameliorates neurological symptoms in mouse disease models. Recombinant human Reelin protein may be useful for the treatment of human diseases, but its properties remain uncharacterized. Here, we report that full-length human Reelin was well secreted from transfected cells and was able to induce Dab1 phosphorylation. Unexpectedly, the central fragment of human Reelin was much less secreted than that of mouse Reelin. Three residues in the sixth Reelin repeat contributed to the secretion inefficiency, and their substitutions with mouse residues increased the secretion without affecting its biological activity. Our findings help efficient production of human Reelin protein for the supplementation therapy. [ABSTRACT FROM AUTHOR]

Introduction: Changes in plasma concentrations of anti-inflammatory cytokines, such as interleukin-6 (IL-6) and IL-10, after acute resistance exercise (RE) have been widely explored. Whether observed changes in plasma cytokine concentration correspond to the activation of anti-inflammatory signaling pathways in immune cells after acute RE is unknown. This study aimed to determine if changes in plasma cytokines after acute RE resulted in the activation of anti- inflammatory signaling pathways in peripheral blood mononuclear cells (PBMC). Methods: Healthy young males (N = 16; age = 23.5 ± 2.7 yr; BMI = 22.4 ± 1.7 kg·m-2) participated in a single session of whole-body RE (4 sets of 4 different exercises at 70% 1-repetition maximum with the last set to failure) and a sedentary control (CON) condition in a randomized crossover design. Blood samples were collected at several time points before and after the exercise bout. Results: Higher plasma IL-6, IL-10, and IL-1 RA concentrations were observed after RE compared with CON. Phosphorylation of STAT3 and protein expression of SOCS3 in PBMC were increased in RE compared with CON. The elevation of plasma IL-6, but not IL-10, coincided with the activation of STAT3 signaling in PBMC. Conclusions: These results highlight a potential mechanism by which RE may exert anti-inflammatory actions in circulating immune cells. [ABSTRACT FROM AUTHOR]

Summary: In Arabidopsis, the enzymatically active lysin motif‐containing receptor‐like kinase (LysM‐RLK) CHITIN ELICITOR RECEPTOR KINASE 1 (CERK1) and the pseudokinases LYSIN MOTIF‐CONTAINING RECEPTOR‐LIKE KINASE 5 (LYK5) and LYK4 are the core components of the canonical chitin receptor complex. CERK1 dimerizes and autophosphorylates upon chitin binding, resulting in activation of chitin signaling.In this study, we clarified and further elucidated the individual contributions of LYK4 and LYK5 to chitin‐dependent signaling using mutant (combination)s and stably transformed Arabidopsis plants expressing fluorescence‐tagged LYK5 and LYK4 variants from their endogenous promoters.Our analyses revealed that LYK5 interacts with CERK1 upon chitin treatment, independently of LYK4 and vice versa. We show that chitin‐induced autophosphorylation of CERK1 is predominantly dependent on LYK5, whereas chitin‐triggered ROS generation is almost exclusively mediated by LYK4. This suggests specific signaling functions of these two co‐receptor proteins apart from their redundant function in mitogen‐activated protein kinase (MAPK) signaling and transcriptional reprogramming.Moreover, we demonstrate that LYK5 is subject to chitin‐induced and CERK1‐dependent ubiquitination, which serves as a signal for chitin‐induced internalization of LYK5. Our experiments provide evidence that a combination of phosphorylation and ubiquitination events controls LYK5 removal from the plasma membrane via endocytosis, which likely contributes to receptor complex desensitization. [ABSTRACT FROM AUTHOR]

Parkinson's disease (PD) is a neurodegenerative disorder caused by the degeneration of dopaminergic neurons in the brain stem. PD is mostly sporadic, but familial PD (FPD) cases are recorded in different studies. The first gene mutation that is linked to FPD is α‐synuclein (α‐syn). It was then found that α‐syn is also accumulated in Lewy body (LB), a classical pathological hallmark in PD patients. Different studies have shown that α‐syn accumulation and aggregation can be a crucial factor contributing to the degeneration of dopaminergic neurons in PD. α‐syn has been found to be degraded by the ubiquitin proteasomal system (UPS) and autophagy–lysosomal pathway (ALP). In this study, we initially explored how α‐syn phosphorylation by GRK6, PLK2 and CK2α would facilitate its degradation in relation to the UPS or ALP. Unexpectedly, we found that the degradation of α‐syn through PLK2 phosphorylation could be modulated by UPS and ALP in a novel mechanism. Specially, attenuation of UPS could increase the amount of PLK2 and then could facilitate the phosphorylation and degradation of α‐syn through ALP. To test this further in vivo, we attenuate the proteasomal activity in a well‐established A53T α‐syn transgenic PD mouse model. We found that attenuation of proteasomal activity in the A53T α‐syn transgenic mice could reduce the accumulation of α‐syn in the striatum and midbrain. Based on our results, this study provides a new insight into how α‐syn is degraded through the UPS and ALP. [ABSTRACT FROM AUTHOR]

Background: Neuroinflammation is widely recognized as a key factor in the pathogenesis of Alzheimer’s disease (AD), alongside ß-amyloid deposition and the formation of neurofibrillary tangles. The NLR family pyrin domain containing 3 (NLRP3) inflammasome, part of the innate immune system, has been implicated in the neuropathology of both preclinical amyloid and tau transgenic models. Activation of the NLRP3 pathway involves an initial priming step, which increases the expression of Nlrp3 and interleukin (IL)-1β, followed by the assembly of the NLRP3 inflammasome complex, comprising NLRP3, ASC, and caspase-1. This assembly leads to the proteolytic maturation of the pro-inflammatory cytokines IL-1β and IL-18. Additionally, the NLRP3 inflammasome induces Gasdermin D (GSDMD) cleavage, forming membrane pores through which IL-1β and IL-18 are secreted. Inhibition of NLRP3 has been shown to enhance plaque clearance by modulating microglial activation. Furthermore, blocking NLRP3 in tau transgenic mice has been found to reduce tau phosphorylation by affecting the activity of certain tau kinases and phosphatases. Methods: In this study, organotypic brain slice cultures from P301S transgenic mice were treated with lipopolysaccharide (LPS) plus nigericin as a positive control or exposed to tau seeds (K18) to evaluate NLRP3 inflammasome activation. The effect of tau seeding on NLRP3 activity was further examined using Meso Scale Discovery (MSD) assays to measure IL1β secretion levels in the presence and absence of NLRP3 inhibitors. The role of NLRP3 activity was investigated in fullbody Nlrp3 knockout mice crossbred with the tau transgenic P301S model. Additionally, full-body and microglia-selective Gsdmd knockout mice were crossbred with P301S mice, and tau pathology and neurodegeneration were evaluated at early and late stages of the disease using immunohistochemistry and biochemical assays. Results: Activation of the NLRP3 pathway was observed in the mouse organotypic slice culture (OSC) model following stimulation with LPS and nigericin or exposure to tau seeds. However, Nlrp3 deficiency did not mitigate tauopathy or neurodegeneration in P301S mice in vivo, showing only a minor effect on plasma neurofilament (NF-L) levels. Consistently, Gsdmd deficiency did not alter tau pathology in P301S mice. Furthermore, neither full-body nor microglia-selective Gsdmd deletion had an impact on neuronal pathology or the release of pro-inflammatory cytokines. Conclusion: The absence of key components of the NLRP3 inflammasome pathway did not yield a beneficial effect on tau pathology or neurodegeneration in the preclinical Tau-P301S mouse model of AD. Nonetheless, organotypic slice cultures could serve as a valuable ex vivo mechanistic model for evaluating NLRP3 pathway activation and pharmacological inhibitors. [ABSTRACT FROM AUTHOR]

Alzheimer’s disease (AD) is a debilitating progressively neurodegenerative disease. The best-characterized hallmark of AD, which is marked by behavioral alterations and cognitive deficits, is the aggregation of deposition of amyloid-beta (Aβ) and hyper-phosphorylated microtubule-associated protein Tau. Despite decades of experimental progress, the control rate of AD remains poor, and more precise deciphering is needed for potential therapeutic targets and signaling pathways involved. In recent years, phosphoinositide 3-kinase (PI3K) and Akt have been recognized for their role in the neuroprotective effect of various agents, and glycogen synthase kinase 3 (GSK3), a downstream enzyme, is also crucial in the tau phosphorylation and Aβ deposition. An overview of the function of PI3K/Akt pathway in the pathophysiology of AD is provided in this review, along with a discussion of recent developments in the pharmaceuticals and herbal remedies that target the PI3K/Akt signaling pathway. In conclusion, despite the challenges and hurdles, cumulative findings of novel targets and agents in the PI3K/Akt signaling axis are expected to hold promise for advancing AD prevention and treatment. [ABSTRACT FROM AUTHOR]

