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Dilated cardiomyopathy (DCM) is one of the major causes of heart failure. Although significant progress has been made in elucidating the underlying mechanisms, further investigation is required for clarifying molecular diagnostic and therapeutic targets. In this study, we found that the mRNA level of protein phosphatase 2 regulatory subunit B' delta (Ppp2r5d) was altered in the peripheral blood plasma of DCM patients. Knockdown of Ppp2r5d in murine cardiomyocytes increased the intracellular levels of reactive oxygen species (ROS) and inhibited adenosine triphosphate (ATP) synthesis. In vivo knockdown of Ppp2r5d in an isoproterenol (ISO)-induced DCM mouse model aggravated the pathogenesis and ultimately led to heart failure. Mechanistically, Ppp2r5d-deficient cardiomyocytes showed an increase in phosphorylation of STAT3 at Y705 and a decrease in phosphorylation of STAT3 at S727. The elevated levels of phosphorylation at Y705 in STAT3 triggered the upregulation of interleukin 6 (IL6) expression. Moreover, the decreased phosphorylation at S727 in STAT3 disrupted mitochondrial electron transport chain function and dysregulated ATP synthesis and ROS levels. These results hereby reveal a novel role for Ppp2r5d in modulating STAT3 pathway in DCM, suggesting it as a potential target for the therapy of the disease. [ABSTRACT FROM AUTHOR]

In eukaryotes, pre‐mRNA splicing is vital for RNA processing and orchestrated by the spliceosome, whose assembly starts with the interaction between U1‐70K and SR proteins. Despite the significance of the U1‐70K/SR interaction, the dynamic nature of the complex and the challenges in obtaining soluble U1‐70K have impeded a comprehensive understanding of the interaction at the structural level for decades. We overcome the U1‐70K solubility issues, enabling us to characterize the interaction between U1‐70K and SRSF1, a representative SR protein. We unveil specific interactions: phosphorylated SRSF1 RS with U1‐70K BAD1, and SRSF1 RRM1 with U1‐70K RRM. The RS/BAD1 interaction plays a dominant role, whereas the interaction between the RRM domains further enhances the stability of the U1‐70K/SRSF1 complex. The RRM interaction involves the C‐terminal extension of U1‐70K RRM and the conserved acid patches on SRSF1 RRM1 that is involved in SRSF1 phase separation. Our circular dichroism spectra reveal that BAD1 adapts an α‐helical conformation and RS is intrinsically disordered. Intriguingly, BAD1 undergoes a conformation switch from α‐helix to β‐strand and random coil upon RS binding. In addition to the regulatory mechanism via SRSF1 phosphorylation, the U1‐70K/SRSF1 interaction is also regulated by U1‐70K BAD1 phosphorylation. We find that U1‐70K phosphorylation inhibits the U1‐70K and SRSF1 interaction. Our structural findings are validated through in vitro splicing assays and in‐cell saturated domain scanning using the CRISPR method, providing new insights into the intricate regulatory mechanisms of pre‐mRNA splicing. [ABSTRACT FROM AUTHOR]

The Hsp70 system is essential for maintaining protein homeostasis and comprises a central Hsp70 and two accessory proteins that belong to the J‐domain protein (JDP) and nucleotide exchange factor families. Posttranslational modifications offer a means to tune the activity of the system. We explore how phosphorylation of specific residues of the J‐domain of DNAJA2, a class A JDP, regulates Hsc70 activity using biochemical and structural approaches. Among these residues, we find that pseudophosphorylation of Y10 and S51 enhances the holding/folding balance of the Hsp70 system, reducing cochaperone collaboration with Hsc70 while maintaining the holding capacity. Truly phosphorylated J domains corroborate phosphomimetic variant effects. Notably, distinct mechanisms underlie functional impacts of these DNAJA2 variants. Pseudophosphorylation of Y10 induces partial disordering of the J domain, whereas the S51E substitution weakens essential DNAJA2‐Hsc70 interactions without a large structural reorganization of the protein. S51 phosphorylation might be class‐specific, as all cytosolic class A human JDPs harbor a phosphorylatable residue at this position. [ABSTRACT FROM AUTHOR]

Background Nephrin is a transmembrane protein with well-established signaling roles in kidney podocytes, and a smaller set of secretory functions in pancreatic β cells are implicated in diabetes. Nephrin signaling is mediated in part through its 3 cytoplasmic YDxV motifs, which can be tyrosine phosphorylated by high glucose and β cell injuries. Although in vitro studies demonstrate these phosphorylated motifs can regulate β cell vesicle trafficking and insulin release, in vivo evidence of their role in this cell type remains to be determined. Methods To further explore the role of nephrin YDxV phosphorylation in β cells, we used a mouse line with tyrosine to phenylalanine substitutions at each YDxV motif (nephrin-Y3F) to inhibit phosphorylation. We assessed islet function via primary islet glucose-stimulated insulin secretion assays and oral glucose tolerance tests. Results Nephrin-Y3F mice successfully developed pancreatic endocrine and exocrine tissues with minimal structural differences. Unexpectedly, male and female nephrin-Y3F mice showed elevated insulin secretion, with a stronger increase observed in male mice. At 8 months of age, no differences in glucose tolerance were observed between wild-type (WT) and nephrin-Y3F mice. However, aged nephrin-Y3F mice (16 months of age) demonstrated more rapid glucose clearance compared to WT controls. Conclusion Taken together, loss of nephrin YDxV phosphorylation does not alter baseline islet function. Instead, our data suggest a mechanism linking impaired nephrin YDxV phosphorylation to improved islet secretory ability with age. Targeting nephrin phosphorylation could provide novel therapeutic opportunities to improve β cell function. [ABSTRACT FROM AUTHOR]

This study aims to explore new flame-retardant composites based on a phosphorus-functionalized cellulose derivative and epoxy/benzoxazine thermosetting resins in order to broaden the use of natural fibers in advanced applications. The study involved the phosphorylation of microcrystalline cellulose followed by its characterization through employing various analytical methods to corroborate the accomplishment of its functionalization. The curing behavior of composites based on the polybenzoxazine/epoxy copolymer reinforced with (1 and 5 wt.%) modified microcrystalline cellulose was hereafter considered. The thermal behavior of these composites was correspondingly investigated using thermogravimetric analysis, where improved thermal stability and the limiting oxygen index were stressed. Flame retardancy tests using the vertical burning test UL 94 and heat of combustion analysis utilizing an oxygen bomb calorimeter were also carried out to deeply examine the possible flame retardancy ability of the considered composites. [ABSTRACT FROM AUTHOR]

Aims: Excessive occlusal force with periodontitis leads to rapid alveolar bone resorption. However, the molecular mechanism by which inflammation and mechanical stress cause bone resorption remains unclear. We examined the role of Piezo1, a mechanosensitive ion channel expressed on osteoblasts, in the changes in the receptor activator of nuclear factor‐kappa B ligand (RANKL)/osteoprotegerin (OPG) ratio in mouse MC3T3‐E1 osteoblast‐like cells under Porphyromonas gingivalis lipopolysaccharide (P.g.‐LPS) and mechanical stress. Methods: To investigate the effect of P.g.‐LPS and mechanical stress on the RANKL/OPG ratio and Piezo1 expression, we stimulated MC3T3‐E1 cells with P.g.‐LPS. After 3 days in culture, shear stress, a form of mechanical stress, was applied to the cells using an orbital shaker. Subsequently, to investigate the role of Piezo1 in the change of RANKL/OPG ratio, we inhibited Piezo1 function by knockdown via Piezo1 siRNA transfection or by adding GsMTx4, a Piezo1 antagonist. Results: The RANKL/OPG ratio significantly increased in MC3T3‐E1 cells cultured in a medium containing P.g.‐LPS and undergoing mechanical stress compared to cells treated with P.g.‐LPS or mechanical stress alone. However, the expression of Piezo1 was not increased by P.g.‐LPS and mechanical stress. In addition, phosphorylation of MEK/ERK was induced in the cells under P.g.‐LPS and mechanical stress. MC3T3‐E1 cells treated with P.g.‐LPS and mechanical stress when cocultured with RAW264.7 cells induced their differentiation into osteoclast‐like cells. The increased RANKL/OPG ratio was suppressed by either Piezo1 knockdown or the addition of GsMTx4. Furthermore, GsMTx4 inhibited the phosphorylation of MEK/ERK. Conclusion: These findings suggest that P.g.‐LPS and Piezo1‐mediated mechanical stress induce MEK/ERK phosphorylation and increase RANKL expression in osteoblasts. Consequently, this leads to the differentiation of osteoclast precursor cells into osteoclasts. [ABSTRACT FROM AUTHOR]

