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Technology in Cancer Research &... 2023Serine metabolism is essential for tumor cells. Endogenous serine arises from de novo synthesis pathways. As the rate-limiting enzyme of this pathway, PHGDH is highly...
Serine metabolism is essential for tumor cells. Endogenous serine arises from de novo synthesis pathways. As the rate-limiting enzyme of this pathway, PHGDH is highly expressed in a variety of tumors including colon cancer. Therefore, targeted inhibition of PHGDH is an important strategy for anti-tumor therapy research. However, the specific gene expression and metabolic pathways regulated by PHGDH in colon cancer are still unclear. Our study was aimed to clarified the role of PHGDH in serine metabolism in colon cancer to provide new knowledge for in-depth understanding of serine metabolism and PHGDH function in colon cancer. In this study, we analyzed the gene expression and metabolic remodeling process of colon cancer cells (SW620) after targeted inhibition of PHGDH by gene transcriptomics and metabolomics. LC-MS analysis was performed in 293T cells to PHGDH gene transcription and protein post-translational modification under depriving exogenous serine. We found that amino acid transporters, amino acid metabolism, lipid synthesis related pathways compensation and other processes are involved in the response process after PHGDH inhibition. And ATF4 mediated the transcriptional expression of PHGDH under exogenous serine deficiency conditions. While LC-MS analysis of post-translational modification revealed that PHGDH produced changes in acetylation sites after serine deprivation that the K289 site was lost, and a new acetylation site K21was produced. Our study performed transcriptomic and metabolomic analysis by inhibiting PHGDH, thus clarifying the role of PHGDH in gene transcription and metabolism in colon cancer cells. The mechanism of high PHGDH expression in colon cancer cells and the acetylation modification that occurs in PHGDH protein were also clarified by serine deprivation. In our study, the role of PHGDH in serine metabolism in colon cancer was clarified by multi-omics analysis to provide new knowledge for in-depth understanding of serine metabolism and PHGDH function in colon cancer.
Topics: Humans; Phosphoglycerate Dehydrogenase; Multiomics; Proteins; Colonic Neoplasms; Serine; Cell Line, Tumor
PubMed: 36707056
DOI: 10.1177/15330338221145994 -
The Journal of General Physiology Apr 2023Phosphoregulation is ubiquitous in biology. Defining the functional roles of individual phosphorylation sites within a multivalent system remains particularly...
Phosphoregulation is ubiquitous in biology. Defining the functional roles of individual phosphorylation sites within a multivalent system remains particularly challenging. We have therefore applied a chemical biology approach to light-control the state of single candidate phosphoserines in the canonical anion channel CFTR while simultaneously measuring channel activity. The data show striking non-equivalency among protein kinase A consensus sites, which vary from <10% to >1,000% changes in channel activity upon phosphorylation. Of note, slow phosphorylation of S813 suggests that this site is rate-limiting to the full activation of CFTR. Further, this approach reveals an unexpected coupling between the phosphorylation of S813 and a nearby site, S795. Overall, these data establish an experimental route to understanding roles of specific phosphoserines within complex phosphoregulatory domains. This strategy may be employed in the study of phosphoregulation of other eukaryotic proteins.
Topics: Cystic Fibrosis Transmembrane Conductance Regulator; Phosphorylation; Anions
PubMed: 36695813
DOI: 10.1085/jgp.202213216 -
Molecular Therapy : the Journal of the... Mar 2023The role of Abraxas 2 (ABRO1 or KIAA0157), a component of the lysine63-linked deubiquitinating system, in the cardiomyocyte proliferation and myocardial regeneration is...
The role of Abraxas 2 (ABRO1 or KIAA0157), a component of the lysine63-linked deubiquitinating system, in the cardiomyocyte proliferation and myocardial regeneration is unknown. Here, we found that ABRO1 regulates cardiomyocyte proliferation and cardiac regeneration in the postnatal heart by targeting METTL3-mediated mA methylation of Psph mRNA. The deletion of ABRO1 increased cardiomyocyte proliferation in hearts and restored the heart function after myocardial injury. On the contrary, ABRO1 overexpression significantly inhibited the neonatal cardiomyocyte proliferation and cardiac regeneration in mouse hearts. The mechanism by which ABRO1 regulates cardiomyocyte proliferation mainly involved METTL3-mediated Psph mRNA methylation and CDK2 phosphorylation. In the early postnatal period, METTL3-dependent mA methylation promotes cardiomyocyte proliferation by hypermethylation of Psph mRNA and upregulating PSPH expression. PSPH dephosphorylates cyclin-dependent kinase 2 (CDK2), a positive regulator of cell cycle, at Thr14/Tyr15 and increases its activity. Upregulation of ABRO1 restricts METTL3 activity and halts the cardiomyocyte proliferation in the postnatal hearts. Thus, our study reveals that ABRO1 is an essential contributor in the cell cycle withdrawal and attenuation of proliferative response in the postnatal cardiomyocytes and could act as a potential target to accelerate cardiomyocyte proliferation and cardiac repair in the adult heart.
