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ACS Central Science Apr 202314-3-3 proteins are dimeric hubs that bind hundreds of phosphorylated "clients" to regulate their function. Installing stable, functional mimics of phosphorylated amino...
14-3-3 proteins are dimeric hubs that bind hundreds of phosphorylated "clients" to regulate their function. Installing stable, functional mimics of phosphorylated amino acids into proteins offers a powerful strategy to study 14-3-3 function in cellular-like environments, but a previous genetic code expansion (GCE) system to translationally install nonhydrolyzable phosphoserine (nhpSer), with the γ-oxygen replaced with CH, site-specifically into proteins has seen limited usage. Here, we achieve a 40-fold improvement in this system by engineering into a six-step biosynthetic pathway that produces nhpSer from phosphoenolpyruvate. Using this autonomous "PermaPhos" expression system, we produce three biologically relevant proteins with nhpSer and confirm that nhpSer mimics the effects of phosphoserine for activating GSK3β phosphorylation of the SARS-CoV-2 nucleocapsid protein, promoting 14-3-3/client complexation, and monomerizing 14-3-3 dimers. Then, to understand the biological function of these phosphorylated 14-3-3ζ monomers (containing nhpSer at Ser58), we isolate its interactome from HEK293T lysates and compare it with that of wild-type 14-3-3ζ. These data identify two new subsets of 14-3-3 client proteins: (i) those that selectively bind dimeric 14-3-3ζ and (ii) those that selectively bind monomeric 14-3-3ζ. We discover that monomeric-but not dimeric-14-3-3ζ interacts with cereblon, an E3 ubiquitin-ligase adaptor protein of pharmacological interest.
PubMed: 37122473
DOI: 10.1021/acscentsci.3c00191 -
Pharmaceutics Apr 2023Compared to metallic hardware, an effective bone adhesive can revolutionize the treatment of clinically challenging situations such as comminuted, articular, and...
Compared to metallic hardware, an effective bone adhesive can revolutionize the treatment of clinically challenging situations such as comminuted, articular, and pediatric fractures. The present study aims to develop such a bio-inspired bone adhesive, based upon a modified mineral-organic adhesive with tetracalcium phosphate (TTCP) and phosphoserine (OPS) by incorporating nanoparticles of polydopamine (nPDA). The optimal formulation, which was screened using in vitro instrumental tensile adhesion tests, was found to be 50%TTCP/50%OPS-2%nPDA with a liquid-to-powder ratio of 0.21 mL/g. This adhesive has a substantially stronger adhesive strength (1.0-1.6 MPa) to bovine cortical bone than the adhesive without nPDA (0.5-0.6 MPa). To simulate a clinical scenario of autograft fixation under low mechanical load, we presented the first in vivo model: a rat fibula glued to the tibia, on which the TTCP/OPS-nPDA adhesive (n = 7) was shown to be effective in stabilizing the graft without displacement (a clinical success rate of 86% and 71% at 5 and 12 weeks, respectively) compared to a sham control (0%). Significant coverage of newly formed bone was particularly observed on the surface of the adhesive, thanks to the osteoinductive property of nPDA. To conclude, the TTCP/OPS-nPDA adhesive fulfilled many clinical requirements for the bone fixation, and potentially could be functionalized via nPDA to offer more biological activities, e.g., anti-infection after antibiotic loading.
PubMed: 37111718
DOI: 10.3390/pharmaceutics15041233 -
Phytomedicine : International Journal... Jul 2023Erchen decoction, a traditional Chinese medicine formula, can reduce the level of oxidative stress for the treatment of dyslipidemia phlegm-dampness retention syndrome...
Erchen decoction to reduce oxidative stress in dyslipidemia phlegm-dampness retention syndrome mice: In vivo mechanism revealed by metabolomics (liquid chromatography-mass spectrometry).
OBJECTIVE
Erchen decoction, a traditional Chinese medicine formula, can reduce the level of oxidative stress for the treatment of dyslipidemia phlegm-dampness retention syndrome (DPDRS); however, studies have not elucidated the mechanism underlying its metabolic action. Here, liquid chromatography-mass spectrometry (LC-MS)-based metabolomic techniques were utilized to characterize the in vivo effects of Erchen decoction in achieving reduction of oxidative stress levels and understand the potential metabolic mechanisms of action.
METHODS
We constructed a DPDRS animal model using a multifactorial composite modeling approach, and Erchen decoction was administered by gavage. We employed LC-MS-based metabolomic techniques in combination with serum-associated factors, gene transcription, methylation detection, and hematoxylin and eosin staining.