GABAergic transmission is influenced by post-translational modifications, like phosphorylation, impacting channel conductance, allosteric modulator sensitivity, and membrane trafficking. O-GlcNAcylation is a post-translational modification involving the O-linked attachment of β-N-acetylglucosamine on serine/threonine residues. Previously we reported an acute increase in O-GlcNAcylation elicits a long-term depression of evoked GABAAR inhibitory postsynaptic currents (eIPSCs) onto hippocampal principal cells. Importantly, O-GlcNAcylation and phosphorylation can co-occur or compete for the same residue; whether they interact in modulating GABAergic IPSCs is unknown. We tested this by recording IPSCs from hippocampal principal cells and pharmacologically increased O-GlcNAcylation, before or after increasing serine phosphorylation using the adenylate cyclase activator, forskolin. Although forskolin had no significant effect on baseline eIPSC amplitude, we found that a prior increase in O-GlcNAcylation unmasks a forskolin-dependent increase in eIPSC amplitude, reversing the O-GlcNAc-induced eIPSC depression. Inhibition of adenylate cyclase or protein kinase A did not prevent the potentiating effect of forskolin, indicating serine phosphorylation is not the mechanism. Surprisingly, increasing O-GlcNAcylation also unmasked a potentiating effect of the neurosteroids 5α-pregnane-3α,21-diol-20-one (THDOC) and progesterone on eIPSC amplitude in about half of the recorded cells, mimicking forskolin. Our findings show that under conditions of heightened O-GlcNAcylation, the neurosteroid site on synaptic GABAARs is possibly accessible to agonists, permitting strengthening of synaptic inhibition. [ABSTRACT FROM AUTHOR]

Phosphoesterification is the only naturally occurring covalent starch modification identified to date, and it has a major impact on overall starch metabolism. The incorporation of phosphate groups mediated by dikinases [α-glucan, water dikinase (GWD), EC 2.7.9.4; phosphoglucan, water dikinase (PWD), EC 2.7.9.5] massively alters the starch granule properties; however, previous studies did not determine whether the starch-related dikinases bind the phosphate to the glucosyl units within the amylopectin molecules in a specific pattern or randomly. In order to answer this challenging question, a number of approaches were initially pursued until a protocol could be established that enabled a massive step forward in the in vitro analysis of phosphorylated glucan chains obtained from starch. For this purpose, phosphorylation by GWD was investigated, including the final state of phosphorylation i.e., the state of substrate saturation when GWD lacks further free hydroxyl groups at OH-C6 for the catalysis of monophosphate esters. Since the separated phosphorylated glucan chains were required for the analysis, isoamylase digestion was performed to cleave the α-1,6-glycosidic bonds and to allow for the removal of the huge number of existing neutral chains by means of anion exchange chromatography. Via Matrix-Assisted Laser Desorption/Ionization–Time of Flight (MALDI-TOF) MS and MALDI-MS/MS, the phosphorylated α-glucan chains were analysed, and the position of the phosphate group within the chain in relation to the reducing end was determined. Here, we demonstrate a protocol that enables the analysis of phosphorylated oligosaccharides, even in small quantities. [ABSTRACT FROM AUTHOR]

Background: Ischemia-reperfusion (I/R) injury is a major factor in liver damage following hepatic resection and liver transplantation, with anesthetics demonstrating the ability to shield organs from this type of injury. Methods: Hypoxia-reoxygenation (H/R) was used to create in vitro I/R hepatocyte cell injury models. The CCK-8 assay, flow cytometer, LDH assay, and ELSIA were utilized to assess hepatocyte injury. The in vivo I/R injury rat model was then built. HE and TUNEL staining were used to assess liver tissue damage. Western-blot was applied to assess the activation of the MAPK/ERK pathway. Results: Remimazolam (RMZL) remarkably improved cell viability and decreased apoptosis in H/R-induced hepatocyte injury. RMZL reduced the release of H/R-induced inflammatory mediators (TNF-α and IL-6) as well as LDH levels. We also discovered that RMZL inhibited p38 and ERK1/2 phosphorylation in vivo and in vitro. The stimulation of MAPK/ERK, on the other hand, abolished RMZL's anti-inflammation effects in H/R-induced hepatocyte injury. Furthermore, RMZL reduced liver tissue injury in I/R rats. Conclusion: RMZL prevented hepatic I/R damage by inhibiting MAPK/ERK signaling. [ABSTRACT FROM AUTHOR]

ATP-citrate lyase (ACLY) is a key enzyme provoking metabolic and epigenetic gene regulation. Molecularly, these functions are exerted by the provision of acetyl-coenzyme A, which is then used as a substrate for de novo lipogenesis or as an acetyl-group donor in acetylation reactions. It has been demonstrated that ACLY activity can be positively regulated via phosphorylation at serine 455 by Akt and protein kinase A. Nonetheless, the impact of phosphorylation on ACLY function in human myeloid cells is poorly understood. In this study we reconstituted ACLY knockout human monocytic THP-1 cells with a wild type ACLY as well as catalytically inactive H760A, and phosphorylation-deficient S455A mutants. Using these cell lines, we determined the impact of ACLY activity and phosphorylation on histone acetylation and pro-inflammatory gene expression in response to lipopolysaccharide (LPS). Our results show that ACLY serine 455 phosphorylation does not influence the proper enzymatic function of ACLY, since both, wild type ACLY and phosphorylation-deficient mutant, exhibited increased cell growth and histone acetylation as compared to cells with a loss of ACLY activity. Transcriptome analysis revealed enhanced expression of pro-inflammatory and interferon response genes in ACLY knockout and H760A THP-1 cells under unstimulated or LPS-treated conditions. At the same time, S455A ACLY-expressing cells showed a phenotype very similar to wild type cells. Contrary to ACLY knockout, pharmacological inhibition of ACLY in THP-1 cells or in primary human macrophages does not enhance LPS-triggered pro-inflammatory gene expression. Our data thus suggest that ACLY retains functionality in the absence of Akt/PKA-mediated phosphorylation in human myeloid cells. Furthermore, loss of ACLY activity may elicit long-term adaptive mechanisms, increasing inflammatory responses. [ABSTRACT FROM AUTHOR]

Herein, we present a radical cascade addition cyclization sequence to access quinoline‐based benzophosphole oxides from ortho‐alkynylated aromatic phosphine oxides using various aryl isonitriles as radical acceptors and inexpensive tert‐butyl‐hydroperoxide (TBHP) as a terminal oxidant in the presence of a catalytic amount of silver acetate. Alternatively, the same cascade can be realized through a sustainable photochemical approach utilizing 1,2,3,5‐tetrakis(carbazol‐9‐yl)‐4,6‐dicyanobenzene (4CzIPN) as an organic photocatalyst at room temperature. The introduced modular approach shows broad functional group tolerance and offers straightforward access to complex P,N‐containing polyheterocyclic arenes. These novel π‐extended benzophosphole oxides exhibit interesting photophysical and electrochemical properties such as absorption in the visible region, emission and reversible reduction at low potentials, which makes them promising for potential materials science applications. The photophysical properties can further be tuned by the addition of external Lewis and Brønsted acids. [ABSTRACT FROM AUTHOR]

Carbon–phosphorus bond formation is of great importance in synthetic chemistry. A versatile strategy for the formation of carbon–phosphorus bonds involves the addition of phosphorus radicals to π-systems. However, the generation methods of phosphorus radicals are primarily restricted to homolytic cleavage of phosphorus–hydrogen bonds with excess peroxides or toxic metal salts as oxidants. Herein, we introduce a photoinduced palladium-catalyzed generation of phosphorus radicals using inexpensive and readily available Cl–phosphine oxides and Cl–phosphates as the phosphorus radical precursors. The key step for this strategy lies in the reductive activation of phosphorus–chloride bonds with the excited palladium complex under visible light irradiation. The involvement of phosphorus radicals has been evidenced by EPR spectroscopy and radical trapping experiments. Based on this strategy, both phosphinyl and phosphonyl groups could be regiospecifically incorporated into simple alkenes and heteroaromatic compounds via radical mediated carbon–phosphorus bond formation. Besides, carbon–phosphorus difunctionalization of alkenes was also achieved to afford phosphorylated oxindoles. [ABSTRACT FROM AUTHOR]

Abnormal accumulation of misfolded and hyperphosphorylated tau protein in brain is the defining feature of several neurodegenerative diseases called tauopathies, including Alzheimer's disease (AD). In AD, this pathological change is reflected by highly specific cerebrospinal fluid (CSF) tau biomarkers, including both phosphorylated and non-phosphorylated variants. Interestingly, despite tau pathology being at the core of all tauopathies, CSF tau biomarkers remain unchanged in certain tauopathies, e.g., progressive supranuclear palsy (PSP), Pick's disease (PiD), and corticobasal neurodegeneration (CBD). To better understand commonalities and differences between tauopathies, we report a multiplex assay combining immunoprecipitation and high-resolution mass spectrometry capable of detecting and quantifying peptides from different tau protein isoforms as well as non-phosphorylated and phosphorylated peptides, including those carrying multiple phosphorylations. We investigated the tau proteoforms in soluble and insoluble fractions of brain tissue from subjects with autopsy-confirmed tauopathies, including sporadic AD (n = 10), PSP (n = 11), PiD (n = 10), and CBD (n = 10), and controls (n = 10). Our results demonstrate that non-phosphorylated tau profiles differ across tauopathies, generally showing high abundance of microtubule-binding region (MTBR)-containing peptides in insoluble protein fractions compared with controls; the AD group showed 12–72 times higher levels of MTBR-containing aggregates. Quantification of tau isoforms showed the 3R being more abundant in PiD and the 4R isoform being more abundant in CBD and PSP in the insoluble fraction. Twenty-three different phosphorylated peptides were quantified. Most phosphorylated peptides were measurable in all investigated tauopathies. All phosphorylated peptides were significantly increased in AD insoluble fraction. However, doubly and triply phosphorylated peptides were significantly increased in AD even in the soluble fraction. Results were replicated using a validation cohort comprising AD (n = 10), CBD (n = 10), and controls (n = 10). Our study demonstrates that abnormal levels of phosphorylation and aggregation do indeed occur in non-AD tauopathies, however, both appear pronouncedly increased in AD, becoming a distinctive characteristic of AD pathology. [ABSTRACT FROM AUTHOR]