Topics: Animals; Mice; Animals, Newborn; Cell Proliferation; Heart; Myocardium; Myocytes, Cardiac; RNA, Messenger; Nuclear Matrix-Associated Proteins; Phosphoric Monoester Hydrolases
PubMed: 36639869
DOI: 10.1016/j.ymthe.2023.01.011 -
Nature Jan 2023Protein phosphorylation is one of the most widespread post-translational modifications in biology. With advances in mass-spectrometry-based phosphoproteomics, 90,000...
Protein phosphorylation is one of the most widespread post-translational modifications in biology. With advances in mass-spectrometry-based phosphoproteomics, 90,000 sites of serine and threonine phosphorylation have so far been identified, and several thousand have been associated with human diseases and biological processes. For the vast majority of phosphorylation events, it is not yet known which of the more than 300 protein serine/threonine (Ser/Thr) kinases encoded in the human genome are responsible. Here we used synthetic peptide libraries to profile the substrate sequence specificity of 303 Ser/Thr kinases, comprising more than 84% of those predicted to be active in humans. Viewed in its entirety, the substrate specificity of the kinome was substantially more diverse than expected and was driven extensively by negative selectivity. We used our kinome-wide dataset to computationally annotate and identify the kinases capable of phosphorylating every reported phosphorylation site in the human Ser/Thr phosphoproteome. For the small minority of phosphosites for which the putative protein kinases involved have been previously reported, our predictions were in excellent agreement. When this approach was applied to examine the signalling response of tissues and cell lines to hormones, growth factors, targeted inhibitors and environmental or genetic perturbations, it revealed unexpected insights into pathway complexity and compensation. Overall, these studies reveal the intrinsic substrate specificity of the human Ser/Thr kinome, illuminate cellular signalling responses and provide a resource to link phosphorylation events to biological pathways.
Topics: Humans; Phosphorylation; Protein Serine-Threonine Kinases; Serine; Substrate Specificity; Threonine; Proteome; Datasets as Topic; Phosphoproteins; Cell Line; Phosphoserine; Phosphothreonine
PubMed: 36631611
DOI: 10.1038/s41586-022-05575-3 -
Nature Communications Jan 2023The heptad repeats of the C-terminal domain (CTD) of RNA polymerase II (Pol II) are extensively modified throughout the transcription cycle. The CTD coordinates RNA...
The heptad repeats of the C-terminal domain (CTD) of RNA polymerase II (Pol II) are extensively modified throughout the transcription cycle. The CTD coordinates RNA synthesis and processing by recruiting transcription regulators as well as RNA capping, splicing and 3'end processing factors. The SPOC domain of PHF3 was recently identified as a CTD reader domain specifically binding to phosphorylated serine-2 residues in adjacent CTD repeats. Here, we establish the SPOC domains of the human proteins DIDO, SHARP (also known as SPEN) and RBM15 as phosphoserine binding modules that can act as CTD readers but also recognize other phosphorylated binding partners. We report the crystal structure of SHARP SPOC in complex with CTD and identify the molecular determinants for its specific binding to phosphorylated serine-5. PHF3 and DIDO SPOC domains preferentially interact with the Pol II elongation complex, while RBM15 and SHARP SPOC domains engage with writers and readers of mA, the most abundant RNA modification. RBM15 positively regulates mA levels and mRNA stability in a SPOC-dependent manner, while SHARP SPOC is essential for its localization to inactive X-chromosomes. Our findings suggest that the SPOC domain is a major interface between the transcription machinery and regulators of transcription and co-transcriptional processes.