The four-carbon non-proteinogenic amino acid γ-aminobutyric acid (GABA) accumulates to high levels in plants in response to various abiotic and biotic stress stimuli, and plays a role in C:N balance, signaling, and as a transport regulator. Expression in Xenopus oocytes and voltage-clamping allowed the characterization of Arabidopsis GAT2 (At5g41800) as a low affinity GABA transporter with a K 0.5 G A B A ~8 mM. l -Alanine and butylamine represented additional substrates. GABA-induced currents were strongly dependent on the membrane potential, reaching the highest affinity and highest transport rates at strongly negative membrane potentials. Mutation of Ser17, previously reported to be phosphorylated in planta, did not result in altered affinity. In a short-term stress experiment, AtGAT2 mRNA levels were up-regulated at low water potential and under osmotic stress (polyethylene glycol and mannitol). Furthermore, AtGAT2 promoter activity was detected in vascular tissues, maturating pollen, and the phloem unloading region of young seeds. Even though this suggested a role for AtGAT2 in long-distance transport and loading of sink organs, under the conditions tested neither AtGAT2 -overexpressing plants, atgat2 or atgat1 T-DNA insertion lines, nor atgat1 atgat2 doubleknockout mutants differed from wild-type plants in growth on GABA, amino acid levels, or resistance to salt and osmotic stress. [ABSTRACT FROM AUTHOR]

In the elderly population, coronary heart disease (CHD) often coexists with hypertension. However, excessive blood pressure reduction can paradoxically increase the incidence of adverse events. Understanding the molecular mechanisms underlying hypertension and CHD in aged populations is crucial for developing targeted therapies and improving clinical outcomes. In this study, we constructed myocardial infarction (MI) and transverse aortic constriction (TAC) modelsY in aged mice to simulate the disease states of CHD and hypertension, respectively. Using integrated proteomic and phosphoproteomic analyses, we investigated the molecular signatures associated with MI and TAC in these models. Our aim was to identify key molecules involved in these conditions and to understand their unique and shared characteristics. Through our comprehensive proteomic and phosphoproteomic analysis, we identified a total of 1583 proteins and 232 phosphorylated proteins. We observed significant upregulation of heart disease markers such as Myh7, Xirp2, and Acta1, indicating the successful establishment of the MI and TAC models. The overlapped differentially expressed proteins (DEPs) and differentially phosphorylated proteins (DPPs) in MI and TAC were involved in heart failure‐related processes including cardiac muscle contraction and hypertrophic cardiomyopathy, further supporting the validity of the models. Among the DEPs, Ppme1 was upregulated in the TAC model but downregulated in the MI model, while Sec31a and Gm56451 displayed the opposite expression patterns. Among the DPPs, Ablim1 and Atp2a2 were found to be significantly upregulated in the TAC model, whereas their expression was markedly reduced in the MI model. In addition, five other DPPs, including REV_Q3TAY5, Cbx3, PITPNB, Eif4b, and A0A1Y7VP73, were elevated in the MI model but decreased in the TAC model. In conclusion, these findings suggest that MI and TAC not only share certain molecular features but also retain their unique characteristics, providing potential biomarkers and therapeutic targets. [ABSTRACT FROM AUTHOR]

Summary Reactive oxygen species (ROS) are among the most important signaling molecules, playing a significant role in plant growth, development, and responses to various environmental stresses. Respiratory burst oxidase homologs (RBOHs) are key enzymes in ROS production. Plants tightly regulate the activation and deactivation of RBOHs through various post‐translational modifications (PTMs), including phosphorylation, ubiquitination, S‐nitrosylation, and persulfidation. These PTMs fine‐tune ROS production, ensuring normal plant growth and development while facilitating rapid responses to abiotic and biotic stresses. This review discusses the effects of different PTMs on RBOH function and their biological relevance. Additionally, we examine the evolutionary conservation of PTM sites and emphasize the complex interplay between multiple PTMs regulating RBOHs. [ABSTRACT FROM AUTHOR]

Contact inhibition of proliferation is a critical cell density control mechanism governed by the Hippo signalling pathway. The biochemical signalling underlying cell density‐dependent cues regulating Hippo signalling and its downstream effectors, YAP, remains poorly understood. Here, we reveal that the tight junction protein ZO‐2 is required for the contact‐mediated inhibition of proliferation. We additionally determined that the well‐established molecular players of this process, namely Hippo kinase LATS1 and YAP, are regulated by ZO‐2 and that the scaffolding function of ZO‐2 promotes the interaction with and phosphorylation of YAP by LATS1. Mechanistically, YAP is phosphorylated when ZO‐2 brings LATS1 and YAP together via its SH3 and PDZ domains, respectively, subsequently leading to the cytoplasmic retention and inactivation of YAP. In conclusion, we demonstrate that ZO‐2 maintains Hippo signalling pathway activation by promoting the stability of LATS1 to inactivate YAP. [ABSTRACT FROM AUTHOR]

Circadian clocks rely on transcriptional/translational feedback loops involving clock genes and their corresponding proteins. While the primary oscillations originate from gene expression, the precise control of clock protein stability plays a pivotal role in establishing the 24-hour circadian rhythms. Most clock proteins are degraded through the ubiquitin/26S proteasome pathway, yet the enzymes responsible for ubiquitination and deubiquitination remain poorly characterised. We identified a missense allele (ubp12-3, S327F) of the UBP12 gene/protein in Arabidopsis. Despite ubp12-3 exhibited a short period phenotype similar to that of a loss-of-function allele, molecular analysis indicated elevated protease activity in ubp12-3. We demonstrated that early flowering of ubp12 mutants is a result of the shortened circadian period rather than a direct alteration of UBP12 function. Analysis of protease activity of non-phosphorylatable (S327A, S327F) and phosphomimetic (S327D) derivatives in bacteria suggested that phosphorylation of serine 327 inhibits UBP12 enzymatic activity, which could explain the over-functioning of S327F in vivo. We showed that phosphomimetic mutations of the conserved serine in the Neurospora and human orthologues reduced ubiquitin cleavage activity suggesting that not only the primary structures of UBP12-like enzymes are phylogenetically conserved across a wide range of species, but also the molecular mechanisms governing their enzymatic activity. [ABSTRACT FROM AUTHOR]

Sepsis-induced myopathy (SIM) has been recognized as a critical risk factor for the development of acquired muscle weakness among patients in the intensive care unit. These individuals frequently encounter inadequate dietary intake and malnutrition. With the aggravation of the severity of the person's condition, leading to increased skeletal muscle protein breakdown and reduced synthesis, which is an urgent problem to be solved in clinical nutritional treatment. Whole milk protein powder (WMPP) has promising bioactive nutrients and holds promising potential for enhancing skeletal muscle mass. The study was designed to delve into the potential effects and mechanisms of WMPP intervention for increaseing skeletal muscle mass on SIM mice. Our results clearly show that the intervention with WMPP can significantly improve the exercise capacity and skeletal muscle mass in SIM mice. It significantly increases the diameter and cross-sectional area (CSA) of skeletal muscle fibers, while effectively reducing the excessive aggregation of collagen fibers and the abnormal accumulation of adipose tissue in the skeletal muscle of SIM mice. Moreover, WMPP intervention also significantly alleviated the oxidative stress status of mitochondria, which subsequently enhanced the expression of mitochondrial metabolic enzymes. The mechanism may be associated with decreased AMPK phosphorylation in skeletal muscle tissue and simultaneously increased phosphorylation of mTOR, p70S6K1, and 4EBP-1 in SIM mice. In summary, the WMPP intervention significantly enhances exercise capacity and skeletal muscle mass while mitigating the oxidative stress status of mitochondria. Furthermore, it regulates skeletal muscle anabolism via the AMPK/mTOR signaling pathway in SIM mice. [ABSTRACT FROM AUTHOR]

SUMMARY The sophisticated regulation of state transition is required to maintain optimal photosynthetic performance under fluctuating light condition, through balancing the absorbed light energy between photosystem II and photosystem I. This exquisite process incorporates phosphorylation and dephosphorylation of light‐harvesting complexes and PSII core subunits, accomplished by thylakoid membrane‐localized kinases and phosphatases that have not been fully identified. In this study, one Chlamydomonas high light response gene, THYLAKOID ENRICHED FRACTION 8 (TEF8), was characterized. The Chlamydomonas tef8 mutant showed high light sensitivity and defective state transition. The enzymatic activity assays showed that TEF8 is a bona fide phosphatase localized in thylakoid membranes. Biochemical assays, including BN‐PAGE, pull‐down, and phosphopeptide mass spectrometry, proved that TEF8 associates with photosystem II and is involved in the dephosphorylation of D2 and CP29 subunits during state 2 to state 1 transition. Taken together, our results identified TEF8 as a thylakoid phosphatase with multiple dephosphorylation targets on photosystem II, and provide new insight into the regulatory mechanism of state transition and high light resistance in Chlamydomonas. [ABSTRACT FROM AUTHOR]

