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Molecular Biology of the Cell Jan 2024α-Synuclein is a presynaptic protein that regulates synaptic vesicle (SV) trafficking. In Parkinson's disease (PD) and dementia with Lewy bodies (DLB), α-synuclein...
α-Synuclein is a presynaptic protein that regulates synaptic vesicle (SV) trafficking. In Parkinson's disease (PD) and dementia with Lewy bodies (DLB), α-synuclein aberrantly accumulates throughout neurons, including at synapses. During neuronal activity, α-synuclein is reversibly phosphorylated at serine 129 (pS129). While pS129 comprises ∼4% of total α-synuclein under physiological conditions, it dramatically increases in PD and DLB brains. The impacts of excess pS129 on synaptic function are currently unknown. We show here that compared with wild-type (WT) α-synuclein, pS129 exhibits increased binding and oligomerization on synaptic membranes and enhanced vesicle "microclustering" in vitro. Moreover, when acutely injected into lamprey reticulospinal axons, excess pS129 α-synuclein robustly localized to synapses and disrupted SV trafficking in an activity-dependent manner, as assessed by ultrastructural analysis. Specifically, pS129 caused a declustering and dispersion of SVs away from the synaptic vicinity, leading to a significant loss of total synaptic membrane. Live imaging further revealed altered SV cycling, as well as microclusters of recently endocytosed SVs moving away from synapses. Thus, excess pS129 caused an activity-dependent inhibition of SV trafficking via altered vesicle clustering/reclustering. This work suggests that accumulation of pS129 at synapses in diseases like PD and DLB could have profound effects on SV dynamics.
Topics: Animals; alpha-Synuclein; Parkinson Disease; Phosphoserine; Synapses; Synaptic Vesicles; Lampreys
PubMed: 37991902
DOI: 10.1091/mbc.E23-07-0269 -
Bio-protocol Nov 2023While site-specific translational encoding of phosphoserine (pSer) into proteins in via genetic code expansion (GCE) technologies has transformed our ability to study...
While site-specific translational encoding of phosphoserine (pSer) into proteins in via genetic code expansion (GCE) technologies has transformed our ability to study phospho-protein structure and function, recombinant phospho-proteins can be dephosphorylated during expression/purification, and their exposure to cellular-like environments such as cell lysates results in rapid reversion back to the non-phosphorylated form. To help overcome these challenges, we developed an efficient and scalable GCE expression system enabling site-specific incorporation of a non-hydrolyzable phosphoserine (nhpSer) mimic into proteins of interest. This nhpSer mimic, with the γ-oxygen of phosphoserine replaced by a methylene (CH) group, is impervious to hydrolysis and recapitulates phosphoserine function even when phosphomimetics aspartate and glutamate do not. Key to this expression system is the co-expression of a biosynthetic pathway that converts the central metabolite phosphoenolpyruvate into non-hydrolyzable phosphoserine (nhpSer) amino acid, which provides a > 40-fold improvement in expression yields compared to media supplementation by increasing bioavailability of nhpSer and enables scalability of expressions. This "PermaPhos" expression system uses the E. coli BL21(DE3) Δ strain and three plasmids that express (i) the protein of interest, (ii) the GCE machinery for translational installation of nhpSer at UAG amber stop codons, and (iii) the nhpSer biosynthetic pathway. Successful expression requires efficient transformation of all three plasmids simultaneously into the expression host, and IPTG is used to induce expression of all components. Permanently phosphorylated proteins made in are particularly useful for discovering phosphorylation-dependent protein-protein interaction networks from cell lysates or transfected cells. Key features • Protocol builds on the nhpSer GCE system by Rogerson et al. (2015), but with a > 40-fold improvement in yields enabled by the nhpSer biosynthetic pathway. • Protein expression uses standard Terrific Broth (TB) media and requires three days to complete. • C-terminal purification tags on target protein are recommended to avoid co-purification of prematurely truncated protein with full-length nhpSer-containing protein. • Phos-tag gel electrophoresis provides a convenient method to confirm accurate nhpSer encoding, as it can distinguish between non-phosphorylated, pSer- and nhpSer-containing variants.
PubMed: 37969748
DOI: 10.21769/BioProtoc.4861 -
Arteriosclerosis, Thrombosis, and... Feb 2024Evolving evidence suggests that besides signaling pathways, platelet activation involves a complex interplay between metabolic pathways to support thrombus growth....
