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Communications Biology Oct 2023Mycobacterium tuberculosis phosphoserine phosphatase MtSerB2 is of interest as a new antituberculosis target due to its essential metabolic role in L-serine biosynthesis...
Mycobacterium tuberculosis phosphoserine phosphatase MtSerB2 is of interest as a new antituberculosis target due to its essential metabolic role in L-serine biosynthesis and effector functions in infected cells. Previous works indicated that MtSerB2 is regulated through an oligomeric transition induced by L-Ser that could serve as a basis for the design of selective allosteric inhibitors. However, the mechanism underlying this transition remains highly elusive due to the lack of experimental structural data. Here we describe a structural, biophysical, and enzymological characterisation of MtSerB2 oligomerisation in the presence and absence of L-Ser. We show that MtSerB2 coexists in dimeric, trimeric, and tetrameric forms of different activity levels interconverting through a conformationally flexible monomeric state, which is not observed in two near-identical mycobacterial orthologs. This morpheein behaviour exhibited by MtSerB2 lays the foundation for future allosteric drug discovery and provides a starting point to the understanding of its peculiar multifunctional moonlighting properties.
Topics: Mycobacterium tuberculosis; Phosphoric Monoester Hydrolases; Catalysis
PubMed: 37817000
DOI: 10.1038/s42003-023-05402-z -
Archives of Biochemistry and Biophysics Aug 2023Mitogen activated protein kinase phosphoserine/threonine/tyrosine-binding protein (MK-STYX) is a dual specificity (DUSP) member of the protein tyrosine phosphatase...
Mitogen activated protein kinase phosphoserine/threonine/tyrosine-binding protein (MK-STYX) is a dual specificity (DUSP) member of the protein tyrosine phosphatase family. It is a pseudophosphatase, which lacks the essential amino acids histidine and cysteine in the catalytic active signature motif (HCXR). We previously reported that MK-STYX interacts with G3BP1 [Ras-GAP (GTPase-activating protein) SH3 (Src homology 3) domain-binding-1] and reduces stress granules, stalled mRNA. To determine how MK-STYX reduces stress granules, truncated domains, CH2 (cell division cycle 25 phosphatase homology 2) and DUSP, of MK-STYX were used. Wild-type MK-STYX and the DUSP domain significantly decreased stressed granules that were induced by sodium arsenite, in which G3BP1 (a stress granule nucleator) was used as the marker. In addition, HEK/293 and HeLa cells co-expressing G3BP1-GFP and mCherry-MK-STYX, mCherry-MK-STYX-CH2, mCherry-MK-STYX-DUSP or mCherry showed that stress granules were significantly decreased in the presence of wild-type MK-STYX and the DUSP domain of MK-STYX. Further characterization of these dynamics in HeLa cells showed that the CH2 domain increased the number of stress granules within a cell, relative to wild-type and DUSP domain of MK-STYX. To further analyze the interaction of G3BP1 and the domains of MK-STYX, coimmunoprecipitation experiments were performed. Cells co-expressing G3BP1-GFP and mCherry, mCherry-MK-STYX, mCherry-MK-STYX-CH2, or mCherry-MK-STYX-DUSP demonstrated that the DUSP domain of MK-STYX interacts with both G3BP1-GFP and endogenous G3BP1, whereas the CH2 domain of MK-STYX did not coimmunoprecipitate with G3BP1. In addition, G3BP1 tyrosine phosphorylation, which is required for stress granule formation, was decreased in the presence of wild-type MK-STYX or the DUSP domain but increased in the presence of CH2. These data highlight a model for how MK-STYX decreases G3BP1-induced stress granules. The DUSP domain of MK-STYX interacts with G3BP1 and negatively alters its tyrosine phosphorylation- decreasing stress granule formation.
Topics: Humans; DNA Helicases; HeLa Cells; Intracellular Signaling Peptides and Proteins; Poly-ADP-Ribose Binding Proteins; RNA Helicases; RNA Recognition Motif Proteins; Stress Granules; Tyrosine
PubMed: 37516290
DOI: 10.1016/j.abb.2023.109702 -
International Journal of Molecular... Nov 2023Apoptotic cells' phosphoserine (PS) groups have a significant immunosuppressive effect. They inhibit proinflammatory signals by interacting with various immune cells,...
BACKGROUND
Apoptotic cells' phosphoserine (PS) groups have a significant immunosuppressive effect. They inhibit proinflammatory signals by interacting with various immune cells, including macrophages, dendritic cells, and CD4 cells. Previously, we synthesized PS-group-immobilized polymers and verified their immunomodulatory effects. Despite its confirmed immunomodulatory potential, the PS group has not been considered as a payload for antibody-drug conjugates (ADCs) in a targeted anti-inflammatory approach.
AIM
We conducted this research to introduce an apoptotic-cell-inspired antibody-drug conjugate for effective immunomodulation.