With both TEMPO and O2 (in air) as the homogeneous redox mediators, BiBrO as the heterogeneous semiconductor photocatalyst, the first example of semi‐heterogeneous photocatalytic decarboxylative phosphorylation of N‐arylglycines with diarylphosphine oxides was established. A series of α‐amino phosphinoxides were efficiently synthesized. [ABSTRACT FROM AUTHOR]

Stomata in leaves regulate gas (carbon dioxide and water vapor) exchange and water transpiration between plants and the atmosphere. SLow Anion Channel 1 (SLAC1) mediates anion efflux from guard cells and plays a crucial role in controlling stomatal aperture. It serves as a central hub for multiple signaling pathways in response to environmental stimuli, with its activity regulated through phosphorylation via various plant protein kinases. However, the molecular mechanism underlying SLAC1 phosphoactivation has remained elusive. Through a combination of protein sequence analyses, AlphaFold-based modeling and electrophysiological studies, we unveiled that the highly conserved motifs on the N-and C-terminal segments of SLAC1 form a cytosolic regulatory domain (CRD) that interacts with the transmembrane domain(TMD), thereby maintaining the channel in an autoinhibited state. Mutations in these conserved motifs destabilize the CRD, releasing autoinhibition in SLAC1 and enabling its transition into an activated state. Our further studies demonstrated that SLAC1 activation undergoes an autoinhibition-release process and subsequent structural changes in the pore helices. These findings provide mechanistic insights into the activation mechanism of SLAC1 and shed light on understanding how SLAC1 controls stomatal closure in response to environmental stimuli. [ABSTRACT FROM AUTHOR]

Simple Summary: Here, we show that the plant-derived product, sanguinarine, inhibited aerobic glycolysis and oxidative phosphorylation; which resulted in energy alternations and necroptosis of tumor cells. Moreover, we identify the PKM2/β-catenin axis as the main target in sanguinarine treatment against HCC development. Backgrounds: Abnormal metabolism is the hallmark of hepatocellular carcinoma. Targeting energy metabolism has become the major focus of cancer therapy. The natural product, sanguinarine, displays remarkable anti-tumor properties by disturbing energy homeostasis; however, the underlying mechanism has not yet been elucidated. Methods: The anticancer activity of sanguinarine was determined using CCK-8 and colony formation assay. Morphological changes of induced cell death were observed under electron microscopy. Necroptosis and apoptosis related markers were detected using western blotting. PKM2 was identified as the target by transcriptome sequencing. Molecular docking assay was used to evaluate the binding affinity of sanguinarine to the PKM2 molecule. Furthermore, Alb-CreERT2; PKM2loxp/loxp; Rosa26RFP mice was used to construct the model of HCC—through the intervention of sanguinarine in vitro and in vivo—to accurately explore the regulation effect of sanguinarine on cancer energy metabolism. Results: Sanguinarine inhibited tumor proliferation, metastasis and induced two modes of cell death. Molecular docking of sanguinarine with PKM2 showed appreciable binding affinity. PKM2 kinase activity and aerobic glycolysis rate declined, and mitochondrial oxidative phosphorylation was inhibited by sanguinarine application; these changes result in energy deficits and lead to necroptosis. Additionally, sanguinarine treatment prevents the translocation of PKM2 into the nucleus and suppresses the interaction of PKM2 with β-catenin; the transcriptional activity of PKM2/β-catenin signaling and its downstream genes were decreased. Conclusions: Sanguinarine showed remarkable anti-HCC activity via regulating energy metabolism by PKM2/β-catenin signaling. On the basis of these investigations, we propose that sanguinarine might be considered as a promising compound for discovery of anti-HCC drugs. [ABSTRACT FROM AUTHOR]

Increased malignancy and poor treatability associated with solid tumour cancers have commonly been attributed to mitochondrial calcium (Ca2+) dysregulation. The mitochondrial Ca2+ uniporter complex (mtCU) is the predominant mode of Ca2+ uptake into the mitochondrial matrix. The main components of mtCU are the pore‐forming mitochondrial Ca2+ uniporter (MCU) subunit, MCU dominant‐negative beta (MCUb) subunit, essential MCU regulator (EMRE) and the gatekeeping mitochondrial Ca2+ uptake 1 and 2 (MICU1 and MICU2) proteins. In this review, we describe mtCU‐mediated mitochondrial Ca2+ dysregulation in solid tumour cancer types, finding enhanced mtCU activity observed in colorectal cancer, breast cancer, oral squamous cell carcinoma, pancreatic cancer, hepatocellular carcinoma and embryonal rhabdomyosarcoma. By contrast, decreased mtCU activity is associated with melanoma, whereas the nature of mtCU dysregulation remains unclear in glioblastoma. Furthermore, we show that numerous polymorphisms associated with cancer may alter phosphorylation sites on the pore forming MCU and MCUb subunits, which cluster at interfaces with EMRE. We highlight downstream/upstream biomolecular modulators of MCU and MCUb that alter mtCU‐mediated mitochondrial Ca2+ uptake and may be used as biomarkers or to aid in the development of novel cancer therapeutics. Additionally, we provide an overview of the current small molecule inhibitors of mtCU that interact with the Asp residue of the critical Asp‐Ile‐Met‐Glu motif or through other allosteric regulatory mechanisms to block Ca2+ permeation. Finally, we describe the relationship between MCU‐ and MCUb‐mediating microRNAs and mitochondrial Ca2+ uptake that should be considered in the discovery of new treatment approaches for cancer. [ABSTRACT FROM AUTHOR]

DNAX-activating protein of 12 kDa (DAP12) is a transmembrane adapter protein expressed in lymphoid and myeloid lineage cells. It interacts with several immunoreceptors forming functional complexes that trigger intracellular signaling pathways. One of the DAP12 associated receptors is the triggering receptor expressed on myeloid cells 2 (TREM2). Mutations in both DAP12 and TREM2 have been linked to neurodegenerative diseases. However, mechanisms involved in the regulation of subcellular trafficking and turnover of these proteins are not well understood. Here, we demonstrate that proteasomal degradation of DAP12 is increased in the absence of TREM2. Interestingly, unassembled DAP12 is also retained in early secretory compartments, including the endoplasmic reticulum (ER) and the ER-Golgi intermediate compartment (ERGIC), thereby preventing its transport to the plasma membrane. We also show that unassembled DAP12 interacts with the retention in ER sorting receptor 1 (RER1). The deletion of endogenous RER1 decreases expression of functional TREM2-DAP12 complexes and membrane proximal signaling, and resulted in almost complete inhibition of phagocytic activity in THP-1 differentiated macrophage-like cells. These results indicate that RER1 acts as an important regulator of DAP12 containing immunoreceptor complexes and immune cell function. [ABSTRACT FROM AUTHOR]

Phosphohistidine (pHis) is a reversible protein post-translational modification (PTM) that is currently poorly understood. The P-N bond in pHis is heat and acid-sensitive, making it more challenging to study than the canonical phosphoamino acids pSer, pThr, and pTyr. As advancements in the development of tools to study pHis have been made, the roles of pHis in cells are slowly being revealed. To date, a handful of enzymes responsible for controlling this modification have been identified, including the histidine kinases NME1 and NME2, as well as the phosphohistidine phosphatases PHPT1, LHPP, and PGAM5. These tools have also identified the substrates of these enzymes, granting new insights into previously unknown regulatory mechanisms. Here, we discuss the cellular function of pHis and how it is regulated on known pHis-containing proteins, as well as cellular mechanisms that regulate the activity of the pHis kinases and phosphatases themselves. We further discuss the role of the pHis kinases and phosphatases as potential tumor promoters or suppressors. Finally, we give an overview of various tools and methods currently used to study pHis biology. Given their breadth of functions, unraveling the role of pHis in mammalian systems promises radical new insights into existing and unexplored areas of cell biology. [ABSTRACT FROM AUTHOR]

Capacitation involves tyrosine phosphorylation (TP) as a key marker. Lifestyle-related factors, such as obesity and smoking, are recognized for their adverse effects on semen quality and male fertility, yet the underlying mechanisms, including their potential impact on TP, remain unclear. Moreover, the effect of sperm cryopreservation on TP at the human sperm population level is unexplored. Flow cytometry analysis of global TP was performed on pre-capacitated, post-capacitated and 1- and 3-hours' incubated fresh and frozen–thawed samples from sperm donors (n = 40). Neither being overweight nor smoking (or both) significantly affected the percentage of sperm showing TP. However, elevated BMI and smoking intensity correlated with heightened basal TP levels (r = 0.226, p = 0.003) and heightened increase in TP after 3 h of incubation (r = 0.185, p = 0.017), respectively. Cryopreservation resulted in increased global TP levels after capacitation but not immediately after thawing. Nonetheless, most donors' thawed samples showed increased TP levels before and after capacitation as well as after incubation. Additionally, phosphorylation patterns in fresh and frozen–thawed samples were similar, indicating consistent sample response to capacitation stimuli despite differences in TP levels. Overall, this study sheds light on the potential impacts of lifestyle factors and cryopreservation on the dynamics of global TP levels during capacitation. [ABSTRACT FROM AUTHOR]