RESULTS
In this study, the constructed animal model of DPDRS had satisfactory quality. Erchen decoction treatment reduced the levels of low-density lipoprotein cholesterol, t total cholesterol and riglyceride; it improved the endothelial structure, increased levels of serum β-nicotinamide adenine dinucleotide phosphate and glutathione concentrations, increased aortic phosphoserine aminotransferase and phosphoserine phosphatase gene expression levels, and decreased aortic phosphoglycerate dehydrogenase methylation level. A total of 64 differential metabolites were obtained using LC-MS assay, and 34 differential metabolic pathways were obtained after enrichment.
CONCLUSIONS
Erchen decoction treatment of DPDRS mice reversed lipid indexes, improved vascular endothelial structure, increased serum and aortic anti-oxidative stress factor concentration and expression levels, and decreased methylation levels, thereby reducing oxidative stress and protecting vascular endothelium. Tricarboxylic acid cycle and metabolic pathways of serum glutamine, serine, tryptophan, pyrimidine, and pyruvate were the most relevant metabolic pathways involved in reducing oxidative stress levels by Erchen decoction during DPDRS treatment; especially, mitochondrial redox homeostasis maintenance in endothelial cells may be crucial. In this work, the therapeutic potential of Erchen decoction for reducing the oxidative stress level in DPDRS was demonstrated; however, its in-depth mechanism is worth further exploration.
Topics: Mice; Animals; Endothelial Cells; Drugs, Chinese Herbal; Metabolomics; Chromatography, Liquid; Mass Spectrometry; Cholesterol, LDL; Dyslipidemias; Oxidative Stress
PubMed: 37087794
DOI: 10.1016/j.phymed.2023.154808 -
Frontiers in Bioengineering and... 2023Bone defects that result from trauma, infection, surgery, or congenital malformation can severely affect the quality of life. To address this clinical problem, a...
Bone defects that result from trauma, infection, surgery, or congenital malformation can severely affect the quality of life. To address this clinical problem, a phosphoserine-loaded chitosan membrane that consists of chitosan membranes serving as the scaffold support to accommodate endogenous stem cells and phosphoserine is synthesized. The introduction of phosphoserine greatly improves the osteogenic effect of the chitosan membranes mutual crosslinking using a crosslinker (EDC, 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide). The morphology of PS-CS membranes was shown by scanning electron microscopy (SEM) to have an interconnected porous structure. The incorporation of phosphoserine into chitosan membranes was confirmed by energy dispersive spectrum (EDS), Fourier Transforms Infrared (FTIR), and X-ray diffraction (XRD) spectrum. The CCK8 assay and Live/Dead staining, Hemolysis analysis, and cell adhesion assay demonstrated that PS-CS membranes had good biocompatibility. The osteogenesis-related gene expression of BMSCs was higher in PS-CS membranes than in CS membranes, which was verified by alkaline phosphatase (ALP) activity, immunofluorescence staining, and real-time quantitative PCR (RT-qPCR). Furthermore, micro-CT and histological analysis of rat cranial bone defect demonstrated that PS-CS membranes dramatically stimulated bone regeneration . Moreover, H&E staining of the main organs (heart, liver, spleen, lung, or kidney) showed no obvious histological abnormalities, revealing that PS-CS membranes were no additional systemic toxicity . Collectively, PS-CS membranes may be a promising candidate for bone tissue engineering.
PubMed: 37034248
DOI: 10.3389/fbioe.2023.1096532 -
The Journal of General Physiology Jun 2023Light-controlled availability for phosphorylation reveals dominant roles of select R-domain serines in CFTR channel activation.
Light-controlled availability for phosphorylation reveals dominant roles of select R-domain serines in CFTR channel activation.
Topics: Phosphoserine; Cystic Fibrosis Transmembrane Conductance Regulator; Ion Channel Gating; Phosphorylation; Serine
PubMed: 37017643
DOI: 10.1085/jgp.202313336 -
International Journal of Molecular... Mar 2023Phosphoserine aminotransferase (PSAT) is a pyridoxal 5'-phosphate-dependent enzyme involved in the second step of the phosphorylated pathway of serine biosynthesis. PSAT...
Phosphoserine aminotransferase (PSAT) is a pyridoxal 5'-phosphate-dependent enzyme involved in the second step of the phosphorylated pathway of serine biosynthesis. PSAT catalyzes the transamination of 3-phosphohydroxypyruvate to 3-phosphoserine using L-glutamate as the amino donor. Although structural studies of PSAT have been performed from archaea and humans, no structural information is available from fungi. Therefore, to elucidate the structural features of fungal PSAT, we determined the crystal structure of PSAT (PSAT) at a resolution of 2.8 Å. The results demonstrated that the PSAT protein was dimeric in its crystal structure. Moreover, the gate-keeping loop of PSAT exhibited a conformation similar to that of other species. Several distinct structural features in the halide-binding and active sites of PSAT were compared with its homologs. Overall, this study contributes to our current understanding of PSAT by identifying the structural features of fungal PSAT for the first time.