Topics: Humans; Phosphorylation; Phosphoserine; RNA Polymerase II; RNA Processing, Post-Transcriptional; RNA Splicing; Transcription, Genetic; Protein Domains; DNA-Binding Proteins; RNA-Binding Proteins
PubMed: 36631525
DOI: 10.1038/s41467-023-35853-1 -
Frontiers in Physiology 2022Phosphorylation of serine residues has been recognized as a pivotal event in the evolution of mineralized tissues in many biological systems. During enamel development,...
Phosphorylation of serine residues has been recognized as a pivotal event in the evolution of mineralized tissues in many biological systems. During enamel development, the extracellular matrix protein amelogenin is most abundant and appears to be critical to the extreme high aspect ratios (length:width) of apatite mineral fibers reaching several millimeters in larger mammalian teeth. A 14-residue peptide (14P2, residues Gly8 to Thr21) was previously identified as a key sequence mediating amelogenin assembly formation, the domain also contains the native single phosphoserine residue (Ser16) of the full-length amelogenin. In this research, 14P2 and its phosphorylated form (p14P2) were investigated at pH 6.0 with various calcium and phosphate ion concentrations, indicating that both peptides could self-assemble into amyloid-like conformation but with differences in structural details. With calcium, the distance between P within the p14P2 self-assemblies is averaged to be 4.4 ± 0.2Å, determined by solid-state NMR P PITHIRDS-CT experiments. Combining with other experimental results, solid-state Nuclear Magnetic Resonance (SSNMR) suggests that the p14P2 self-assemblies are in parallel in-register -sheet conformation and divalent calcium ions most likely connect two adjacent peptide chains by binding to the phosphate group of Ser16 and the carboxylate of Glu18 side-chain. This study on the interactions between calcium ions and amelogenin-derived peptides provides insights on how amelogenin may self-assemble in the presence of calcium ions in early enamel development.
PubMed: 36589425
DOI: 10.3389/fphys.2022.1063970 -
Protein Science : a Publication of the... Feb 2023Amelogenin constitutes ~90% of the enamel matrix in the secretory stage of amelogenesis, a still poorly understood process that results in the formation of the hardest...
High-yield recombinant bacterial expression of C-, N-labeled, serine-16 phosphorylated, murine amelogenin using a modified third generation genetic code expansion protocol.
Amelogenin constitutes ~90% of the enamel matrix in the secretory stage of amelogenesis, a still poorly understood process that results in the formation of the hardest and most mineralized tissue in vertebrates-enamel. Most biophysical research with amelogenin uses recombinant protein expressed in Escherichia coli. In addition to providing copious amounts of protein, recombinant expression allows C- and N-labeling for detailed structural studies using NMR spectroscopy. However, native amelogenin is phosphorylated at one position, Ser-16 in murine amelogenin, and there is mounting evidence that Ser-16 phosphorylation is important. Using a modified genetic code expansion protocol we have expressed and purified uniformly C-, N-labeled murine amelogenin (pS16M179) with ~95% of the protein being correctly phosphorylated. Homogeneous phosphorylation was achieved using commercially available, enriched, C-, N-labeled media, and protein expression was induced with isopropyl β-D-1-thiogalactopyranoside at 310 K. Phosphoserine incorporation was verified from one-dimensional P NMR spectra, comparison of H- N HSQC spectra, Phos-tag SDS PAGE, and mass spectrometry. Phosphorus-31 NMR spectra for pS16M179 under conditions known to trigger amelogenin self-assembly into nanospheres confirm nanosphere models with buried N-termini. Lambda phosphatase treatment of these nanospheres results in the dephosphorylation of pS16M179, confirming that smaller oligomers and monomers with exposed N-termini are in equilibrium with nanospheres. Such C-, N-labeling of amelogenin with accurately encoded phosphoserine incorporation will accelerate biomineralization research to understand amelogenesis and stimulate the expanded use of genetic code expansion protocols to introduce phosphorylated amino acids into proteins.
Topics: Animals; Mice; Amelogenin; Escherichia coli; Genetic Code; Phosphoserine; Recombinant Proteins; Serine
PubMed: 36585836
DOI: 10.1002/pro.4560 -
BMC Medicine Dec 2022The pathogenesis of immunoglobulin G4-related disease (IgG4-RD) remains unclear. IgG4-RD often mimics other diseases, including pancreatic cancer (PC) and Sjogren's...