Psilocybin, the natural hallucinogen from Psilocybe (magic) mushrooms, is a highly promising drug candidate for the treatment of depression and several other mental health conditions. Biosynthesis of psilocybin from the amino acid l‐tryptophan involves four strictly sequential modifications. The third of these, ATP‐dependent phosphorylation of the intermediate 4‐hydroxytryptamine, is catalysed by PsiK. Here we present a crystallographic analysis and a structure‐based mutagenesis study of this kinase, providing insight into its mode of substrate recognition. The results of our work will support future bioengineering efforts aimed at generating variants of psilocybin with enhanced therapeutic properties. [ABSTRACT FROM AUTHOR]

Kinesin-5 motors play an essential role during mitotic spindle assembly in many organisms 1,2,3,4,5,6,7,8,9,10,11 : they crosslink antiparallel spindle microtubules, step toward plus ends, and slide the microtubules apart. 12,13,14,15,16,17 This activity separates the spindle poles and chromosomes. Kinesin-5s are not only plus-end-directed but can walk or be carried toward MT minus ends, 18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34 where they show enhanced localization. 3,5,7,27,29,32 The kinesin-5 C-terminal tail interacts with and regulates the motor, affecting structure, motility, and sliding force of purified kinesin-5 35,36,37 along with motility and spindle assembly in cells. 27,38,39 The tail contains phosphorylation sites, particularly in the conserved BimC box. 6,7,40,41,42,43,44 Nine mitotic tail phosphorylation sites were identified in the kinesin-5 motor of the fission yeast Schizosaccharomyces pombe , 45,46,47,48 suggesting that multi-site phosphorylation may regulate kinesin-5s. Here, we show that mutating all nine sites to either alanine or glutamate causes temperature-sensitive lethality due to a failure of bipolar spindle assembly. We characterize kinesin-5 localization and sliding force in the spindle based on Cut7-dependent microtubule minus-end protrusions in cells lacking kinesin-14 motors. 39,49,50,51,52 Imaging and computational modeling show that Cut7p simultaneously moves toward the minus ends of protrusion MTs and the plus ends of spindle midzone MTs. Phosphorylation mutants show dramatic decreases in protrusions and sliding force. Comparison to a model of force to create protrusions suggests that tail truncation and phosphorylation mutants decrease Cut7p sliding force similarly to tail-truncated human Eg5. 36 Our results show that C-terminal tail phosphorylation is required for kinesin-5/Cut7 sliding force and bipolar spindle assembly in fission yeast. [Display omitted] • Phosphorylation site mutations in the kinesin-5 tail affect motor function • Phosphorylation site mutants show impaired mitotic spindle assembly and elongation • Kinesin-5 sliding force is reduced in phosphorylation mutants • Computational modeling reproduces kinesin-5 accumulation in protrusions Jones et al. show that phosphorylation of the kinesin-5 C-terminal tail is required for proper spindle assembly in fission yeast. Mutations in multiple tail phosphorylation sites impair both spindle function and motor sliding force, similarly to full tail truncations of fission yeast and (by comparison to a model of force) human kinesin-5s. [ABSTRACT FROM AUTHOR]

SUMOylation regulates numerous cellular stress responses, yet targets in the apoptotic machinery remain elusive. We show that a single, DNA damage-induced monoSUMOylation event controls PIDDosome (PIDD1/RAIDD/caspase-2) formation and apoptotic death in response to unresolved DNA interstrand crosslinks (ICLs). SUMO-1 conjugation occurs on conserved K879 in the PIDD1 death domain (DD); is catalyzed by PIAS1 and countered by SENP3; and is triggered by ATR phosphorylation of neighboring T788 in the PIDD1 DD, which enables PIAS1 docking. Phospho/SUMO-PIDD1 proteins are captured by nucleolar RAIDD monomers via a SUMO-interacting motif (SIM) in the RAIDD DD, thus compartmentalizing nascent PIDDosomes for caspase-2 recruitment. Denying SUMOylation or the SUMO-SIM interaction spares the onset of PIDDosome assembly but blocks its completion, thus eliminating the apoptotic response to ICL repair failure. Conversely, removal of SENP3 forces apoptosis, even in cells with tolerable ICL levels. SUMO-mediated PIDDosome control is also seen in response to DNA breaks but not supernumerary centrosomes. These results illuminate PIDDosome formation in space and time and identify a direct role for SUMOylation in the assembly of a major pro-apoptotic device. After DNA damage a caspase-2 activating PIDDosome can be formed. Here the authors identify sequential post-translational modifications of PIDD1 which orchestrate PIDDosome formation in space and time. [ABSTRACT FROM AUTHOR]

The intrinsically disordered carboxy-terminal domain (CTD) of the largest subunit of RNA Polymerase II (RNAPII) consists of multiple tandem repeats of the consensus heptapeptide Y1-S2-P3-T4-S5-P6-S7. The CTD promotes liquid-liquid phase-separation (LLPS) of RNAPII in vivo. However, understanding the role of the conserved heptad residues in LLPS is hampered by the lack of direct biochemical characterization of the CTD. Here, we generated a systematic array of CTD variants to unravel the sequence-encoded molecular grammar underlying the LLPS of the human CTD. Using in vitro experiments and molecular dynamics simulations, we report that the aromaticity of tyrosine and cis-trans isomerization of prolines govern CTD phase-separation. The cis conformation of prolines and β-turns in the SPXX motif contribute to a more compact CTD ensemble, enhancing interactions among CTD residues. We further demonstrate that prolines and tyrosine in the CTD consensus sequence are required for phosphorylation by Cyclin-dependent kinase 7 (CDK7). Under phase-separation conditions, CDK7 associates with the surface of the CTD droplets, drastically accelerating phosphorylation and promoting the release of hyperphosphorylated CTD from the droplets. Our results highlight the importance of conformationally restricted local structures within spacer regions, separating uniformly spaced tyrosine stickers of the CTD heptads, which are required for CTD phase-separation. The authors explore sequence-encoded molecular grammar underlying the liquid liquid phase-separation of the human RNA polymerase II C-terminal domain and demonstrate that phase-separation appears to accelerate on phosphorylation by CDK7. [ABSTRACT FROM AUTHOR]

This article reviews the latest research progress on the role of mitochondrial autophagy receptor FUN14 domain containing 1 (FUNDC1) in mitochondrial events and kidney disease. FUNDC1 is a protein located in the outer membrane of mitochondria, which maintains the function and quality of mitochondria by regulating mitochondrial autophagy, that is, the selective degradation process of mitochondria. The structural characteristics of FUNDC1 enable it to respond to intracellular signal changes and regulate the activity of mitochondrial autophagy through phosphorylation and dephosphorylation. During phosphorylation, unc-51-like kinase 1 (ULK1) promotes the activation of mitophagy by phosphorylating Ser17 of FUNDC1. In contrast, Src and CK2 kinases inhibit the interaction between FUNDC1 and LC3 by phosphorylating Tyr18 and Ser13, thereby inhibiting mitophagy. During dephosphorylation, PGAM5 phosphatase enhances the interaction between FUNDC1 and LC3 by dephosphorylating Ser13, thereby activating mitophagy. BCL2L1 inhibits the activity of PGAM5 by interacting with PGAM5, thereby preventing the dephosphorylation of FUNDC1 and inhibiting mitophagy. FUNDC1 plays an important role in mitochondrial events, participating in mitochondrial fission, maintaining the homeostasis of iron and proteins in mitochondrial matrix, and mediating crosstalk between mitochondria, endoplasmic reticulum and lysosomes, which have important effects on cell energy metabolism and programmed death. In the aspect of kidney disease, the abnormal function of FUNDC1 is closely related to the occurrence and development of many diseases. In acute kidney injury (AKI), cardiorenal syndrome (CRS), diabetic nephropathy (DN), chronic kidney disease (CKD), renal fibrosis (RF) and renal anemia, FUNDC1-mediated imbalance of mitophagy may be one of the key factors in disease progression. Therefore, in-depth study of the regulatory mechanism and function of FUNDC1 is of great significance for understanding the pathogenesis of renal disease and developing new treatment strategies. [ABSTRACT FROM AUTHOR]