BACKGROUND
Evolving evidence suggests that besides signaling pathways, platelet activation involves a complex interplay between metabolic pathways to support thrombus growth. Selective targeting of metabolic checkpoints may inhibit platelet activation and provide a novel antiplatelet strategy. We, therefore, examined global metabolic changes that occur during the transition of human platelets from resting to an activated state to identify metabolites and associated pathways that contribute to platelet activation.
METHODS
We performed metabolic profiling of resting and convulxin-stimulated human platelet samples. The differential levels, pathway analysis, and PCA (principal component analysis) were performed using Metaboanalyst. Metascape was used for metabolite network construction.
RESULTS
Of the 401 metabolites identified, 202 metabolites were significantly upregulated, and 2 metabolites were downregulated in activated platelets. Of all the metabolites, lipids scored highly and constituted ≈50% of the identification. During activation, aerobic glycolysis supports energy demand and provides glycolytic intermediates required by metabolic pathways. Consistent with this, an important category of metabolites was carbohydrates, particularly the glycolysis intermediates that were significantly upregulated compared with resting platelets. We found that lysophospholipids such as 1-palmitoyl-GPA (glycero-3-phosphatidic acid), 1-stearoyl-GPS (glycero-3-phosphoserine), 1-palmitoyl-GPI (glycerophosphoinositol), 1-stearoyl-GPI, and 1-oleoyl-GPI were upregulated in activated platelets. We speculated that platelet activation could be linked to 1-carbon metabolism, a set of biochemical pathways that involve the transfer and use of 1-carbon units from amino acids, for cellular processes, including nucleotide and lysophospholipid synthesis. In alignment, based on pathway enrichment and network-based prioritization, the metabolites from amino acid metabolism, including serine, glutamate, and branched-chain amino acid pathway were upregulated in activated platelets, which might be supplemented by the high levels of glycolytic intermediates.
CONCLUSIONS
Metabolic analysis of resting and activated platelets revealed that glycolysis and 1-carbon metabolism are necessary to support platelet activation.
Topics: Humans; Blood Platelets; Glycolysis; Phosphorylation; Platelet Activation; Signal Transduction
PubMed: 37942614
DOI: 10.1161/ATVBAHA.123.319821 -
Frontiers in Molecular Biosciences 2023This study aims to test the hypothesis that increased ketone body production resulting from a ketogenic diet (KD) will correlate with reductions in pro-inflammatory...
This study aims to test the hypothesis that increased ketone body production resulting from a ketogenic diet (KD) will correlate with reductions in pro-inflammatory cytokines and lipid subspecies and improved clinical outcomes in adults treated with an adjunctive ketogenic diet for super-refractory status epilepticus (SRSE). Adults (18 years or older) were treated with a 4:1 (fat: carbohydrate and protein) ratio of enteral KD as adjunctive therapy to pharmacologic seizure suppression in SRSE. Blood and urine samples and clinical measurements were collected at baseline ( = 10), after 1 week ( = 8), and after 2 weeks of KD ( = 5). In addition, urine acetoacetate, serum -hydroxybutyrate, lipidomics, pro-inflammatory cytokines (IL-1β and IL-6), chemokines (CCL3, CCL4, and CXCL13), and clinical measurements were obtained at these three time points. Univariate and multivariate data analyses were performed to determine the correlation between ketone body production and circulating lipids, inflammatory biomarkers, and clinical outcomes. Changes in lipids included an increase in ceramides, mono-hexosylceramide, sphingomyelin, phosphocholine, and phosphoserines, and there was a significant reduction in pro-inflammatory mediators, IL-6 and CXCL13, seen at 1 and 2 weeks of KD. Higher blood -hydroxybutyrate levels at baseline correlated with better clinical outcomes; however, ketone body production did not correlate with other variables during treatment. Higher chemokine CCL3 levels following treatment correlated with a longer stay in the intensive care unit and a higher modified Rankin Scale score (worse neurologic disability) at discharge and 6-month follow up. Adults receiving an adjunctive enteral ketogenic diet for super-refractory status epilepticus exhibit alterations in select pro-inflammatory cytokines and lipid species that may predict their response to treatment.
PubMed: 37936721
DOI: 10.3389/fmolb.2023.1173039 -
Nature Communications Nov 2023Adjuvants and antigen delivery kinetics can profoundly influence B cell responses and should be critically considered in rational vaccine design, particularly for...