METHOD
Poly(2-hydroxyethyl methacrylate--2-methacryloyloxyethyl phosphorylserine) (p(HEMA--MPS)) was synthesized as a payload using RAFT polymerization, and goat anti-mouse IgG was selected as a model antibody, which was conjugated with the synthesized p(HEMA--MPS) via 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide/-Hydroxysuccinimide (EDC/NHS) reaction. The antibody-binding affinity, anti-inflammatory potential, and cytotoxicity measurements were evaluated.
RESULTS
We successfully synthesized ADCs with a significant anti-inflammatory effect and optimized the antibody-polymer ratio to achieve the highest antibody-binding affinity.
CONCLUSION
We successfully introduced p(HEMA--MPS) to IgG without decreasing the anti-inflammatory potential of the polymer while maintaining its targeting ability. We suggest that the antibody-polymer ratio be appropriately adjusted for effective therapy. In the future, this technology can be applied to therapeutic antibodies, such as Tocilizumab or Abatacept.
Topics: Immunoconjugates; Polymers; Immunoglobulin G; Anti-Inflammatory Agents
PubMed: 38003225
DOI: 10.3390/ijms242216036 -
BioRxiv : the Preprint Server For... Jun 2024Protein post-translational modifications, such as phosphorylation, are important regulatory signals for diverse cellular functions. In particular, intrinsically...
UNLABELLED
Protein post-translational modifications, such as phosphorylation, are important regulatory signals for diverse cellular functions. In particular, intrinsically disordered protein regions (IDRs) are subject to phosphorylation as a means to modulate their interactions and functions. Toward understanding the relationship between phosphorylation in IDRs and specific functional outcomes, we must consider how phosphorylation affects the IDR conformational ensemble. Various experimental techniques are suited to interrogate the features of IDR ensembles; molecular simulations can provide complementary insights and even illuminate ensemble features that may be experimentally inaccessible. Therefore, we sought to expand the tools available to study phosphorylated IDRs by all-atom Monte Carlo simulations. To this end, we implemented parameters for phosphoserine (pSer) and phosphothreonine (pThr) into the OPLS version of the continuum solvent model, ABSINTH, and assessed their performance in all-atom simulations compared to published findings. We simulated short (< 20 residues) and long (> 80 residues) phospho-IDRs that, collectively, survey both local and global phosphorylation-induced changes to the ensemble. Our simulations of four well-studied phospho-IDRs show near-quantitative agreement with published findings for these systems via metrics including changes to radius of gyration, transient helicity, and persistence length. We also leveraged the inherent advantage of sequence control in molecular simulations to explore the conformational effects of diverse combinations of phospho-sites in two multi-phosphorylated IDRs. Our results support and expand on prior observations that connect phosphorylation to changes in the IDR conformational ensemble. Herein, we describe phosphorylation as a means to alter sequence chemistry, net charge and charge patterning, and intramolecular interactions, which can collectively modulate the local and global IDR ensemble features.
SIGNIFICANCE
Spatially and temporally controlled phosphorylation in disordered protein regions is critical to many facets of protein function and broader cellular health. Intrinsically disordered protein regions (IDRs) are overrepresented as targets of phosphorylation, but the structural and functional consequences of such modifications remain elusive for many systems. Toward rigorous modeling of phosphorylated IDRs using all-atom simulations, we present new parameters for phosphoserine and phosphothreonine for the ABSINTH implicit solvent paradigm. Through the study of four example phospho-IDRs, we demonstrate excellent agreement between our phospho-IDR simulations and published datasets.
PubMed: 38915510
DOI: 10.1101/2024.06.10.598315 -
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 -
The Oncologist Mar 2024To date, no study has systematically explored the potential role of serum metabolites and lipids in the diagnosis of small cell lung cancer (SCLC). Therefore, we aimed...
BACKGROUND
To date, no study has systematically explored the potential role of serum metabolites and lipids in the diagnosis of small cell lung cancer (SCLC). Therefore, we aimed to conduct a case-cohort study that included 191 cases of SCLC, 91 patients with lung adenocarcinoma, 82 patients with squamous cell carcinoma, and 97 healthy controls.
METHODS
Metabolomics and lipidomics were applied to analyze different metabolites and lipids in the serum of these patients. The SCLC diagnosis model (d-model) was constructed using an integrated machine learning technology and a training cohort (n = 323) and was validated in a testing cohort (n=138).
RESULTS
Eight metabolites, including 1-mristoyl-sn-glycero-3-phosphocholine, 16b-hydroxyestradiol, 3-phosphoserine, cholesteryl sulfate, D-lyxose, dioctyl phthalate, DL-lactate and Leu-Phe, were successfully selected to distinguish SCLC from controls. The d-model was constructed based on these 8 metabolites and showed improved diagnostic performance for SCLC, with the area under curve (AUC) of 0.933 in the training cohort and 0.922 in the testing cohort. Importantly, the d-model still had an excellent diagnostic performance after adjusting the stage and related clinical variables and, combined with the progastrin-releasing peptide (ProGRP), showed the best diagnostic performance with 0.975 of AUC for limited-stage patients.
CONCLUSION
This study is the first to analyze the difference between metabolomics and lipidomics and to construct a d-model to detect SCLC using integrated machine learning. This study may be of great significance for the screening and early diagnosis of SCLC patients.