Background: Epigenetic dysregulation affecting oncogenic transcription and DNA damage response is a hallmark of cancer. The histone demethylase KDM4B, a factor regulating these processes, plays important roles in estrogen receptor-mediated transcription and DNA repair in breast cancer. However, how oncogenic phospho-signal transduction affects epigenetic regulation is not fully understood. Here we found that KDM4B phosphorylation by ribosomal S6 kinase (RSK), a downstream effector of the Ras/MAPK pathway, is critical for the function of KDM4B in response to DNA damage. Methods: KDM4B-knockout breast cancer cell lines were generated via CRISPR/Cas9-mediated gene editing. Re-expression of wild-type or phospho-site mutated KDM4B in knockout cells was performed by lentivirus-mediated gene transfer. Gene knockdown was achieved by RNA interference. DNA double-strand breaks (DSBs) were induced by ionizing radiation or laser-microirradiation. Protein accumulation at DSB sites was analyzed by immunofluorescence. KDM4B phosphorylation by RSK was assessed by in vitro and in vivo kinase assays. Gene and protein expression levels were analyzed by RT‒PCR and western blotting. The sensitivity of cells to ionizing radiation was examined by a clonogenic survival assay. Results: RSK phosphorylated KDM4B at Ser666, and inhibition of the phosphorylation by RSK depletion or RSK inhibitors abrogated KDM4B accumulation at the sites of DNA double-strand breaks (DSBs). DSB repair was significantly delayed in KDM4B-knockout cells or cells treated with RSK inhibitors. The replacement of endogenous KDM4B with the phosphomimetic mutant S666D restored KDM4B accumulation and DSB repair that had been inhibited by RSK inhibitors, suggesting a critical role for RSK at the specific serine residue of KDM4B in the effect of RSK inhibitors on DSB repair. As a consequence of these aberrant responses, inhibition of KDM4B phosphorylation increased the sensitivity of the cells to ionizing radiation. Conclusions: Overall, the present study uncovered a novel function of RSK on the DNA damage response, which provides an additional role of its inhibitor in cancer therapy. [ABSTRACT FROM AUTHOR]

The insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) is a conserved RNA-binding protein that regulates RNA stability, localization and translation. IGF2BP1 is part of various ribonucleoprotein (RNP) condensates. However, the mechanism that regulates its assembly into condensates remains unknown. By using proteomics, we demonstrate that phosphorylation of IGF2BP1 at S181 in a disordered linker is regulated in a stress-dependent manner. Phosphomimetic mutations in two disordered linkers, S181E and Y396E, modulate RNP condensate formation by IGF2BP1 without impacting its binding affinity for RNA. Intriguingly, the S181E mutant, which lies in linker 1, impairs IGF2BP1 condensate formation in vitro and in cells, whereas a Y396E mutant in the second linker increases condensate size and dynamics. Structural approaches show that the first linker binds RNAs nonspecifically through its RGG/RG motif, an interaction weakened in the S181E mutant. Notably, linker 2 interacts with IGF2BP1's folded domains and these interactions are partially impaired in the Y396E mutant. Importantly, the phosphomimetic mutants impact IGF2BP1's interaction with RNAs and remodel the transcriptome in cells. Our data reveal how phosphorylation modulates low-affinity interaction networks in disordered linkers to regulate RNP condensate formation and RNA metabolism. How phosphorylation regulates the RNA-binding protein IGF2BP1 remained largely unclear. Here, the authors discovered that phosphorylation of IGF2BP1 in disordered linkers regulates its assembly into ribonucleoprotein granules and impacts metabolism of its target RNAs. [ABSTRACT FROM AUTHOR]

p57Kip2 is a member of the cyclin-dependent kinase (CDK) Interacting Protein/Kinase Inhibitory Protein (CIP/Kip) family that also includes p21Cip1/WAF1 and p27Kip1. Different from its siblings, few data are available about the p57Kip2 protein, especially in humans. Structurally, p57Kip2 is an intrinsically unstructured protein, a characteristic that confers functional flexibility with multiple transient interactions influencing the metabolism and roles of the protein. Being an IUP, its localization, stability, and binding to functional partners might be strongly modulated by post-translational modifications, especially phosphorylation. In this work, we investigated by two-dimensional analysis the phosphorylation pattern of p57Kip2 in different cellular models, revealing how the human protein appears to be extensively phosphorylated, compared to p21Cip1/WAF1 and p27Kip1. We further observed clear differences in the phosphoisoforms distributed in the cytosolic and nuclear compartments in asynchronous and synchronized cells. Particularly, the unmodified form is detectable only in the nucleus, while the more acidic forms are present in the cytoplasm. Most importantly, we found that the phosphorylation state of p57Kip2 influences the binding with some p57Kip2 partners, such as CDKs, LIMK1 and CRM1. Thus, it is necessary to completely identify the phosphorylated residues of the protein to fully unravel the roles of this CIP/Kip protein, which are still partially identified. [ABSTRACT FROM AUTHOR]

SUMMARY: The casein kinase II (CK2) complex consists of catalytic (α) and regulatory (β) subunits and is highly conserved throughout eukaryotes. Plant CK2 plays critical roles in multiple physiological processes; however, its function in plant immunity remains obscure. In this study, we demonstrated that the unique chloroplast‐localized CK2 α subunit (CPCK2) is a negative regulator of Arabidopsis thaliana innate immunity. cpck2 mutants displayed enhanced resistance against the fungal pathogen powdery mildew, Golovinomyces cichoracearum and the virulent bacterial pathogen, Pseudomonas syringae pv. tomato (Pto) DC3000. Moreover, the cpck2‐1 mutant accumulated higher salicylic acid (SA) levels and mutations that disabled SA biosynthesis or signaling inhibited cpck2‐1‐mediated disease resistance. CPCK2 interacted with the chloroplast‐localized carbonic anhydrase (CA), SA‐binding protein 3 (SABP3), which was required for cpck2‐mediated immunity. Significantly, CPCK2 phosphorylated SABP3, which promoted S‐nitrosylation of this enzyme. It has previously been established that S‐nitrosylation of SABP3 reduces both its SA binding function and its CA activity, which compromises the immune‐related function of SABP3. Taken together, our results establish CPCK2 as a negative regulator of SA accumulation and associated immunity. Importantly, our findings unveil a mechanism by which CPCK2 negatively regulates plant immunity by promoting S‐nitrosylation of SABP3 through phosphorylation, which provides the first example in plants of S‐nitrosylation being promoted by cognate phosphorylation. Significance Statement: CPCK2, a chloroplast‐localized casein kinase II α subunit, plays a negative role in plant innate immunity. CPCK2 directly interacted with and phosphorylated SA‐binding protein 3 (SABP3) to enhance the S‐nitrosylation status of SABP3, implying a potential mechanism by which CPCK2 negatively regulates plant immunity by promoting S‐nitrosylation of SABP3 through phosphorylation, which is the first example in plants of S‐nitrosylation being promoted by cognate phosphorylation. [ABSTRACT FROM AUTHOR]

Summary: The integrity of wheat (Triticum aestivum) production is increasingly jeopardized by the fungal pathogen Blumeria graminis f. sp. tritici (Bgt), particularly amid the vicissitudes of climate change.Here, we delineated the role of a wheat transcription factor, TaNAC1, which precipitates cellular apoptosis and fortifies resistance against Bgt. Utilizing BiFC, co‐immunoprecipitation, protein quantification, luciferase report assays, we determined that cytoplasmic TaNAC1‐7A undergoes phosphorylation at the S184/S258 sites by TaCDPK20, facilitating its nuclear translocation. This migration appears to prime further phosphorylation by TaMPK1, thereby enhancing transcriptional regulatory activity. Notably, the apoptotic activity of phosphorylated TaNAC1‐7A is negatively modulated by the nuclear protein phosphatase PP2Ac.Furthermore, activation of TaNAC1 phosphorylation initiates transcription of downstream genes TaSec1a and TaCAMTA4, through binding to the C[T/G]T[N7]A[A/C]G nucleic acid motif. Suppression of TaNAC1, TaCDPK20, and TaMPK1 in wheat compromises its resistance to Bgt strain E09, whereas overexpression of TaNAC1 and silencing of PP2Ac markedly elevate resistance levels.Our results reveal the pivotal role of TaNAC1 in basal resistance which is mediated by its effects on homotypic fusion, vacuolar protein sorting, and the expression of defense‐related genes. The findings highlight the potential through targeting TaNAC1 and its regulators as a strategy for improving wheat's resistance to fungal pathogens. [ABSTRACT FROM AUTHOR]