The rice blast fungus Magnaporthe oryzae stands as a formidable adversary to one of the world's most important crops, rice, which feeds over half of the global population. Its ability to rapidly evolve and adapt underscores the urgent need for a comprehensive understanding of its infection strategies. In a large-scale study published in Cell, Cruz-Mireles et al. (Cell 187:2557-73, 2024) utilized phosphoproteomics to globally map the phosphorylation landscape during the infection-related development by M. oryzae, identifying 2062 activated phosphoproteins carrying 8005 phosphosites. A subset of these phosphosites were conserved in the proteins of diverse fungal pathogens and appeared to be associated with biotrophic and hemibiotrophic infection. Thirty-two of these phosphoproteins are regulated by pathogenicity mitogen-activated kinase 1 (Pmk1), a central component of the MAPK signaling pathway, including VTi 1–2 suppressor, whose regulation by Pmk1 is essential for rice blast disease. Together, this global phosphorylation atlas offers a rich tapestry of potential therapeutic targets for developing green agrochemicals to control fungal diseases of plants. [ABSTRACT FROM AUTHOR]

The Mec1/ATR kinase is crucial for genome stability, yet the mechanism by which it prevents gross chromosomal rearrangements (GCRs) remains unknown. Here we find that in cells with deficient Mec1 signaling, GCRs accumulate due to the deregulation of multiple steps in homologous recombination (HR). Mec1 primarily suppresses GCRs through its role in activating the canonical checkpoint kinase Rad53, which ensures the proper control of DNA end resection. Upon loss of Rad53 signaling and resection control, Mec1 becomes hyperactivated and triggers a salvage pathway in which the Sgs1 helicase is recruited to sites of DNA lesions via the 911-Dpb11 scaffolds and phosphorylated by Mec1 to favor heteroduplex rejection and limit HR-driven GCR accumulation. Fusing an ssDNA recognition domain to Sgs1 bypasses the requirement of Mec1 signaling for GCR suppression and nearly eliminates D-loop formation, thus preventing non-allelic recombination events. We propose that Mec1 regulates multiple steps of HR to prevent GCRs while ensuring balanced HR usage when needed for promoting tolerance to replication stress. Synopsis: Chromosomal rearrangements (GCR) are a hallmark of many cancers, but how cells prevent these deleterious events remains incompletely understood. This work analyzes how the DNA-damage signaling kinase Mec1/ATR suppresses chromosomal rearrangements, uncovering an intricate interplay between the checkpoint and the Sgs1 helicase. In yeast cells lacking downstream DNA damage checkpoint signaling, Mec1 is hyperactivated and targets the Sgs1 helicase. Recruitment of Sgs1 helicase and its phosphorylation by Mec1 are crucial for GCR suppression in cells lacking downstream DNA damage checkpoint signaling. Engineered Sgs1 recruitment suppresses GCRs and reduces D-loop levels in cells defective for Mec1 signaling. Phosphorylation of Sgs1 by Mec1 regulates its ability to reject recombination between homeologous sequences. Phosphorylation of Sgs1 by Mec1 regulates its ability to reject recombination between homeologous sequences, showing an intricate interplay between the yeast DNA damage checkpoint and the Sgs1 helicase. [ABSTRACT FROM AUTHOR]

More than 650 reversible and irreversible post-translational modifications (PTMs) of proteins have been listed so far. Canonical PTMs of proteins consist of the covalent addition of functional or chemical groups on target backbone aminoacids or the cleavage of the protein itself, giving rise to modified proteins with specific properties in terms of stability, solubility, cell distribution, activity, or interactions with other biomolecules. PTMs of protein contribute to cell homeostatic processes, enabling basal cell functions, allowing the cell to respond and adapt to variations of its environment, and globally maintaining the constancy of the milieu interieur (the body's inner environment) to sustain human health. Abnormal protein PTMs are, however, associated with several disease states, such as cancers, metabolic disorders, or neurodegenerative diseases. Abnormal PTMs alter the functional properties of the protein or even cause a loss of protein function. One example of dramatic PTMs concerns the cellular prion protein (PrPC), a GPI-anchored signaling molecule at the plasma membrane, whose irreversible post-translational conformational conversion (PTCC) into pathogenic prions (PrPSc) provokes neurodegeneration. PrPC PTCC into PrPSc is an additional type of PTM that affects the tridimensional structure and physiological function of PrPC and generates a protein conformer with neurotoxic properties. PrPC PTCC into PrPSc in neurons is the first step of a deleterious sequence of events at the root of a group of neurodegenerative disorders affecting both humans (Creutzfeldt-Jakob diseases for the most representative diseases) and animals (scrapie in sheep, bovine spongiform encephalopathy in cow, and chronic wasting disease in elk and deer). There are currently no therapies to block PrPC PTCC into PrPSc and stop neurodegeneration in prion diseases. Here, we review known PrPC PTMs that influence PrPC conversion into PrPSc. We summarized how PrPC PTCC into PrPSc impacts the PrPC interactome at the plasma membrane and the downstream intracellular controlled protein effectors, whose abnormal activation or trafficking caused by altered PTMs promotes neurodegeneration. We discussed these effectors as candidate drug targets for prion diseases and possibly other neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Hedgehog (Hh) signaling, an evolutionarily conserved pathway, plays an essential role in development and tumorigenesis, making it a promising drug target. Multiple negative regulators are known to govern Hh signaling; however, how activated Smoothened (SMO) participates in the activation of downstream GLI2 and GLI3 remains unclear. Herein, we identified the ciliary kinase DYRK2 as a positive regulator of the GLI2 and GLI3 transcription factors for Hh signaling. Transcriptome and interactome analyses demonstrated that DYRK2 phosphorylates GLI2 and GLI3 on evolutionarily conserved serine residues at the ciliary base, in response to activation of the Hh pathway. This phosphorylation induces the dissociation of GLI2/GLI3 from suppressor, SUFU, and their translocation into the nucleus. Loss of Dyrk2 in mice causes skeletal malformation, but neural tube development remains normal. Notably, DYRK2-mediated phosphorylation orchestrates limb development by controlling cell proliferation. Taken together, the ciliary kinase DYRK2 governs the activation of Hh signaling through the regulation of two processes: phosphorylation of GLI2 and GLI3 downstream of SMO and cilia formation. Thus, our findings of a unique regulatory mechanism of Hh signaling expand understanding of the control of Hh-associated diseases. [ABSTRACT FROM AUTHOR]

Objective: The present study aimed to investigate the underlying mechanism of mechanical stimulation in regulating osteogenic differentiation. Materials and methods: Osteoblasts were exposed to compressive force (0–4 g/cm2) for 1–3 days or CGRP for 1 or 3 days. Expression of receptor activity modifying protein 1 (RAMP1), the transcription factor RUNX2, osteocalcin, p38 and p-p38 were analyzed by western blotting. Calcium mineralization was analyzed by alizarin red straining. Results: Using compressive force treatments, low magnitudes (1 and 2 g/cm2) of compressive force for 24 h promoted osteoblast differentiation and mineral deposition whereas higher magnitudes (3 and 4 g/cm2) did not produce osteogenic effect. Through western blot assay, we observed that the receptor activity-modifying protein 1 (RAMP1) expression was upregulated, and p38 mitogen-activated protein kinase (MAPK) was phosphorylated during low magnitudes compressive force-promoted osteoblast differentiation. Further investigation of a calcitonin gene-related peptide (CGRP) peptide incubation, a ligand for RAMP1, showed that CGRP at concentration of 25 and 50 ng/ml could increase expression levels of RUNX2 and osteocalcin, and percentage of mineralization, suggesting its osteogenic potential. In addition, with the same conditions, CGRP also significantly upregulated RAMP1 and phosphorylated p38 expression levels. Also, the combination of compressive forces (1 and 2 g/cm2) with 50 ng/ml CGRP trended to increase RAMP1 expression, p38 activity, and osteogenic marker RUNX2 levels, as well as percentage of mineralization compared to compressive force alone. This suggest that RAMP1 possibly acts as an upstream regulator of p38 signaling during osteogenic differentiation. Conclusion: These findings suggest that CGRP-RAMP1/p38MAPK signaling implicates in osteoblast differentiation in response to optimal magnitude of compressive force. This study helps to define the underlying mechanism of compressive stimulation and may also enhance the application of compressive stimulation or CGRP peptide as an alternative approach for accelerating tooth movement in orthodontic treatment. [ABSTRACT FROM AUTHOR]