Adjuvants and antigen delivery kinetics can profoundly influence B cell responses and should be critically considered in rational vaccine design, particularly for difficult neutralizing antibody targets such as human immunodeficiency virus (HIV). Antigen kinetics can change depending on the delivery method. To promote extended immunogen bioavailability and to present antigen in a multivalent form, native-HIV Env trimers are modified with short phosphoserine peptide linkers that promote tight binding to aluminum hydroxide (pSer:alum). Here we explore the use of a combined adjuvant approach that incorporates pSer:alum-mediated antigen delivery with potent adjuvants (SMNP, 3M-052) in an extensive head-to-head comparison study with conventional alum to assess germinal center (GC) and humoral immune responses. Priming with pSer:alum plus SMNP induces additive effects that enhance the magnitude and persistence of GCs, which correlate with better GC-T cell help. Autologous HIV-neutralizing antibody titers are improved in SMNP-immunized animals after two immunizations. Over 9 months after priming immunization of pSer:alum with either SMNP or 3M-052, robust Env-specific bone marrow plasma cells (BM B) are observed. Furthermore, pSer-modification of Env trimer reduce targeting towards immunodominant non-neutralizing epitopes. The study shows that a combined adjuvant approach can augment humoral immunity by modulating immunodominance and shows promise for clinical translation.
Topics: Animals; Immunity, Humoral; Germinal Center; Adjuvants, Immunologic; Antigens; Primates; Antibodies, Neutralizing; HIV Infections; HIV Antibodies; env Gene Products, Human Immunodeficiency Virus
PubMed: 37925510
DOI: 10.1038/s41467-023-42923-x -
Frontiers in Bioscience (Landmark... Oct 2023Current evidence suggests that phosphoserine aminotransferase 1 () is overexpressed in various tumors. Herein, we investigate the significance of in non-small cell lung...
PURPOSE
Current evidence suggests that phosphoserine aminotransferase 1 () is overexpressed in various tumors. Herein, we investigate the significance of in non-small cell lung cancer (NSCLC) and its correlation with immune infiltration.
METHODS
The expression profile of in NSCLC patients and related clinical information was obtained from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA-NSCLC) databases. and experimental validation were conducted to assess the role of in NSCLC. Gene set enrichment analysis (GSEA) was performed to investigate the disparities in biological functions between groups with high and low expression. Additionally, the biological characteristics and immune cell infiltration were compared between these two groups. We also assessed whether expression could predict the sensitivity of NSCLC patients to immunotherapy using the immunophenotype score (IPS) and an anti-PD-L1 immunotherapy cohort (IMvig-or210). Furthermore, the difference in drug sensitivity between -high and -low expression cell lines was investigated.
RESULTS
Analysis of transcriptional expression profiles using TCGA data revealed overexpression of in NSCLC tissues correlated with poor overall survival (OS). GSEA results showed enrichment of DNA recombination and repair, nucleotide biosynthesis, and the P53 signaling pathway in the -high group. Experimental validation demonstrated that the knockdown of suppressed cell proliferation, migration, and invasion of NSCLC. Immune cell infiltration analysis revealed an immune-activated tumor microenvironment in the -low group. It was also observed that -low cell lines were more likely to benefit from immunotherapy and several chemotherapy drugs.
CONCLUSIONS
has enormous potential for applications in the prediction of NSCLC patient outcomes and provides the foothold for more precise individualized treatment of this patient population.
Topics: Humans; Carcinoma, Non-Small-Cell Lung; Cell Line; Cell Proliferation; Immunotherapy; Lung Neoplasms; Tumor Microenvironment
PubMed: 37919070
DOI: 10.31083/j.fbl2810243 -
Journal of Virology Nov 2023The infectious bursal disease virus is still an important agent which largely threatens global poultry farming industry economics. VP3 is a multifunctional scaffold...
The infectious bursal disease virus is still an important agent which largely threatens global poultry farming industry economics. VP3 is a multifunctional scaffold structural protein that is involved in virus morphogenesis and the regulation of diverse cellular signaling pathways. However, little is known about the roles of VP3 phosphorylation during the IBDV life cycle. In this study, we determined that IBDV infection induced the upregulation of Cdc7 expression and phosphorylated the VP3 Ser13 site to promote viral replication. Moreover, we confirmed that the negative charge addition of phosphoserine on VP3 at the S13 site was essential for IBDV proliferation. This study provides novel insight into the molecular mechanisms of VP3 phosphorylation-mediated regulation of IBDV replication.