CCAAT/enhancer‐binding protein α (C/EBPα), a key myeloid transcription factor, drives myeloid differentiation from blast cells by regulating the expression of granulocyte colony stimulating factor receptor and C/EBPε as required for promoting granulocyte differentiation. Here, we show that serine/threonine‐protein kinase NLK, also known as Nemo‐like kinase, physically associates with C/EBPα and phosphorylates it at multiple sites, including Ser21, Thr226, Thr230 and S234, leading to its ubiquitin‐mediated degradation. Individual phospho‐point mutants of C/EBPα could be phosphorylated by NLK, but a mutant with all phosphorylatable residues replaced by alanine resisted phosphorylation and degradation by NLK, as did the single point mutants. Furthermore, although ectopic expression of NLK enhanced phosphorylation of C/EBPα levels, it markedly inhibited total C/EBPα protein levels. Conversely, NLK depletion inhibited endogenous C/EBPα phosphorylation but enhanced its total protein levels in several acute myeloid leukemia (AML) cell lines and in peripheral blood mononuclear cells isolated from number of AML patient samples. Importantly, NLK depletion in peripheral blood mononuclear cells from primary AML patients not only restored C/EBPα protein levels, but also induced myeloid differentiation, suggesting that NLK could be therapeutically targeted to restore C/EBPα to resolve differentiation arrest in AML. [ABSTRACT FROM AUTHOR]

Phosphorylation is a key post-translational modification regulating protein function and biological outcomes. However, the phosphorylation dynamics orchestrating mammalian oocyte development remains poorly understood. In the present study, we apply high-resolution mass spectrometry-based phosphoproteomics to obtain the first global in vivo quantification of mouse oocyte phosphorylation. Of more than 8000 phosphosites, 75% significantly oscillate and 64% exhibit marked upregulation during meiotic maturation, indicative of the dominant regulatory role. Moreover, we identify numerous novel phosphosites on oocyte proteins and a few highly conserved phosphosites in oocytes from different species. Through functional perturbations, we demonstrate that phosphorylation status of specific sites participates in modulating critical events including metabolism, translation, and RNA processing during meiosis. Finally, we combine inhibitor screening and enzyme-substrate network prediction to discover previously unexplored kinases and phosphatases that are essential for oocyte maturation. In sum, our data define landscape of the oocyte phosphoproteome, enabling in-depth mechanistic insights into developmental control of germ cells. Synopsis: Oocytes plays a central role in the success of reproduction, yet the phosphorylation dynamics during mammalian oocyte development remain poorly understood. This resource article describes the comprehensive stage-resolved phosphoproteome and proteome landscape of mouse oocytes, and reveals novel molecular mechanisms controlling meiotic maturation. The phosphorylation landscape of oocytes during in vivo maturation is delineated. Novel phosphosites in oocytes and conserved phosphosites across multiple species are identified. Key kinases orchestrating the regulation of oocyte meiosis are pinpointed Phosphatase dynamics during meiotic maturation are deciphered. This resource article provides the most comprehensive stage-resolved phosphoproteome and proteome landscape of mouse oocytes to date, revealing novel molecular control mechanisms. [ABSTRACT FROM AUTHOR]

ABSTRACT Actin depolymerizing factors (ADFs), like other actin‐binding proteins (ABPs), are modified by phosphorylation to regulate the dynamics of the actin filaments, thereby functioning in various processes throughout the plant lifecycle. In this study, we found that the Arabidopsis thaliana cytoplasmic kinase AGC1.7 interacts with ADF7 in vitro and in vivo. AGC1.7 phosphorylates ADF7 at its Ser‐6, Ser‐103 and Ser‐104 residues in vitro, while replacing these residues with alanine promotes ADF7‐mediated actin depolymerization in vitro. Expression of the phosphorylation‐mimetic mutant protein ADF7S6/103/104D driven by the pollen‐specific LAT52 promoter fully rescues the defects in germination rate, silique length and seeds per silique in both adf7‐2 and agc1.5 agc1.7 (agcdm) mutants. Our data establish a model whereby AGC1.7‐mediated ADF7 phosphorylation plays an important role in pollen germination and pollen tube growth. [ABSTRACT FROM AUTHOR]

Increasing cyclic GMP activates 26S proteasomes via phosphorylation by Protein Kinase G and stimulates the intracellular degradation of misfolded proteins. Therefore, agents that raise cGMP may be useful therapeutics against neurodegenerative diseases and other diseases in which protein degradation is reduced and misfolded proteins accumulate, including Charcot Marie Tooth 1A and 1B peripheral neuropathies, for which there are no treatments. Here we increased cGMP in the S63del mouse model of CMT1B by treating for three weeks with either the phosphodiesterase 5 inhibitor tadalafil, or the brain-penetrant soluble guanylyl cyclase stimulator CYR119. Both molecules activated proteasomes in the affected peripheral nerves, reduced polyubiquitinated proteins, and improved myelin thickness and nerve conduction. CYR119 increased cGMP more than tadalafil in the peripheral nerves of S63del mice and elicited greater biochemical and functional improvements. To determine whether raising cGMP could be beneficial in other neuropathies, we first showed that polyubiquitinated proteins and the disease-causing protein accumulate in the sciatic nerves of the C3 mouse model of CMT1A. Treatment of these mice with CYR119 reduced the levels of polyubiquitinated proteins and the disease-causing protein, presumably by increasing their degradation, and improved myelination, nerve conduction, and motor coordination. Thus, pharmacological agents that increase cGMP are promising treatments for CMT1 neuropathies and may be useful against other proteotoxic and neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

During the embryonic developmental stage in vertebrates, internal organs are arranged along the left–right axis. Disruptions in this process can result in congenital diseases or laterality disorders. The molecular mechanisms of left–right asymmetry in vertebrate development remain largely unclear. Due to its straightforward structure, zebrafish has become a favoured model for studying early laterality events. Here, we demonstrate that growth and development factor 11 (Gdf11) is essential for left–right development via TGF‐β signalling. Morphological analysis showed that gdf11 morphants and mutants displayed clear heart and liver laterality disorders in a Nodal signal‐dependent manner. Additionally, we found that Kupffer's vesicle formation and ciliogenesis were impaired following gdf11 deletion. We also observed that Gdf11 may form a heterodimer with Spaw, which promotes Smad2/3 phosphorylation and activates TGF‐β signalling. Subsequently, Gdf11 promotes left–right laterality by stimulating Foxj1a and its target gene expression. In summary, we reveal a critical role of Gdf11 in left–right patterning, providing fundamental insights into the developmental process of left–right asymmetry. [ABSTRACT FROM AUTHOR]

The complex network governing self-renewal in epidermal stem cells (EPSCs) is only partially defined. FOXM1 is one of the main players in this network, but the upstream signals regulating its activity remain to be elucidated. In this study, we identify cyclin-dependent kinase 1 (CDK1) as the principal kinase controlling FOXM1 activity in human primary keratinocytes. Mass spectrometry identified CDK1 as a key hub in a stem cell-associated protein network, showing its upregulation and interaction with essential self renewal-related markers. CDK1 phosphorylates FOXM1 at specific residues, stabilizing the protein and enhancing its nuclear localization and transcriptional activity, promoting self-renewal. Additionally, FOXM1 binds to the CDK1 promoter, inducing its expression. We identify the CDK1-FOXM1 feedforward loop as a critical axis sustaining EPSCs during in vitro cultivation. Understanding the upstream regulators of FOXM1 activity offers new insights into the biochemical mechanisms underlying self-renewal and differentiation in human primary keratinocytes. [ABSTRACT FROM AUTHOR]

Racemisation without Consequence: MOM deprotection during a routine synthesis of phosphoric acid 1 promoted partial racemisation of the product BINOL 5. Surprisingly, however, after phosphorylation of the partially racemised BINOL, enantiopure acid 1 was isolated. Further inspection revealed that during phosphorylation, unhydrolysed racemic phosphorochloridate 6 precipitated, thus restoring homochirality in the product phosphoric acid 1. Moreover, MOM deprotection partial racemisation was avoided by conducting reactions at lower temperatures for no longer than the required deprotection time, and/or by application of other deprotection conditions from the literature. [ABSTRACT FROM AUTHOR]