Background: Artemisia argyi is a traditional herbal medicine belonging to the genus Artemisia that plays an important role in suppressing inflammation. However, the chemical constituents and underlying mechanisms of its therapeutic potential in neuroinflammation are still incompletely understood, and warrant further investigation. Methods: Several column chromatography were employed to isolate and purify chemical constituents from Artemisia argyi, and modern spectroscopy techniques were used to elucidate their chemical structures. The screening of monomeric compounds with nitric oxide inhibition led to the identification of the most effective bioactive compound, which was subsequently confirmed for its anti-inflammatory capability through qRT‒PCR. Predictions of compound-target interactions were made using the PharmMapper webserver and the TargetNet database, and an integrative protein-protein interaction network was constructed by intersecting the predicted targets with neuroinflammation-related targets. Topological analysis was performed to identify core targets, and molecular docking and molecular dynamics simulations were utilized to validate the findings. The result of the molecular simulations was experimentally validated through drug affinity responsive target stability (DARTS) and Western blot experiments. Results: Seventeen sesquiterpenoids, including fifteen known sesquiterpenoids and two newly discovered guaiane-type sesquiterpenoids (argyinolide S and argyinolide T) were isolated from Artemisia argyi. Bioactivity screening revealed that argyinolide S (AS) possessed the most potent anti-inflammatory activity. However, argyinolide T (AT) showed weak anti-inflammatory activity, so AS was the target compound for further study. AS may regulate neuroinflammation through its modulation of eleven core targets: protein kinase B 1 (AKT1), epidermal growth factor receptor (EGFR), proto-oncogene tyrosine-protein Kinase (FYN), Janus Kinase (JAK) 1, mitogen-activated protein (MAP) Kinase 1,8 and 14, matrix metalloproteinase 9 (MMP9), ras-related C3 botulinum toxin substrate 1 (RAC1), nuclear factor kappa-B p65 (RELA), and retinoid X receptor alpha (RXRA). Molecular dynamics simulations and DARTS experiments confirmed the stable binding of AS to JAK1, and Western blot experiments demonstrated the ability of AS to inhibit the phosphorylation of downstream Signal transducer and activator of transcription 3 (STAT3) mediated by JAK1. Conclusions: The sesquiterpenoid compounds isolated from Artemisia argyi, exhibit significant inhibitory effects on inflammation in C57BL/6 murine microglia cells (BV-2). Among these compounds, AS, a newly discovered guaiane-type sesquiterpenoid in Artemisia argyi, has been demonstrated to effectively inhibit the occurrence of neuroinflammation by targeting JAK1. [ABSTRACT FROM AUTHOR]

Background: To explore the role of GAB2 in pan-cancer based on bioinformatics analysis. Methods: Based on TCGA and GTEx databases, we used TIMER2.0 online analysis tool and R language to analyze the expression of GAB2 in pan-cancer. We used Kaplan–Meier Plotter to analyze the relationship between GAB2 and OS and RFS in pan-cancer. We utilized the CPTAC database to examine the expression of phosphorylated GAB2 in pan-cancer. We investigated the effects of mutation features on the occurrence and development of human cancers by cBioPortal and COSMIC. Using the database, we conducted an analysis of molecular compounds that have the potential to interact with GAB2 through molecular docking. Moreover, we use the TIMER to explore the relationship between GAB2 and immune cell infiltration, and draw relevant heatmaps by R language. Results: GAB2 was abnormally expressed in various tumors and was associated with prognosis. There were differences in the expression of GAB2 phosphorylation in tumor tissues and corresponding normal tissues among different types of tumors. GAB2 interacts with Docetaxel and was associated with immune cell infiltration in various tumors. Conclusion: GAB2 participates in regulating immune infiltration and affects the prognosis of patients. GAB2 may serve as a potential tumor marker. [ABSTRACT FROM AUTHOR]

In this study, an anti-solvent method is employed to encapsulate linseed oil (LO) into phosphorylated ethyl cellulose (P-EC), successfully preparing microcapsules (LO@P-EC). Scanning electron microscopy and Fourier transform infrared spectroscopy experimental results indicate the introduction of phosphoric groups into P-EC, and the successful encapsulation of LO within the microcapsules, which are spherical with an average diameter of 145 μm. Hartmann tests reveal that the introduction of phosphate groups reduces the microcapsule flame propagation height by 43.75% and the average flame propagation speed by 55.67%. Thermodynamic analyses based on the CoatsRedfern equation are performed to determine the changes in activation energy during thermal degradation. Furthermore, fitting analysis shows that the apparent activation energy of microcapsules with shell material of P-EC significantly increases, confirming the evident thermodynamic inhibition of microcapsule deflagration by P-EC. This is mainly attributed to the formation of a char structure by the P-EC shell during combustion, which hinders the progress of the combustion reaction. When immersed in 3.5 wt% NaCl corrosive solution for 15 days, the microcapsule-filled epoxy resin (LO@P-EC/EP) coating exhibits excellent corrosion resistance, which is mainly attributed to the excellent self-healing properties of the microcapsule core LO. [ABSTRACT FROM AUTHOR]

Summary Identification and quantification of phosphorylation sites are essential for biological interpretation of a phosphoproteomics experiment. For data independent acquisition mass spectrometry-based (DIA-MS) phosphoproteomics, extracting a site-level report from the output of current processing software is not straightforward as multiple peptides might contribute to a single site, multiple phosphorylation sites can occur on the same peptides, and protein isoforms complicate site specification. Currently only limited support is available from a commercial software package via a platform-specific solution with a rather simple site quantification method. Here, we present sitereport, a software tool implemented in an extendable Python package called msproteomics to report phosphosites and phosphopeptides from a DIA-MS phosphoproteomics experiment with a proven quantification method called MaxLFQ. We demonstrate the use of sitereport for downstream data analysis at site level, allowing benchmarking different DIA-MS processing software tools. Availability and implementation sitereport is available as a command line tool in the Python package msproteomics, released under the Apache License 2.0 and available from the Python Package Index (PyPI) at https://pypi.org/project/msproteomics and GitHub at https://github.com/tvpham/msproteomics. [ABSTRACT FROM AUTHOR]

Simple Summary: Post-translational modifications (PTMs) strongly influence the stability and function of proteins. These modifications have been reported to affect wild-type (wt) p53 as well as the mutant forms of this protein (mutp53), often detected in cancer cells. Thus, PTMs may be key regulators of the oncosuppressor activity of wtp53 and of the pro-oncogenic functions that some mutant forms of p53 may acquire, in terms of tumor survival, progression and resistance to anti-cancer therapies. As treatments that specifically target mutp53 do not exist, manipulating PTMs may represent a promising approach to achieve this goal and even to reactivate the wt functions of mutant proteins, in some cases, as reported in this review. Wild-type (wt) p53 and mutant forms (mutp53) play a key but opposite role in carcinogenesis. wtP53 acts as an oncosuppressor, preventing oncogenic transformation, while mutp53, which loses this property, may instead favor this process. This suggests that a better understanding of the mechanisms activating wtp53 while inhibiting mutp53 may help to design more effective anti-cancer treatments. In this review, we examine possible PTMs with which both wt- and mutp53 can be decorated and discuss how their manipulation could represent a possible strategy to control the stability and function of these proteins, focusing in particular on mutp53. The impact of ubiquitination, phosphorylation, acetylation, and methylation of p53, in the context of several solid and hematologic cancers, will be discussed. Finally, we will describe some of the recent studies reporting that wt- and mutp53 may influence the expression and activity of enzymes responsible for epigenetic changes such as acetylation, methylation, and microRNA regulation and the possible consequences of such changes. [ABSTRACT FROM AUTHOR]

Exercise has long been known for its beneficial effects on insulin sensitivity (IS) and glucose handling with both moderate-intensity continuous (MIC) exercise and resistance exercise (RE) inducing beneficial effects. In recent years, low-load, high-repetition (LLHR) RE has emerged as a strategy to increase muscle mass and strength to levels similar to traditional RE; however, the effects of LLHR RE on glucose handling has yet to be investigated. The purpose of this trial was to compare the acute effects of LLHR RE to MIC exercise on post-exercise glycemic control and insulin sensitivity in males and females. Twenty-four (n = 12/sex) participants completed acute bouts of MIC exercise (30 min at 65% V̇O₂peak) and LLHR (3 circuits, 6 exercises/circuit, 25–35 repetitions/exercise/circuit) matched for time with muscle biopsies immediately pre and post exercise and an oral glucose tolerance test (OGTT) 90 min following exercise. Blood glucose concentrations (p = 0.002, ηp2 = 0.37), glucose AUC (p = 0.002, ηp2 = 0.35) and max glucose concentration (p = 0.003, ηp2 = 0.34) were lower during the post exercise OGTT following LLHR RE compared to MIC exercise. There was a main effect of trial on TBC1D1 Ser237 phosphorylation (p = 0.04, ηp2 = 0.19) such that it was greater following MIC exercise compared to LLHR RE. Furthermore, phosphorylated ACC Ser79 increased following MIC exercise with no change following LLHR RE (p < 0.001, ηp2 = 0.50). Phosphorylation of PTEN Ser380 was greater in males than females during LLHR RE (p = 0.01, ηp2 = 0.27). These findings suggest that LLHR RE is a feasible exercise modality to improve post-exercise glycemic control in both males and females. Trial registration number: NCT06217679. [ABSTRACT FROM AUTHOR]