Topics: Animals; Avibirnavirus; Birnaviridae Infections; Capsid Proteins; Cell Cycle Proteins; Chickens; Infectious bursal disease virus; Phosphorylation; Protein Serine-Threonine Kinases; Viral Structural Proteins; Virus Replication
PubMed: 37902398
DOI: 10.1128/jvi.01125-23 -
Endocrine Regulations Jan 2023Serine synthesis as well as endoplasmic reticulum stress and hypoxia are important factors of malignant tumor growth including glioblastoma. Previous studies have shown...
Serine synthesis as well as endoplasmic reticulum stress and hypoxia are important factors of malignant tumor growth including glioblastoma. Previous studies have shown that the knockdown of ERN1 (endoplasmic reticulum to nucleus signaling) significantly suppressed the glioblastoma cell proliferation and modified the hypoxia regulation. The present study is aimed to investigate the impact of hypoxia on the expression of (phosphoglycerate dehydrogenase), (phosphoserine aminotransferase 1), (phosphoserine phosphatase), (activating transcription factor 4), and (serine hydroxymethyltransferase 1) in U87MG glioblastoma cells in relation to knockdown of ERN1 with the intent to reveal the role of ERN1 signaling pathway on the endoplasmic reticulum stress-dependent regulation of expression of these genes. The control U87MG glioblastoma cells (transfected by empty vector) and ERN1 knockdown cells (transfected by dominant-negative ERN1) were exposed to hypoxia introduced by dimethyloxalylglycine for 4 h. RNA was extracted from cells and reverse transcribed. The expression level of , , , , and genes was studied by real-time qPCR and normalized to ACTB. It was found that hypoxia up-regulated the expression level of , , and genes in control U87MG cells, but and genes expression was down-regulated. The expression of , , and genes in glioblastoma cells with knockdown of ERN1 signaling protein was more sensitive to hypoxia, especially gene. At the same time, the expression of gene in ERN1 knockdown cells was resistant to hypoxia. The expression of gene, encoding the enzyme responsible for conversion of serine to glycine, showed similar negative sensitivity to hypoxia in both control and ERN1 knockdown glioblastoma cells. The results of the present study demonstrate that the expression of genes responsible for serine synthesis is sensitive to hypoxia in gene-specific manner and that ERN1 knockdown significantly modifies the impact of hypoxia on the expression of , , , and genes in glioblastoma cells and reflects the ERN1-mediated reprograming of hypoxic regulation at gene expression level.
Topics: Humans; Protein Serine-Threonine Kinases; Glioblastoma; Cell Hypoxia; Serine; Endoribonucleases; Hypoxia; Cell Line, Tumor; Gene Expression Regulation, Neoplastic
PubMed: 37823569
DOI: 10.2478/enr-2023-0028 -
Frontiers in Microbiology 2023has been isolated from extreme environments such as hot springs, volcanoes, as well as pasteurized acidic beverages, because it can tolerate extreme temperatures and...
INTRODUCTION
has been isolated from extreme environments such as hot springs, volcanoes, as well as pasteurized acidic beverages, because it can tolerate extreme temperatures and acidity. In our previous study, was isolated during the enrichment of methane oxidizing bacteria from Yellowstone Hot Spring samples.
METHODS
Physiological characterization and genomic exploration of two new isolates, AL01A and AL05G, are the main focus of this study to identify their potential relationships with a thermoacidophilic methanotroph () isolated from the same hot spring sediments.
RESULTS AND DISCUSSION
In the present study, both isolates showed optimal growth at pH 3.5 and 55°C, and contain ω-alicyclic fatty acids as a major lipid ( 60%) in the bacterial membrane. Genomic analysis of these strains revealed specific genes and pathways that the methanotroph genome does not have in the intermediary carbon metabolism pathway such as (phosphoserine aminotransferase), (phosphosulfolactate synthase), and DAK (glycerone kinase). Both strains were also found to contain transporter systems for extracellular sulfate (ABC transporter), suggesting that they could play an important role in sulfur metabolism in this extreme environment. Genomic analysis of vitamin metabolism revealed and are able to complement each other's nutritional deficiencies, resulting in a mutually beneficial relationship, especially in vitamin B(thiamin), B (niacin), and B (biotin) metabolism. These findings provide insights into the role of isolates in geothermal environments and their unique metabolic adaptations to these environments.
PubMed: 37822751
DOI: 10.3389/fmicb.2023.1232587 -
Bio-protocol Sep 2023
PubMed: 37818481
DOI: 10.21769/BioProtoc.4860