Background: Chronic migraine is a severe and common neurological disorder, yet its precise physiological mechanisms remain unclear. The IGF1/IGF1r signaling pathway plays a crucial role in pain modulation. Studies have shown that IGF1, by binding to its receptor IGF1r, activates a series of downstream signaling cascades involved in neuronal survival, proliferation, autophagy and functional regulation. The activation of these pathways can influence nociceptive transmission. Furthermore, alterations in IGF1/IGF1r signaling are closely associated with the development of various chronic pain conditions. Therefore, understanding the specific mechanisms by which this pathway contributes to pain is of significant importance for the development of novel pain treatment strategies. In this study, we investigated the role of IGF1/IGF1r and its potential mechanisms in a mouse model of chronic migraine. Methods: Chronic migraine was induced in mice by repeated intraperitoneal injections of nitroglycerin. Mechanical and thermal hypersensitivity responses were assessed using Von Frey filaments and radiant heat, respectively. To determine the role of IGF1/IGF1r in chronic migraine (CM), we examined the effects of the IGF1 receptor antagonist ppp (Picropodophyllin) on pain behaviors and the expression of calcitonin gene-related peptide (CGRP) and c-Fos. Result: In the nitroglycerin-induced chronic migraine model in mice, neuronal secretion of IGF1 is elevated within the trigeminal nucleus caudalis (TNC). Increased phosphorylation of the IGF1 receptor occurs, predominantly co-localizing with neurons. Treatment with ppp alleviated basal mechanical hypersensitivity and acute mechanical allodynia. Furthermore, ppp ameliorated autophagic dysfunction and reduced the expression of CGRP and c-Fos. Conclusion: Our findings demonstrate that in the chronic migraine (CM) model in mice, there is a significant increase in IGF1 expression in the TNC region. This upregulation of IGF1 leads to enhanced phosphorylation of IGF1 receptors on neurons. Targeting and inhibiting this signaling pathway may offer potential preventive strategies for mitigating the progression of chronic migraine. [ABSTRACT FROM AUTHOR]

• PD-O-α-Syn and MSA-O-α-Syn are differentially phosphorylated at serine 129. • PD-O-α-Syn and MSA-O-α-Syn have different cytotoxicity and seeding activity. • PD-O-α-Syn and MSA-O-α-Syn cause distinct dopaminergic damage and motor disorders. Previous studies have shown that α-synuclein (α-Syn) aggregates derived from the brains of patients with Parkinson's disease (PD) and multiple system atrophy (MSA) exhibit different phosphorylation, cytotoxicity, and seeding activity. However, the mechanism underlying the differences remains poorly understood. Here, recombinant human α-Syn was incubated in the plasma of patients with PD and MSA, and the oligomers formed in the plasma (PD-O-α-Syn and MSA-O-α-Syn) were purified and analyzed for their phosphorylation, cytotoxicity and seeding activity. In vitro assays revealed that both PD-O-α-Syn and MSA-O-α-Syn were phosphorylated at serine 129. However, the phosphorylation degree of MSA-O-α-Syn was significantly higher than that of PD-O-α-Syn. In addition, MSA-O-α-Syn exhibited stronger cytotoxicity and seeding activity compared with PD-O-α-Syn. In vivo experiments showed that mice receiving intrastriatal inoculation of MSA-O-α-Syn developed more severe motor dysfunction and dopaminergic degeneration than mice receiving intrastriatal inoculation of PD-O-α-Syn. Compared with the mice inoculated with PD-O-α-Syn, the mice inoculated with MSA-O-α-Syn accumulated more phosphorylated and oligomerized α-Syn in the striatum and brain regions (substantia nigra, hippocampus and prefrontal cortex) away from the inoculated site. The results obtained suggest that α-Syn oligomers formed in PD and MSA plasma are different in phosphorylation, cytotoxicity, and seeding activity. [ABSTRACT FROM AUTHOR]

Mcm10 plays an essential role in the activation of replicative helicase CMG through the cell cycle-regulated interaction with the prototype MCM double hexamer in Saccharomyces cerevisiae. In this study, we reported that Mcm10 is phosphorylated by S-phase cyclin-dependent kinases (S-CDKs) at S66, which enhances Mcm10--MCM association during the S phase. S66A single mutation or even deletion of whole N-terminus (a.a. 1-128) only causes mild growth defects. Nevertheless, S66 becomes indispensable in the absence of the Mcm10 C-terminus ((a.a. 463-571), the major MCM-binding domain. Using a two-degron strategy to efficiently deplete Mcm10, we show that mcm10-S66AΔC has a severe defect in proceeding into the S phase. Notably, both lethality and S-phase deficiency can be rescued by artificially tethering mcm10-S66AΔC to MCM. These findings illustrate how the Mcm10-MCM association is regulated as a crucial event in DNA replication initiation. [ABSTRACT FROM AUTHOR]

Summary: Cassava bacterial blight significantly affects cassava yield worldwide, while major cassava cultivars are susceptible to this disease. Therefore, it is crucial to identify cassava disease resistance gene networks and defence molecules for the genetic improvement of cassava cultivars. In this study, we found that MeHB16 transcription factor as a differentially expressed gene in cassava cultivars with contrasting disease resistance, positively modulated disease resistance by modulating defence molecule lignin accumulation. Further investigation showed that MeHB16 physically interacted with itself via the leucine‐Zippe domain (L‐Zip), which was necessary for the transcriptional activation of downstream lignin biosynthesis genes. In addition, protein kinase MeKIN10 directly interacted with MeHB16 to promote its phosphorylation at Ser6, which in turn enhanced MeHB16 self‐association and downstream lignin biosynthesis. In summary, this study revealed the molecular network of MeKIN10‐mediated MeHB16 protein phosphorylation improved cassava bacterial blight resistance by fine‐tuning lignin biosynthesis and provides candidate genes and the defence molecule for improving cassava disease resistance. [ABSTRACT FROM AUTHOR]

The canonical Wnt signaling pathway plays crucial roles in cell fate decisions as well as in pathogenesis of various diseases. Previously, we reported Caprin-2 as a new regulator of canonical Wnt signaling through a mechanism of facilitating LRP5/6 phosphorylation. Here, we resolved the crystal structure of the N-terminal homologous region 1 (HR1) domain of human Caprin-2. HR1 domain is so far only observed in Caprin-2 and its homologous protein Caprin-1, and the function of this domain remains largely mysterious. Here, the structure showed that HR1 domain of human Caprin-2 forms a homo-dimer and exhibits an overall structure roughly resembling the appearance of a pair of scissors. Moreover, we found that residues R200 and R201, which located at a basic cluster within the N-terminal "blades" region, are critical for Caprin-2's localization to the plasma membrane. In line with this, mutations targeting these two residues decrease Caprin-2's activity in the canonical Wnt signaling. Overall, we characterized a previously unknown "scissors"-like structure of the fulllength HR1 domain and revealed its function in mediating Caprin-2's localization to the plasma membrane. [ABSTRACT FROM AUTHOR]

The phosphorylation of Ser15 in the transactivation domain (TAD) of the tumor suppressor protein 53 (p53) by ataxia-telangiectasia mutated (ATM) kinase is a crucial step in the tumor suppressor function of p53. An understanding of the factors that affect the rate of Ser15 phosphorylation may provide new strategies for the manipulation of the ATM-p53 pathway in cancer therapy. In this study, the effect of electrostatic interactions between ATM and p53 was investigated by measuring the phosphorylation of Ser15 at varying pH ranges from 5 to 9. To achieve this, two different kinase assay methods were utilized: the ELISA technique, which directly quantifies the phosphorylated Ser15, and the Universal Kinase Assay, which assesses the formation of ADP. The results revealed that Ser15 phosphorylation was pH-dependent, with higher phosphorylation rates observed in the alkaline range. To ascertain whether the lower phosphorylation rates observed at acidic pH were due to protein denaturation, a pH-dependent solubility profile was generated using the CamSol server. The obtained results demonstrated comparable solubility rates within the pH range of the kinase assays performed. Furthermore, the significance of negatively charged residues in TAD1-39 was evaluated by substituting Asp and Glu residues with hydrophobic and uncharged hydrophilic residues in TAD1-39 using ChimeraX and subsequently comparing their interactions with the ATM using the protein-protein docking server HADDOCK2.4. The results of the docking simulations indicated that the alteration of negatively charged residues with uncharged ones resulted in a reduction in the efficiency of the interaction between the ATM and TAD1-39 . In conclusion, it can be stated that electrostatic interactions between the ATM and TAD are important for optimal Ser15 phosphorylation. [ABSTRACT FROM AUTHOR]

Simple Summary: Phosphorylation is a crucial process that regulates various cellular activities. Detecting phosphorylation sites, especially in cells infected by the SARS-CoV-2 virus, is challenging due to technical limitations. To address this, we developed GBMPhos, an advanced tool combining deep learning techniques, to accurately identify these sites. GBMPhos outperformed traditional methods and current state-of-the-art approaches in identifying phosphorylation sites. We have developed a free web server, which helps researchers gain a better understanding of protein modifications during a SARS-CoV-2 infection, potentially aiding in the development of therapeutic strategies and contributing to the fight against COVID-19. Phosphorylation, a reversible and widespread post-translational modification of proteins, is essential for numerous cellular processes. However, due to technical limitations, large-scale detection of phosphorylation sites, especially those infected by SARS-CoV-2, remains a challenging task. To address this gap, we propose a method called GBMPhos, a novel method that combines convolutional neural networks (CNNs) for extracting local features, gating mechanisms to selectively focus on relevant information, and a bi-directional gated recurrent unit (Bi-GRU) to capture long-range dependencies within protein sequences. GBMPhos leverages a comprehensive set of features, including sequence encoding, physicochemical properties, and structural information, to provide an in-depth analysis of phosphorylation sites. We conducted an extensive comparison of GBMPhos with traditional machine learning algorithms and state-of-the-art methods. Experimental results demonstrate the superiority of GBMPhos over existing methods. The visualization analysis further highlights its effectiveness and efficiency. Additionally, we have established a free web server platform to help researchers explore phosphorylation in SARS-CoV-2 infections. The source code of GBMPhos is publicly available on GitHub. [ABSTRACT FROM AUTHOR]