Background: Most Parkinson's disease (PD) loci have shown low prevalence in the Indian population, highlighting the need for further research. Objective: The aim of this study was to characterize a novel phosphatase tensin homolog‐induced serine/threonine kinase 1 (PINK1) mutation causing PD in an Indian family. Methods: Exome sequencing of a well‐characterized Indian family with PD. A novel PINK1 mutation was studied by in silico modeling using AlphaFold2, expression of mutant PINK1 in human cells depleted of functional endogenous PINK1, followed by quantitative image analysis and biochemical assessment. Results: We identified a homozygous chr1:20648535–20648535 T>C on GRCh38 (p.F385S) mutation in exon 6 of PINK1, which was absent in 1029 genomes from India and in other known databases. PINK1 F385S lies within the highly conserved DFG motif, destabilizes its active state, and impairs phosphorylation of ubiquitin at serine 65 and proper engagement of parkin upon mitochondrial depolarization. Conclusions: We characterized a novel nonconservative mutation in the DFG motif of PINK1, which causes loss of its ubiquitin kinase activity and inhibition of mitophagy. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society. [ABSTRACT FROM AUTHOR]

Adenosine N6-methylation (m6A) has been shown to be associated with the cell proliferation. The role of RNA methyltransferase 3 (METTL3), a key enzyme catalyzing m6A, in mediating m6A modification in lung epithelial cell proliferation remains unclear. This study aims to explore the effects and mechanisms of METTL3-mediated m6A modification in regulating lung epithelial cell proliferation. Results showed that knockdown of METTL3 significantly inhibited cell growth in lung epithelial cells, while overexpression of METTL3 promoted cell proliferation (P<0.05). Further Western blotting results demonstrated that the expression levels of the key proteins PCNA involved in cell growth and proliferation were significantly downregulated in METTL3 knockdown lung epithelial cells, along with a significant decrease in phosphorylation levels of Akt and mTOR (P<0.05). Immunofluorescence staining revealed a significant decrease in m6A modification levels in METTL3 knockdown lung epithelial cells (P<0.05). Real-time quantitative PCR and Western blotting results indicated that the mRNA and protein expression levels of PDK1, an upstream regulator of the Akt-mTOR signaling pathway, were significantly decreased in METTL3 knockdown lung epithelial cells (P<0.05). Mechanistically, m6A-IP-qPCR and RIP-qPCR results further demonstrated that METTL3 catalyzed m6A modification in the 3′ UTR region of PDK1 mRNA, which was then recognized by YTH N6-methyladenosine RNA-binding protein 1 (YTHDF1), enhancing the stability of its mRNA. In conclusion, this study reveals that METTL3 promotes cell proliferation by enhancing PDK1 m6A modification, thereby activating the Akt-mTOR signaling pathway. It provides evidence for a novel role of METTL3 in epithelial cell proliferation and offers new therapeutic targets for lung epithelial cell injury repair. [ABSTRACT FROM AUTHOR]

• Upcycled canola meal extract (CME) showed anti-photoaging effects in hairless mice. • CME reduced the number, mean length, and mean depth of wrinkles. • CME inhibited the increase of epidermal thickness induced by UVB radiation. • CME increased catalase activity and hydroxyproline content in mouse skin. • CME inhibited the phosphorylation of MAPKs such as ERK, JNK, and p38. • CME reduced MMP-2, -9, and -13 production in mice in response to UVB radiation. Canola meal, a by-product of processing canola into oil, reportedly contains high amounts of phenolic compounds and proteins. However, as canola meal is primarily used as feed for livestock, advances in multiple research fields are required to broaden its potential applications. Photoaging is caused by continuous exposure to ultraviolet (UV) radiation from sunlight. UV radiation generates reactive oxygen species and destroys collagen in the skin, thickening the epidermis, reducing elasticity, and causing wrinkles. We hypothesized that canola meal extract (CME) can mitigate the damage to skin associated with wrinkles induced by exposure to UVB radiation. To evaluate the anti-wrinkle effect, we administered CME orally to 40 female Hos:HR-1 hairless mice divided into 5 groups: (1) control mice, (2) a UVB group, and (3−5) CME-treated groups (CME−250, 500, and 1000 mg/kg body weight/day, respectively). All groups except the controls were irradiated with UVB 3 times a week to create wrinkles due to photoaging. CME administration inhibited the increase of the number, mean length, and mean depth of wrinkles induced by UVB radiation as assessed using a skin replica. Histopathological image analysis revealed that CME administration resulted in a decrease in epidermal thickness and an increase in collagen content, while increasing catalase activity and hydroxyproline content in skin tissues. CME administration inhibited the phosphorylation of mitogen-activated protein kinase and decreased the production of collagenase and gelatinase. These results suggest that CME, an upcycled material, has the potential to develop into a healthful and functional food ingredient with anti-wrinkling effects. Administration of CME to hairless mouse decreased the number, length, and depth of wrinkles and epidermal thickness. CME administration also increased antioxidant enzyme activity and hydroxyproline content in skin tissue and inhibited phosphorylation of MAPKs, reducing the production of collagenase and gelatinase. These results suggest that CME mitigates skin wrinkles caused by UVB radiation. CME, canola meal extract; MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase; UVB, ultraviolet B. [Display omitted] [ABSTRACT FROM AUTHOR]

Activation of PINK1 and Parkin in response to mitochondrial damage initiates a response that includes phosphorylation of RAB7A at Ser72. Rubicon is a RAB7A binding negative regulator of autophagy. The structure of the Rubicon:RAB7A complex suggests that phosphorylation of RAB7A at Ser72 would block Rubicon binding. Indeed, in vitro phosphorylation of RAB7A by TBK1 abrogates Rubicon:RAB7A binding. Pacer, a positive regulator of autophagy, has an RH domain with a basic triad predicted to bind an introduced phosphate. Consistent with this, Pacer-RH binds to phosho-RAB7A but not to unphosphorylated RAB7A. In cells, mitochondrial depolarization reduces Rubicon:RAB7A colocalization whilst recruiting Pacer to phospho- RAB7A--positive puncta. Pacer knockout reduces Parkin mitophagy with little effect on bulk autophagy or Parkin-independent mitophagy. Rescue of Parkin-dependent mitophagy requires the intact pRAB7A phosphate-binding basic triad of Pacer. Together these structural and functional data support a model in which the TBK1-dependent phosphorylation of RAB7A serves as a switch, promoting mitophagy by relieving Rubicon inhibition and favoring Pacer activation. [ABSTRACT FROM AUTHOR]

The micronutrient iron plays a crucial role in the growth and development of plants, necessitating meticulous regulation for its absorption by plants. Prior research has demonstrated that the transcription factor MxZR3.1 restricts iron absorption in apple rootstocks; however, the precise mechanism by which MxZR3.1 contributes to the regulation of iron homoeostasis in apple rootstocks remains unexplored. Here, MxMPK3‐2, a protein kinase, was discovered to interact with MxZR3.1. Y2H, bimolecular fluorescence complementation and pull down experiments were used to confirm the interaction. Phosphorylation and cell semi‐degradation tests have shown that MxZR3.1 can be used as a substrate of MxMPK3‐2, which leads to the MxZR3.1 protein being more stable. In addition, through tobacco transient transformation (LUC and GUS) experiments, it was confirmed that MxZR3.1 significantly inhibited the activity of the MxHA2 promoter, while MxMPK3‐2 mediated phosphorylation at the Ser94 site of MxZR3.1 further inhibited the activity of the MxHA2 promoter. It is tightly controlled to absorb iron during normal growth and development of apple rootstocks due to the regulatory effect of the MxMPK3‐2‐MxZR3.1 module on MxHA2 transcription level. Consequently, this research has revealed the molecular basis of how the MxMPK3‐2‐MxZR3.1 module in apple rootstocks controls iron homoeostasis by regulating the MxHA2 promoter's activity. Summary Statement: This study unveils the regulatory mechanism of iron absorption in apple rootstocks, elucidating the interaction between the protein kinase MxMPK3‐2 and the transcription factor MxZR3.1. The MxMPK3‐2‐MxZR3.1 module tightly controls iron homoeostasis by stabilizing MxZR3.1 and inhibiting the MxHA2 promoter during apple rootstock development. [ABSTRACT FROM AUTHOR]

Fusarium spp., a necrotrophic soil‐borne pathogen, causes root rot disease on many crops. CERK1, as a typical pattern recognition receptor, has been widely studied. However, the function of CERK1 during plant–Fusarium interaction has not been well described. We determined that MdCERK1 is a susceptibility gene in the apple‐Fusarium solani (Fs) interaction, and jasmonic acid (JA) plays a crucial role in this process. MdCERK1 directly targets and phosphorylates the lipoxygenase MdLOX2.1, an enzyme initiating the JA biosynthesis, at positions Ser326 and Thr327. These phosphorylations inhibit its translocation from the cytosol to the chloroplasts, leading to a compromised JA biosynthesis. Fs upregulates MdCERK1 expression during infection. In turn, when the JA level is low, the apple MdWRKY71, a transcriptional repressor of MdCERK1, is markedly upregulated and phosphorylated at Thr99 and Thr102 residues by the MAP kinase MdMMK2. The phosphorylation of MdWRKY71 enhances its transcription inhibition on MdCERK1. Taken together, MdCERK1 plays a novel role in limiting JA biosynthesis. There seems to be an arms race between apple and Fs, in which Fs activates MdCERK1 expression to reduce the JA level, while apple senses the low JA level and activates the MdMMK2‐MdWRKY71 module to elevate JA level by inhibiting MdCERK1 expression. Summary Statement: Chitin Elicitor Receptor Kinase 1, CERK1 plays a well‐known role in plants to initiate the primary defence upon pathogens infection. In addition to this, this study discovered that apple CERK1 interacts with and phosphorylates the lipoxygenase MdLOX2.1 to block the JA biosynthesis. Moreover, the apple MMK2‐WRKY71 module negatively regulates MdCERK1 expression to maintain the JA level. [ABSTRACT FROM AUTHOR]