Topics: Humans; Molecular Structure; Saccharomyces cerevisiae; Amino Acid Sequence; Transaminases; Pyridoxal Phosphate; Crystallography, X-Ray
PubMed: 36982214
DOI: 10.3390/ijms24065139 -
International Journal of Molecular... Mar 2023Ethanol consumption influences cardiovascular functions. In humans, acute consumption of ethanol causes dose-dependent tachycardia. Our previous study showed that...
Ethanol consumption influences cardiovascular functions. In humans, acute consumption of ethanol causes dose-dependent tachycardia. Our previous study showed that ethanol-induced tachycardia might involve decreased nitric oxide (NO) signaling in the brain's medulla. NMDA receptors, another important target of ethanol, are one of the upstream signals of nitric oxide. Reports showed the modulation of NMDA receptor function by estrogen or estrogen receptors. The present study aims to examine the hypothesis that depletion of estrogen by ovariectomy (OVX) might modulate ethanol-induced tachycardia by regulating NMDA receptor function and NO signaling in the cardiovascular regulatory nucleus of the brain. Ethanol (3.2 g/kg, 40% /, 10 mL/kg) or saline (10 mL/kg) was administered by oral gavage in sham or OVX female Sprague-Dawley (SD) rats. The blood pressure (BP) and heart rate (HR) were measured using the tail-cuff method. The levels of phosphoserine 896 of the GluN1 subunit (pGluN1-serine 896) and NMDA GluN1 subunits (GluN1) were determined by immunohistochemistry. The expressions of nitric oxide synthase (NOS) and estrogen receptors in the tissue were measured by Western blotting. Nitric oxide contents were measured as total nitrate-nitrite by colorimetric assay kit. In a 2-h observation, there was no significant change in BP between the saline and ethanol groups. However, compared with saline, ethanol caused an increase in HR (tachycardia) in sham control or OVX rats. Interestingly, ethanol produced more significant tachycardia in the OVX group than in the sham control group. Nitric oxide levels were lower in the area of the rostral ventrolateral medulla (RVLM) 60 min following ethanol administration in OVX compared with sham control, without significant changes in the expression of NOS and estrogen receptors (ERα and ERβ). In addition, a decrease in the immunoreactivity of pGluN1-serine 896, without significant changes in GluN1, was found in neurons of RVLM 40 min following ethanol administration in OVX compared with sham control. Our results suggest that depletion of estradiol (E2) by OVX might exacerbate the tachycardia following ethanol administration, the underlying mechanism of which might be associated with decreased NMDA receptor function and NO level in the RVLM.
Topics: Humans; Rats; Female; Animals; Ethanol; Receptors, N-Methyl-D-Aspartate; Nitric Oxide; Rats, Sprague-Dawley; Receptors, Estrogen; Blood Pressure; Tachycardia; Estrogens; Nitric Oxide Synthase; Ovariectomy; Serine
PubMed: 36982161
DOI: 10.3390/ijms24065087 -
Journal of Materials Science. Materials... Mar 2023Present surgical situations require a bone adhesive which has not yet been developed for use in clinical applications. Recently, phosphoserine modified cements (PMC)...
Present surgical situations require a bone adhesive which has not yet been developed for use in clinical applications. Recently, phosphoserine modified cements (PMC) based on mixtures of o-phosphoserine (OPLS) and calcium phosphates, such as tetracalcium phosphate (TTCP) or α-tricalcium phosphate (α-TCP) as well as chelate setting magnesium phosphate cements have gained increasing popularity for their use as mineral bone adhesives. Here, we investigated new mineral-organic bone cements based on phosphoserine and magnesium phosphates or oxides, which possess excellent adhesive properties. These were analyzed by X-ray diffraction, Fourier infrared spectroscopy and electron microscopy and subjected to mechanical tests to determine the bond strength to bone after ageing at physiological conditions. The novel biomineral adhesives demonstrate excellent bond strength to bone with approximately 6.6-7.3 MPa under shear load. The adhesives are also promising due to their cohesive failure pattern and ductile character. In this context, the new adhesive cements are superior to currently prevailing bone adhesives. Future efforts on bone adhesives made from phosphoserine and Mg appear to be very worthwhile.