Background: Infertility is a global reproductive health burden. Assisted reproductive technologies (ARTs) have been widely used to help patients become pregnant. Few embryos develop to the blastocyst stage with ARTs, leading to relatively low live birth rates. Protein modifications play crucial roles in nearly every aspect of cell biology, including reproductive processes. The aim of this study was to explore the characteristics of protein modifications during embryonic development. Methods: Proteomic data from humans and mice were acquired from the integrated proteome resources (iProX) of ProteomeXchange (PXD024267) and a tandem mass tag (TMT)‐mass spectrometry dataset. Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were applied for functional annotation. Protein–protein interactions (PPIs) of the modification‐related genes were revealed by the STRING database. Modified proteins during mouse embryogenesis were visualized through heatmaps of hierarchically clustering using k‐means. Results: We identified modification‐related proteins in human embryo development and characterized them through heatmaps, GO analysis, KEGG analysis, and PPI network analysis. We found that the 4‐cell stage to the 8‐cell stage might be the demarcation period for modification‐related protein expression patterns during embryo development. Using quantitative mass spectrometry, we elucidated the methylation, acetylation, and ubiquitination events that occur during mouse embryogenesis to validate our findings in human embryonic development to some extent. Conclusions: The results of our study suggest that the posttranslational modifications (PTMs) of human preimplantation embryos might exhibit the same trends as those in mice to exert synergistic and fine‐tuned regulatory effects during embryonic development. [ABSTRACT FROM AUTHOR]

Introduction: The study aimed to investigate to what extent acute endurance exercise, especially eccentric exercise and cardiorespiratory fitness, affects the metabolic profile of CD4+ cells. Methods: Fifteen male, healthy adults aged between 20 and 33 yr with a maximal oxygen uptake (V̇O2max) between 44 and 63 mL·kg−1 ·min−1 performed a downhill run (DR) and a level run (LR) for 45 min at 70% of their V̇O2max on a treadmill in a crossover design. Blood samples were taken before (T0), directly after (T1), 3 h after (T3), and 24 h (T24) after each exercise for analyzing leukocyte numbers and cytokine levels. Isolated CD4+ cells were incubated for 4 h in autologous resting versus 3 h after exercise serum (T3 DR and T3 LR), and subsequently, cellular respiration, transcriptomic, and metabolomics profiles were measured. Results: The systemic immune inflammation index increased significantly after DR and LR at T1 and T3 (P < 0.001). In contrast, the transcriptomic and metabolic profile of CD4+ cells showed no significant alterations after incubation in T3 exercise serum. However, cardiorespiratory fitness positively correlated with the maximal mitochondrial respiration in CD4+ cells after incubation with T3 LR serum (r = 0.617, P = 0.033) and with gene expression of oxidative phosphorylation and levels of different metabolites. Similarly, V̇O2max was associated with an anti-inflammatory profile on RNA level. Lower lactate, methylmalonic acid, and D-gluconic acid levels were found in CD4+ cells of participants with a high V̇O2max (P < 0.001). Conclusions: Acute exercise leads to a mild proinflammatory milieu with only small changes in the metabolic homeostasis of CD4+ cells. High cardiorespiratory fitness is associated with a metabolic shift to oxidative phosphorylation in CD4+ cells. Functional relevance of this metabolic shift needs to be further investigated. [ABSTRACT FROM AUTHOR]

Plants can perceive a slight upsurge in ambient temperature and respond by undergoing morphological changes, such as elongated hypocotyls and early flowering. The dynamic functioning of PHYTOCHROME INTERACTING FACTOR4 (PIF4) in thermomorphogenesis is well established, although the complete regulatory pathway involved in thermosensing remains elusive. We establish that an increase in temperature from 22 to 28 °C induces upregulation and activation of MITOGEN-ACTIVATED PROTEIN KINASE 4 (MPK4) in Arabidopsis (Arabidopsis thaliana), subsequently leading to the phosphorylation of PIF4. Phosphorylated PIF4 represses the expression of ACTIN-RELATED PROTEIN 6 (ARP6), which is required for mediating the deposition of histone variant H2A.Z at its target loci. Furthermore, we demonstrate that variations in ARP6 expression in PIF4 phosphor-null and phosphor-mimetic seedlings affect hypocotyl growth at 22 and 28 °C by modulating the regulation of ARP6-mediated H2A.Z deposition at the loci of genes involved in elongating hypocotyl cells. Interestingly, the expression of MPK4 is also controlled by H2A.Z deposition in a temperature-dependent manner. Taken together, these findings highlight the regulatory mechanism of thermosensing by which MPK4-mediated phosphorylation of PIF4 affects ARP6-mediated H2A.Z deposition at the genes involved in hypocotyl cell elongation. Regulatory mechanism of elevated temperature-mediated thermosensing. [ABSTRACT FROM AUTHOR]

Angiomiotin (AMOT) family comprises three members: AMOT, AMOT‐like protein 1 (AMOTL1), and AMOT‐like protein 2 (AMOTL2). AMOTL2 is widely expressed in endothelial cells, epithelial cells, and various cancer cells. Specifically, AMOTL2 predominantly localizes in the cytoplasm and nucleus in human normal cells, whereas associates with cell–cell junctions and actin cytoskeleton in non‐human cells, and locates at cell junctions or within the recycling endosomes in cancer cells. AMOTL2 is implicated in regulation of tube formation, cell polarity, and shape, although the specific impact on tumorigenesis remains to be conclusively determined. It has been shown that AMOTL2 enhances tumor growth and metastasis in pancreatic, breast, and colon cancer, however inhibits cell proliferation and migration in lung, hepatocellular cancer, and glioblastoma. In addition to its role in cell shape and cytoskeletal dynamics through co‐localization with F‐actin, AMOTL2 modulates the transcription of Yes‐associated protein (YAP) by binding to it, thereby affecting its phosphorylation and cellular sequestration. Furthermore, the stability and cellular localization of AMOTL2, influenced by its phosphorylation and ubiquitination mediated by specific proteins, affects its cellular function. Additionally, we observe that AMOTL2 is predominantly downregulated in some tumors, but significantly elevated in colorectal adenocarcinoma (COAD). Moreover, overall analysis, GSEA and ROC curve analysis indicate that AMOTL2 exerts as an oncogenic protein in COAD by modulating Wnt pathway, participating in synthesis of collagen formation, and interacting with extracellular matrix receptor. In addition, AMOTL2 potentially regulates the distribution of immune cells infiltration in COAD. In summary, AMOTL2 probably functions as an oncogene in COAD. Consequently, further in‐depth mechanistic research is required to elucidate the precise roles of AMOTL2 in various cancers. [ABSTRACT FROM AUTHOR]