Background: Aberrant expression of adipocyte enhancer‐binding protein 1 (AEBP1) has been demonstrated to be involved in the tumorigenesis and progression of numerous cancers. This study was aimed to investigate the mechanism of AEBP1 in the development of cervical cancer. Methods: The expression of AEBP1 in cervical cancer was assessed by immunohistochemistry. The function of AEBP1 on cell proliferation, migration, and invasion was determined by methyl thiazolyl tetrazolium assay, colony formation, and transwell assay. The activation of related signaling pathway was determined by western blot. The bioinformatics analysis was performed by Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Results: Higher protein expression of AEBP1 was observed in patients with cervical cancer. Overexpressed AEBP1 promoted cell proliferation, migration, and invasion abilities in cervical cancer cells. Moreover, the research manifested that AEBP1 activated the phosphorylation of STAT3. GO and KEGG analysis showed that genes positively related to AEBP1 were highly enriched in functions like epithelial cell proliferation, muscle cell migration, myoblast migration, smooth muscle tissue development, ECM–receptor interaction, transcriptional misregulation in cancer, and proteoglycans in cancer. While genes negatively related to AEBP1 were associated with immunity, including inflammatory response, external‐stimulus response, neutrophil, granulocyte, and macrophage chemotaxis. Conclusions: This study suggested that AEBP1 acts as an oncogened and might be a potential therapeutic target for the treatment of cervical cancer. [ABSTRACT FROM AUTHOR]

Summary: Plants grow rapidly for maximal production under optimal conditions; however, they adopt a slower growth strategy to maintain survival when facing environmental stresses. As salt stress restricts crop architecture and grain yield, identifying genetic variations associated with growth and yield responses to salinity is critical for breeding optimal crop varieties. OsDSK2a is a pivotal modulator of plant growth and salt tolerance via the modulation of gibberellic acid (GA) metabolism; however, its regulation remains unclear. Here, we showed that OsDSK2a can be phosphorylated at the second amino acid (S2) to maintain its stability. The gene‐edited mutant osdsk2aS2G showed decreased plant height and enhanced salt tolerance. SnRK1A modulated OsDSK2a‐S2 phosphorylation and played a substantial role in GA metabolism. Genetic analysis indicated that SnRK1A functions upstream of OsDSK2a and affects plant growth and salt tolerance. Moreover, SnRK1A activity was suppressed under salt stress, resulting in decreased phosphorylation and abundance of OsDSK2a. Thus, SnRK1A preserves the stability of OsDSK2a to maintain plant growth under normal conditions, and reduces the abundance of OsDSK2a to limit growth under salt stress. Haplotype analysis using 3 K‐RG data identified a natural variation in OsDSK2a‐S2. The allele of OsDSK2a‐G downregulates plant height and improves salt‐inhibited grain yield. Thus, our findings revealed a new mechanism for OsDSK2a stability and provided a valuable target for crop breeding to overcome yield limitations under salinity stress. [ABSTRACT FROM AUTHOR]

Summary: In maize, two pyruvate orthophosphate dikinase (PPDK) regulatory proteins, ZmPDRP1 and ZmPDRP2, are respectively specific to the chloroplast of mesophyll cells (MCs) and bundle sheath cells (BSCs). Functionally, ZmPDRP1/2 catalyse both phosphorylation/inactivation and dephosphorylation/activation of ZmPPDK, which is implicated as a major rate‐limiting enzyme in C4 photosynthesis of maize. Our study here showed that maize plants lacking ZmPDRP1 or silencing of ZmPDRP1/2 confer resistance to a prevalent potyvirus sugarcane mosaic virus (SCMV). We verified that the C‐terminal domain (CTD) of ZmPDRP1 plays a key role in promoting viral infection while independent of enzyme activity. Intriguingly, ZmPDRP1 and ZmPDRP2 re‐localize to cytoplasmic viral replication complexes (VRCs) following SCMV infection. We identified that SCMV‐encoded cytoplasmic inclusions protein CI targets directly ZmPDRP1 or ZmPDRP2 or their CTDs, leading to their re‐localization to cytoplasmic VRCs. Moreover, we found that CI could be degraded by the 26S proteasome system, while ZmPDRP1 and ZmPDRP2 could up‐regulate the accumulation level of CI through their CTDs by a yet unknown mechanism. Most importantly, with genetic, cell biological and biochemical approaches, we provide evidence that BSCs‐specific ZmPDRP2 could accumulate in MCs of Zmpdrp1 knockout (KO) lines, revealing a unique regulatory mechanism crossing different cell types to maintain balanced ZmPPDK phosphorylation, thereby to keep maize normal growth. Together, our findings uncover the genetic link of the two cell‐specific maize PDRPs, both of which are co‐opted to VRCs to promote viral protein accumulation for robust virus infection. [ABSTRACT FROM AUTHOR]

The human Origin Recognition Complex (ORC) is required not only for the initiation of DNA replication, but is also implicated in diverse cellular functions, including chromatin organization, centrosome biology, and cytokinesis. The smallest subunit of ORC, Orc6, is poorly conserved amongst eukaryotes. Recent studies from our laboratory have suggested that human Orc6 is not required for replication licensing, but is needed for S-phase progression. Further, ATR-dependent phosphorylation of Orc6 at T229 is implicated in DNA damage response during S-phase. In this study, we demonstrate that the CDK-dependent phosphorylation of Orc6 at T195 occurs during mitosis. While the phosphorylation at T195 does not seem to be required to exit mitosis, cells expressing the phosphomimetic T195E mutant of Orc6 impede S-phase progression. Moreover, the phosphorylated form of Orc6 associates with ORC more robustly, and Orc6 shows enhanced association with the ORC outside of G1, supporting the view that Orc6 may prevent the role of Orc1-5 in licensing outside of G1. Finally, Orc6 and the phosphorylated Orc6 localize to the nucleolar organizing centers and regulate ribosome biogenesis. Our results suggest that phosphorylated Orc6 at T195 prevents replication. [ABSTRACT FROM AUTHOR]

Polysaccharides are a kind of biological macromolecule, which widely exists in plants, fungi, animals, microorganisms, and other organisms. Polysaccharides exhibit various biological activities such as immunological regulation, antioxidation, antitumor, antiinflammatory, regulating intestinal flora, and protecting liver. These biological activities depend on their structure and physicochemical properties including monosaccharide composition, molecular weight (Mw), spatial configuration, etc. However, biological activities of polysaccharides are hardly comparable to synthetic drugs due to their inherent physicochemical properties. Therefore, structural modification of polysaccharides is an effective way to enhance their biological activities and promote the presentation of bioactivity. At present, phosphorylation modification is a common chemical modification method, which can effectively improve the biological activities of polysaccharides. However, there are limited reports on the synthesis, structure identification, and biological activities of phosphorylated polysaccharides. In view of this, the synthesis methods, structure identification methods, and biological activities of phosphorylated polysaccharides are reviewed in this paper. The findings can provide useful guidance for the research, production, and application of phosphorylated polysaccharides in functional foods and therapeutic agents. [ABSTRACT FROM AUTHOR]

Cassava starch has not been widely exploited in modified starch sector, despite its beneficial and peculiar properties including bland taste and flavor, easy extractability, and good paste clarity. In this study, cassava starch is phosphorylated using sodium tripolyphosphate (STPP) and sodium trimetaphosphate (STMP) at different pH levels according to a central composite design and optimized reaction conditions for maximum phosphorus content. The granule morphology, crystalline pattern, pasting properties, physicochemical properties, and in vitro starch digestibility of the modified starches are determined. The reaction conditions have significant effects on the P content. The highest P content (0.160%) is obtained for the sample synthesized using 8% of STMP and 4% of STPP (based on starch dry weight) at a pH of 9.0. X‐ray diffraction analysis revealed no noticeable change in crystalline properties of the modified starches in comparison to native starch. Samples with lower %P have similar granule morphology as the native starch, whereas at higher levels of P, granule rupture and agglomeration are noticed. Wide differences in the pasting and swelling properties are observed among the modified starches. Crosslinked starches formed at higher levels of pH show highly reduced paste viscosity and restricted in vitro starch digestibility. [ABSTRACT FROM AUTHOR]

Many intrinsically disordered regions (IDRs) can be phosphorylated. Phosphorylation of IDRs can change their conformation and functions. Structural rearrangements of IDRs can influence the interaction of proteins with RNA. IDR phosphorylation can selectively alter global protein–RNA binding profiles. Due to their capacity to mediate repetitive protein interactions, intrinsically disordered regions (IDRs) are crucial for the formation of various types of protein–RNA complexes. The functions of IDRs are strongly modulated by post-translational modifications (PTMs). Phosphorylation is the most common and well-studied modification of IDRs, which can alter homomeric or heteromeric interactions of proteins and impact their ability to phase separate. Moreover, phosphorylation can influence the RNA-binding properties of proteins, and recent studies demonstrated its selective impact on the global profiles of protein–RNA binding and regulation. These findings highlight the need for further integrative approaches to understand how signalling remodels protein–RNA networks in cells. [ABSTRACT FROM AUTHOR]