Topics: Bone Cements; Phosphoserine; Magnesium; Oxides; Adhesives; Calcium Phosphates; Phosphates; Minerals; Materials Testing; Microscopy, Electron, Scanning
PubMed: 36964421
DOI: 10.1007/s10856-023-06714-6 -
Frontiers in Molecular Neuroscience 2023This study aims to prove that the variants in gene are associated with neurodevelopmental disorders (NDD) with developmental delay (DD) and infantile spasm (IS) and to...
OBJECTIVE
This study aims to prove that the variants in gene are associated with neurodevelopmental disorders (NDD) with developmental delay (DD) and infantile spasm (IS) and to determine the genotype-phenotype correlations.
METHODS
Trio-based exome sequencing (ES) was performed on the four families enrolled in this study. We collected and systematically reviewed the four probands' clinical data, magnetic resonance images (MRI), and electroencephalography (EEG). We also carried out bioinformatics analysis by integrating published exome/genome sequencing data and human brain transcriptomic data.
RESULTS
We described four patients whose median age of seizure onset was 5 months. The primary manifestation was infantile spasms with typical hypsarrhythmia on EEG. Developmental delays or intellectual disabilities varied among the four individuals. Three missense variants in gene were identified from four families, including chr5:66438324 (c.2693T > C: p.Ile898Thr) z, chr5:66459419 (c.4412C > T: p.Thr1471Ile), and chr5:66462662 (c.7655C > G:p.Ser2552Trp). The missense variant p.Ile898Thr is mapped to the AGC-kinase C-terminal with phosphatase activity. The other variant p.Ser2552Trp is located in a phosphoserine-modified residue which may affect cell membrane stability and signal transduction. Besides, the variant p.Thr1471Ile is a recurrent site screened out in two unrelated patients. Compared to private mutations (found only in a single family or a small population) of in the gnomAD non-neuro subset, all variants were predicted to be damaging or probably damaging through different bioinformatic analyses. Significantly higher CADD scores of the variant p.Thr1471Ile indicate more deleteriousness of the recurrent site. And the affected amino acids are highly conserved across multiple species. According to the Brainspan Atlas database, is expressed primarily in the mediodorsal nucleus of the thalamus and medial prefrontal cortex during the prenatal period, potentially contributing to embryonic brain development.
CONCLUSION
Our results revealed that the variants of gene might lead to neurodevelopmental disorders with developmental delay and infantile spasm. Thus, variants should be considered the potential candidate gene in patients with neurodevelopmental disorders clinically marked by infantile spasms.
PubMed: 36910266
DOI: 10.3389/fnmol.2023.1097553 -
Scientific Reports Mar 2023Phosphoserine aminotransferase 1 (PSAT1) has been associated with the occurrence and development of various carcinomas; however, its function in uterine corpus...
Phosphoserine aminotransferase 1 (PSAT1) has been associated with the occurrence and development of various carcinomas; however, its function in uterine corpus endometrial carcinoma (UCEC) is unknown. We aimed to explore the relationship between PSAT1 and UCEC using The Cancer Genome Atlas database and functional experiments. PSAT1 expression levels in UCEC were employed using the paired sample t-test, Wilcoxon rank-sum test, the Clinical Proteomic Tumor Analysis Consortium database, and the Human Protein Atlas database, while survival curves were constructed using the Kaplan-Meier plotter. We performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis to explore the possible functions and related pathways of PSAT1. Furthermore, single-sample gene set enrichment analysis was performed to detect the relationship between PSAT1 and tumor immune infiltration. StarBase and quantitative PCR were used to predict and verify the interactions between miRNAs and PSAT1. The Cell Counting Kit-8, EdU assay, clone formation assay, western blotting and flow cytometry were used to evaluate cell proliferation. Finally, Transwell and Wound healing assays were used to assess cell invasion and migration. Our study found that PSAT1 was significantly overexpressed in UCEC, and this high expression was associated with a worse prognosis. A high level of PSAT1 expression was associated with a late clinical stage and, histological type. In addition, the results of GO and KEGG enrichment analysis showed that PSAT1 was mainly involved in the regulation of cell growth, immune system and cell cycle in UCEC. In addition, PSAT1 expression was positively correlated with Th2 cells and negatively correlated with Th17 cells. Furthermore, we also found that miR-195-5P negatively regulated the expression of PSAT1 in UCEC. Finally, the knockdown of PSAT1 resulted in the inhibition of cell proliferation, migration, and invasion in vitro. Overall, PSAT1 was identified as a potential target for the diagnosis and immunotherapy of UCEC.
Topics: Female; Humans; Carcinoma, Endometrioid; Cell Proliferation; Databases, Protein; Down-Regulation; Endometrial Neoplasms; Proteomics; Transaminases
PubMed: 36906716
DOI: 10.1038/s41598-023-31325-0