Topics: Humans; Small Cell Lung Carcinoma; Lung Neoplasms; Carcinoma, Non-Small-Cell Lung; Cohort Studies; Biomarkers, Tumor; Lipids
PubMed: 37706531
DOI: 10.1093/oncolo/oyad261 -
Scientific Reports May 2024The non-essential amino acid L-serine is involved in a number of metabolic pathways and in the brain its level is largely due to the biosynthesis from the glycolytic...
The non-essential amino acid L-serine is involved in a number of metabolic pathways and in the brain its level is largely due to the biosynthesis from the glycolytic intermediate D-3-phosphoglycerate by the phosphorylated pathway (PP). This cytosolic pathway is made by three enzymes proposed to generate a reversible metabolon named the "serinosome". Phosphoserine phosphatase (PSP) catalyses the last and irreversible step, representing the driving force pushing L-serine synthesis. Genetic defects of the PP enzymes result in strong neurological phenotypes. Recently, we identified the homozygous missense variant [NM_004577.4: c.398A > G p.(Asn133Ser)] in the PSPH, the PSP encoding gene, in two siblings with a neurodevelopmental syndrome and a myelopathy. The recombinant Asn133Ser enzyme does not show significant alterations in protein conformation and dimeric oligomerization state, as well as in enzymatic activity and functionality of the reconstructed PP. However, the Asn133Ser variant is less stable than wild-type PSP, a feature also apparent at cellular level. Studies on patients' fibroblasts also highlight a strong decrease in the level of the enzymes of the PP, a partial nuclear and perinuclear localization of variant PSP and a stronger perinuclear aggregates formation. We propose that these alterations contribute to the formation of a dysfunctional serinosome and thus to the observed reduction of L-serine, glycine and D-serine levels (the latter playing a crucial role in modulating NMDA receptors). The characterization of patients harbouring the Asn133Ser PSP substitution allows to go deep into the molecular mechanisms related to L-serine deficit and to suggest treatments to cope with the observed amino acids alterations.
Topics: Humans; Serine; Mutation, Missense; Phosphoric Monoester Hydrolases; Fibroblasts; Male; Neurodevelopmental Disorders; Female
PubMed: 38816452
DOI: 10.1038/s41598-024-63164-y -
Molecular Oncology Apr 2024Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a limited number of known driver mutations but considerable cancer cell heterogeneity....
Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a limited number of known driver mutations but considerable cancer cell heterogeneity. Phosphoproteomics provides a direct read-out of aberrant signaling and the resultant clinically relevant phenotype. Mass spectrometry (MS)-based proteomics and phosphoproteomics were applied to 42 PDAC tumors. Data encompassed over 19 936 phosphoserine or phosphothreonine (pS/T; in 5412 phosphoproteins) and 1208 phosphotyrosine (pY; in 501 phosphoproteins) sites and a total of 3756 proteins. Proteome data identified three distinct subtypes with tumor intrinsic and stromal features. Subsequently, three phospho-subtypes were apparent: two tumor intrinsic (Phos1/2) and one stromal (Phos3), resembling known PDAC molecular subtypes. Kinase activity was analyzed by the Integrative iNferred Kinase Activity (INKA) scoring. Phospho-subtypes displayed differential phosphorylation signals and kinase activity, such as FGR and GSK3 activation in Phos1, SRC kinase family and EPHA2 in Phos2, and EGFR, INSR, MET, ABL1, HIPK1, JAK, and PRKCD in Phos3. Kinase activity analysis of an external PDAC cohort supported our findings and underscored the importance of PI3K/AKT and ERK pathways, among others. Interestingly, unfavorable patient prognosis correlated with higher RTK, PAK2, STK10, and CDK7 activity and high proliferation, whereas long survival was associated with MYLK and PTK6 activity, which was previously unknown. Subtype-associated activity profiles can guide therapeutic combination approaches in tumor and stroma-enriched tissues, and emphasize the critical role of parallel signaling pathways. In addition, kinase activity profiling identifies potential disease markers with prognostic significance.
PubMed: 38650175
DOI: 10.1002/1878-0261.13625 -
MicroPublication Biology 2024Abnormal synaptic aggregation of α-synuclein is linked to cognitive deficits in Parkinson's disease (PD). While the impacts of excess α-synuclein on synaptic function...
Abnormal synaptic aggregation of α-synuclein is linked to cognitive deficits in Parkinson's disease (PD). While the impacts of excess α-synuclein on synaptic function are well established, comparatively less is known about the effects on local mitochondria. Here, we examined morphological features of synaptic mitochondria treated with wild type (WT) or phosphoserine 129 (pS129) α-synuclein, a variant with prominent synaptic accumulation in PD. Acute introduction of pS129 α-synuclein to lamprey synapses caused an activity-dependent swelling and bursting of mitochondria, which did not occur with WT α-synuclein. These pS129-induced effects on mitochondria likely contribute to the synaptic deficits observed in PD.
PubMed: 38854632
DOI: 10.17912/micropub.biology.001206 -
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