The addition of heat stress to resistance exercise or heated resistance exercise (HRE) is growing in popularity as emerging evidence indicates altered neuromuscular function and an amplification of several mechanistic targets of protein synthesis. Studies demonstrating increased protein synthesis activity have shown temperature-dependent mammalian target of rapamycin phosphorylation, supplemental calcium release, augmented heat shock protein expression, and altered immune and hormone activity. These intriguing observations have largely stemmed from myotube, isolated muscle fiber, or rodent models using passive heating alone or in combination with immobilization or injury models. A growing number of translational studies in humans show comparable results employing local tissue or whole-body heat with and without resistance exercise. While few, these translational studies are immensely valuable as they are most applicable to sport and exercise. As such, this brief narrative review aims to discuss evidence primarily from human HRE studies detailing the neuro)muscular, hormonal, and molecular responses to HRE and subsequent strength and hypertrophy adaptations. Much remains unknown in this exciting new area of inquiry from both a mechanistic and functional perspective warranting continued research. [ABSTRACT FROM AUTHOR]

The dystrophin protein has well‐characterized roles in force transmission and maintaining membrane integrity during muscle contraction. Studies have reported decreased expression of dystrophin in atrophying muscles during wasting conditions, and that restoration of dystrophin can attenuate atrophy, suggesting a role in maintaining muscle mass. Phosphorylation of S3059 within the cysteine‐rich region of dystrophin enhances binding between dystrophin and β‐dystroglycan, and mimicking phosphorylation at this site by site‐directed mutagenesis attenuates myotube atrophy in vitro. To determine whether dystrophin phosphorylation can attenuate muscle wasting in vivo, CRISPR‐Cas9 was used to generate mice with whole body mutations of S3059 to either alanine (DmdS3059A) or glutamate (DmdS3059E), to mimic a loss of, or constitutive phosphorylation of S3059, on all endogenous dystrophin isoforms, respectively. Sciatic nerve transection was performed on these mice to determine whether phosphorylation of dystrophin S3059 could attenuate denervation atrophy. At 14 days post denervation, atrophy of tibialis anterior (TA) but not gastrocnemius or soleus muscles, was partially attenuated in DmdS3059E mice relative to WT mice. Attenuation of atrophy was associated with increased expression of β‐dystroglycan in TA muscles of DmdS3059E mice. Dystrophin S3059 phosphorylation can partially attenuate denervation‐induced atrophy, but may have more significant impact in less severe modes of muscle wasting. [ABSTRACT FROM AUTHOR]

Objectives: To assess the role of TNF‐α/TNFR2 axis on promoting angiogenesis in oral squamous cell carcinoma (OSCC) cells and uncover the underlying mechanisms. Materials and Methods: The expression of TNFR2 and CD31 in OSCC tissues was examined; gene expression relationship between TNF‐α/TNFR2 and angiogenic markers or signaling molecules was analyzed; the expression of angiogenic markers, signaling molecules, TNFR1, and TNFR2 in TNF‐α‐stimulated OSCC cells treated with or without TNFR2 neutralizing antibody (TNFR2 Nab) were assessed; the concentration of angiogenic markers in the supernatant of OSCC cells was detected; conditioned mediums of OSCC cells treated with TNF‐α or TNF‐α + TNFR2 Nab were applied to human umbilical vein endothelial cells (HUVECs), followed by tube formation and cell migration assays. Results: Significantly elevated expression of TNFR2 and CD31 in OSCC tissues was observed. A positive gene expression correlation was identified between TNF‐α/TNFR2 and angiogenic markers or signaling molecules. TNFR2 Nab inhibited the effects of TNF‐α on enhancing the expression of angiogenic factors and TNFR2, the phosphorylation of the Akt/mTOR signaling pathway, HUVECs migration, and tube formation. Conclusions: TNFR2 Nab counteracts the effect of TNF‐α on OSCC cells through the TNFR2/Akt/mTOR axis, indicating that blocking TNFR2 might be a promising strategy against cancer. [ABSTRACT FROM AUTHOR]

Objectives: Connexin43 (Cx43) is involved in the inflammation of many tissue types. Dental caries is infectious disease resulting from mineralized tissue dissolution by a specific bacterial population, causing pulp inflammation. However, Cx43's role in dental pulp remains unclear. Here, we investigated the function of Cx43 during pulp inflammation. Materials and Methods: We constructed a dentin injury model in Sprague–Dawley rats to investigate changes in Cx43 expression during pulp inflammation. Cx43 was inhibited in human dental pulp cells (hDPCs) that had been stimulated with lipopolysaccharide (LPS) to investigate the effect of Cx43 on inflammatory response. Promotion of TLR4‐NF‐κB pathway activity and special Cx43 channel inhibitors were used to clarify the function of Cx43 in hDPCs. Results: Dentin injury led to low‐level inflammation in dental pulp. Following dentin injury, Cx43 expression initially decreased before gradually recovering to normal levels. Cx43 inhibition reduced LPS‐induced expression of inflammatory cytokines and NF‐κB pathway activity. Promotion of NF‐κB pathway activity counteracted the effect of Cx43 in hDPCs. Furthermore, inhibition of Cx43 hemichannels reduced LPS‐induced inflammatory cytokine expression. Conclusions: Cx43 is involved in inflammation of dental pulp, while its inhibition reduced LPS‐induced inflammation in hDPCs through NF‐κB pathway via blockage of hemichannels. [ABSTRACT FROM AUTHOR]

Objective: The objective of this investigation was to examine the presence of interleukin (IL)‐13 and its receptor IL‐13Rα2 in the tissues of oral submucous fibrosis (OSF), investigate their biological functions, and explore the underlying mechanisms involved in the development of OSF. Materials and Methods: The expression of IL‐13 and IL‐13Rα2 in the oral mucosa of patients with OSF and normal individuals was determined through immunohistochemistry and reverse transcription–quantitative polymerase chain reaction (RT–qPCR). Primary fibroblasts (FBs) were extracted through enzymatic digestion and then cultured. Immunofluorescence was employed to identify the FB cultures and the location of IL‐13Rα2. The effects of IL‐13/IL‐13Rα2/PI3K/AKT/mTOR on the migration, proliferation, and secretion of fiber‐related proteins of FBs were explored via the wound healing assay, CCK‐8 assay, EDU assay, and RT–qPCR. The impact of IL‐13Rα2 silencing and PI3K/AKT inhibition on the effect of IL‐13 on FBs was analyzed by RT–qPCR and Western blotting. Results: IL‐13 and IL‐13Rα2 were highly expressed in OSF. Primary FBs were successfully extracted and cultured. IL‐13Rα2 was found to be localized in myofibroblasts. IL‐13 promoted the proliferation, migration, and secretion of fibril‐associated proteins in FBs. The proliferation, migration, and secretion of fibril‐associated proteins of FBs were decreased following IL‐13Rα2 silencing and inhibition of the PI3K/AKT/mTOR pathway. Conclusion: IL‐13 may promote the proliferation, migration, and secretion of fiber‐related proteins of FBs through the PI3K/AKT/mTOR pathway by targeting IL‐13Rα2. [ABSTRACT FROM AUTHOR]

Simple Summary: Prostate cancer (PCa) is the most common cancer in males in the UK, resulting in more than 12,000 deaths per year in the UK. A large proportion of patients stop responding to currently available treatments; consequently, identifying alternative treatment options is key to prolonging the life of these men and improving survival rates. Protein kinases regulate signalling pathways, which are often dysregulated in cancer, leading to tumour growth. Additionally, kinase driven phosphorylation regulates androgen receptor activity, which is a key driver of PCa. Understanding how these signalling pathways contribute towards PCa is critical in the development of novel treatment options. The kinase, casein kinase 1 alpha (CK1α), has potential as a PCa therapeutic. Here we describe the current literature supporting this hypothesis and identify where further research is needed to fully understand the functional role and therapeutic potential of CK1α in PCa. The androgen receptor (AR) is a key driver of prostate cancer (PCa) and, as such, current mainstay treatments target this molecule. However, resistance commonly arises to these therapies and, therefore, additional targets must be evaluated to improve patient outcomes. Consequently, alternative approaches for indirectly targeting the AR are sought. AR crosstalk with other signalling pathways, including several protein kinase signalling cascades, has been identified as a potential route to combat therapy resistance. The casein kinase 1 (CK1) family of protein kinases phosphorylate a multitude of substrates, allowing them to regulate a diverse range of pathways from the cell cycle to DNA damage repair. As well as its role in several signalling pathways that are de-regulated in PCa, mutational data suggest its potential to promote prostate carcinogenesis. CK1α is one isoform predicted to regulate AR activity via phosphorylation and has been implicated in the progression of several other cancer types. In this review, we explore how the normal biological function of CK1 is de-regulated in cancer, the impact on signalling pathways and how this contributes towards prostate tumourigenesis, with a particular focus on the CK1α isoform as a novel therapeutic target for PCa. [ABSTRACT FROM AUTHOR]