Objective To explore the mechanism of Xinkang Prescription (Descurainiae Semen Lepidii Semen, Armeniacae Semen Amarum, Poria, Astragali Radix, Citri Reticulatae Pericarpium, Sparanii Rhizoma) for purging the lung and promoting diuresis in treating heart failure by regulating phosphorylation of phospholamban (PLN). Methods Forty-eight C57BL/6J mice were randomly divided into sham-operation group, model group, low-, medium- and high- dose Xinkang Prescription groups (0.455, 0.91, 1.82 g·kg-1), as well as Entresto group (25 mg·kg-1), with eight mice in each group. The model of ischemic heart failure was established by ligating the left anterior descending coronary artery in mice. After the model was successfully replicated, mice were orally administered with the above-mentioned dosages of Xinkang Prescription and Entresto once a day for four weeks, while sham-operation group and model group were given 0.9% sodium chloride solution by gavage at the same time. Echocardiography was used to detect the cardiac function of the mice in each group, including left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular end-diastolic dimension (LVEDD) and left ventricular end systolic diameter (LVESD). Hematoxylin-eosin (HE) staining was used to observe the pathological changes in cardiac tissue of mice. qRT-PCR was used to detect the mRNA expression of BNP. Western Blot and Jess were used to detect the expression of PLN, p-Thr17-PLN, p-Ser16-PLN, sarcoplasmic reticulum calcium ATPase 2a (SERCA2a), protein kinase A (PKA), p-PKA, Ca2+/calmodulin-dependent kinaseⅡ (CaMKⅡ) and p-CaMKⅡin cardiac tissue, and to calculate the ratio of SERCA2a/PLN. The SERCA2a activity was determined by the inorganic phosphorus method. Results Compared with the sham-operation group, the model group showed a significant decrease in LVEF and LVFS (P＜0.01) and a significant increase in LVEDD and LVESD (P＜0.01). HE staining showed the fibril of cardiac muscle broke and disarranged, accompanied by inflammatory cell infiltration. The mRNA expression of BNP was significantly up-regulated (P＜ 0.01), and the protein expressions of p-Ser16-PLN, p-Thr17-PLN, p-PKA, the ratio of SERCA2a/PLN and SERCA2a activity were significantly down-regulated (P＜0.01), while the expression of p-CaMK Ⅱ was up-regulated (P＜0.01). Compared with the model group, LVEF and LVFS in medium-, high-dose Xinkang Prescription groups were significantly increased (P＜0.01), while LVEDD and LVESD were significantly decreased (P＜0.01). HE staining showed significant improvement in the pathological damage of cardiac tissue. The expression level of BNP was significantly decreased (P＜0.01), while the protein expressions of p-Ser16-PLN, p-Thr17-PLN, p-PKA, the ratio of SERCA2a/PLN and SERCA2a activity were significantly increased (P＜0.01) . The protein expression of p-CaMKⅡ was remarkably decreased (P＜0.05). Conclusion Xinkang Prescription can effectively improve cardiac function of mice with heart failure, which may be related to enhance phosphorylation levels of phospholamban. [ABSTRACT FROM AUTHOR]

A general approach is presented for synthesizing alkyne-modified nucleoside triphosphates via the Sonogashira cross-coupling reaction of unprotected halogenated 2ʹ-deoxynucleoside, followed by monophosphorylation and the reaction of the corresponding phosphoromorpholidate with tributylammonium pyrophosphate. A highly efficient approach for the milligram-scale synthesis of base-modified nucleoside triphosphates with an amino acid-like side chain was developed. The present chemical method outweighs the other reported methods of a base-modified nucleoside triphosphates synthesis in terms of it being a protection-free strategy, the shortening of reaction steps, and increased yields (about 70%). The resulting 8-alkynylated dATP was tested as a substrate for DNA polymerases in a primer extension reaction. [ABSTRACT FROM AUTHOR]

Microtubule affinity‐regulating kinase 2 (MARK2) is a Ser/Thr protein kinase that regulates cell polarity and immune responses. Here, we report that Orf9b, one of the accessory proteins encoded in the SARS‐CoV‐2 genome, increases MARK2 activity via interaction with the autoinhibitory KAI domain. We found that co‐expression of Orf9b enhances the kinase activity of MARK2 in HEK293 cells. Orf9b does not bind to or enhance the activity of the mutant form of MARK2 lacking the KA1 domain. Orf9b lowers inhibitory phosphorylation of MARK2 at T595 while mutation experiments indicate that this site is dispensable for Orf9b‐mediated enhancement of MARK2 activity. Our results suggest that Orf9b enhances MARK2 activity by binding the autoinhibitory KA1 domain, which closely interacts with the kinase domain. [ABSTRACT FROM AUTHOR]

Simple Summary: Cancer chemotherapy is dominated by cytotoxic drugs that non-selectively kill proliferating cells by blocking DNA replication and inducing apoptosis. Some natural anticancer products are less toxic to normal, slow-replicating cells but kill aggressive tumor cells. The combination of two structural isomers of pentacyclic triterpenes, oleanolic acid and ursolic acid, showed potent cytotoxic activity in human breast cancer cells in vitro by inducing cytotoxic autophagy at a significantly lower dose than previously reported. They were nontoxic to breast epithelial cells. Their activity was mainly due to the inhibition of AKT-mediated cell survival and the inhibition of the mammalian target of rapamycin (mTOR) signaling pathway, a central regulator of cell metabolism, growth, and proliferation. The compounds were equally effective against ER+ and triple-negative breast cancer cells. Combining low doses of nontoxic inhibitors of PI3k kinase increased the activities of these compounds. In contrast, inhibition of autophagy by 3-methyl adenine annulled their activity, demonstrating that cytotoxic autophagy may be the dominant mechanism of cell death. These compounds have potential for cancer therapy in neoadjuvant or adjuvant settings. Background: Oleanolic acid (OA) and Ursolic acid (UA) are bioactive triterpenoids. Reported activities vary with the dose used for testing their activities in vitro. Studies using doses of ≥20 µM showed apoptosis activities in cancer cells. However, reported drug levels in circulation achieved by oral administration of UA and OA are ≤2 µM, thus limiting their use for treatment or delivering a combination treatment. Materials and Methods: The present report demonstrates the efficacy of OA, UA, and OA + UA on tumor cell-specific cytotoxicity at low doses (5 µM to 10 µM) in breast cancer (BrCa) cell lines MCF7 and MDA-MB231. Results: The data show that both OA and UA killed BrCa cells at low doses, but were significantly less toxic to MCF-12A, a non-tumorigenic cell line. Moreover, OA + UA at ≤10 µM was lethal to BrCa cells. Mechanistic studies unraveled the significant absence of apoptosis, but their cytotoxicity was due to the induction of excessive autophagy at a OA + UA dose of 5 µM each. A link to drug-induced cytotoxic autophagy was established by demonstrating a lack of their cytotoxicity by silencing the autophagy-targeting genes (ATGs), which prevented OA-, UA-, or OA + UA-induced cell death. Further, UA or OA + UA treatment of BrCa cells caused an inhibition of PI3 kinase-mediated phosphorylation of Akt/mTOR, the key pathways that regulate cancer cell survival, metabolism, and proliferation. Discussion: Combinations of a PI3K inhibitor (LY294002) with OA, UA, or OA + UA synergistically inhibited BrCa cell survival. Therefore, the dominance of cytotoxic autophagy by inhibiting PI3K-mediated autophagy may be the primary mechanism of PTT-induced anticancer activity in BrCa cells. Conclusion: These results suggest it would be worthwhile testing combined OA and UA in clinical settings. [ABSTRACT FROM AUTHOR]

Simple Summary: The role of the GPER in cancer is controversial due to its dual anti and protumor effects. Elevated GPER expression in cervical cancer has been associated with improved survival outcomes. In cervical cancer cell lines, the selective GPER agonist G-1 induces cell cycle arrest and apoptosis, although the precise molecular mechanisms behind these effects are not fully understood. This study explores the impact of GPER activation by G-1 on the transcriptome, cell migration, and invasion in SiHa cells, as well as in non-tumorigenic keratinocytes transduced with HPV16 E6 or E7 oncogenes. G-1 has been shown to exert antitumor effects by activating apoptotic pathways and modulating migration and invasion processes. The findings support the GPER as a promising prognostic marker and suggest that G-1 could be a valuable therapeutic tool for treating cervical cancer. Background/Objectives: Estrogens and HPV are necessary for cervical cancer (CC) development. The levels of the G protein-coupled estrogen receptor (GPER) increase as CC progresses, and HPV oncoproteins promote GPER expression. The role of this receptor is controversial due to its anti- and pro-tumor effects. This study aimed to determine the effect of GPER activation, using its agonist G-1, on the transcriptome, cell migration, and invasion in SiHa cells and non-tumorigenic keratinocytes transduced with the HPV16 E6 or E7 oncogenes. Methods: Transcriptome analysis was performed to identify G-1-enriched pathways in SiHa cells. We evaluated cell migration, invasion, and the expression of associated proteins in SiHa, HaCaT-16E6, and HaCaT-16E7 cells using various assays. Results: Transcriptome analysis revealed pathways associated with proliferation/apoptosis (TNF-α signaling, UV radiation response, mitotic spindle formation, G2/M cell cycle, UPR, and IL-6/JAK/STAT), cellular metabolism (oxidative phosphorylation), and cell migration (angiogenesis, EMT, and TGF-α signaling) in SiHa cells. Key differentially expressed genes included PTGS2 (pro/antitumor), FOSL1, TNFRSF9, IL1B, DIO2, and PHLDA1 (antitumor), along with under-expressed genes with pro-tumor effects that may inhibit proliferation. Additionally, DKK1 overexpression suggested inhibition of cell migration. G-1 increased vimentin expression in SiHa cells and reduced it in HaCaT-16E6 and HaCaT-16E7 cells. However, G-1 did not affect α-SMA expression or cell migration in any of the cell lines but increased invasion in HaCaT-16E7 cells. Conclusions: GPER is a promising prognostic marker due to its ability to activate apoptosis and inhibit proliferation without promoting migration/invasion in CC cells. G-1 could potentially be a tool in the treatment of this neoplasia. [ABSTRACT FROM AUTHOR]