BACKGROUND
The pathogenesis of immunoglobulin G4-related disease (IgG4-RD) remains unclear. IgG4-RD often mimics other diseases, including pancreatic cancer (PC) and Sjogren's syndrome (SS), which may easily lead to misdiagnosis. This study was performed to explore the metabolite changes and potential biomarkers of IgG4-RD and other misdiagnosed diseases.
METHODS
Untargeted liquid chromatography-tandem mass spectrometry metabolomics profiling of plasma samples from a cohort comprising healthy controls (HCs) and patients with IgG4-RD (n = 87), PC (n = 33), and SS (n = 31) was performed. A random forest machine learning model was used to verify the relevance of the identified metabolites in the diagnosis of different diseases and the prediction of disease prognosis.
RESULTS
The ATP-binding cassette transporter pathway was found to be most closely related to IgG4-RD, which was significantly up-regulated in the IgG4-RD group than in all the matched groups. Five metabolites were proved to be valuable biomarkers for IgG4-RD. Caftaric acid, maltotetraose, D-glutamic acid, 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphoserine, and hydroxyproline were useful in distinguishing between IgG4-RD, PC, SS, and HC [area under the curve (AUC) = 1]. A combination of phenylalanine betaine, 1-(1z-hexadecenyl)-sn-glycero-3-phosphocholine, Pi 40:8, uracil, and N1-methyl-2-pyridone-5-carboxamide showed a moderate value in predicting relapse in patients with IgG4-RD (AUC = 0.8).
CONCLUSIONS
Our findings revealed the metabolite changes of IgG4-RD and provide new insights for deepening our understanding of IgG4-RD despite the lack of validation in external cohorts. Metabolomic biomarkers have significance in the clinical diagnosis and disease prognosis of IgG4-RD.
Topics: Humans; Sjogren's Syndrome; Immunoglobulin G4-Related Disease; Prognosis; Biomarkers; Pancreatic Neoplasms
PubMed: 36575511
DOI: 10.1186/s12916-022-02700-x -
Antioxidants (Basel, Switzerland) Dec 2022Both genetic and environmental factors increase risk for Parkinson's disease. Many of the known genetic factors influence α-synuclein aggregation or degradation,...
Both genetic and environmental factors increase risk for Parkinson's disease. Many of the known genetic factors influence α-synuclein aggregation or degradation, whereas most of the identified environmental factors produce oxidative stress. Studies using in vitro approaches have identified mechanisms by which oxidative stress can accelerate the formation of α-synuclein aggregates, but there is a paucity of evidence supporting the importance of these processes over extended time periods in brain. To assess this issue, we evaluated α-synuclein aggregates in brains of three transgenic mouse strains: hSyn mice, which overexpress human α-synuclein in neurons and spontaneously develop α-synuclein aggregates; EAAT3 mice, which exhibit a neuron-specific impairment in cysteine uptake and resultant neuron-selective chronic oxidative stress; and double-transgenic hSyn/EAAT3 mice. Aggregate formation was evaluated by quantitative immunohistochemistry for phosphoserine 129 α-synuclein and by an α-synuclein proximity ligation assay. Both methods showed that the double transgenic hSyn/EAAT3 mice exhibited a significantly higher α-synuclein aggregate density than littermate hSyn mice in each brain region examined. Negligible aggregate formation was observed in the EAAT3 mouse strain, suggesting a synergistic rather than additive interaction between the two genotypes. A similar pattern of results was observed in assessments of motor function: the pole test and rotarod test. Together, these observations indicate that chronic, low-grade neuronal oxidative stress promotes α-synuclein aggregate formation in vivo. This process may contribute to the mechanism by which environmentally induced oxidative stress contributes to α-synuclein pathology in idiopathic Parkinson's disease.
PubMed: 36552674
DOI: 10.3390/antiox11122466 -
Synthetic and Systems Biotechnology Mar 2023Studies on the mechanism of protein phosphorylation and therapeutic interventions of its related molecular processes are limited by the difficulty in the production of...
Studies on the mechanism of protein phosphorylation and therapeutic interventions of its related molecular processes are limited by the difficulty in the production of purpose-built phosphoproteins harboring site-specific phosphorylated amino acids or their nonhydrolyzable analogs. Here we address this limitation by customizing the cell-free protein synthesis (CFPS) machinery via chassis strain selection and orthogonal translation system (OTS) reconfiguration screening. The suited chassis strains and reconfigured OTS combinations with high orthogonality were consequently picked out for individualized phosphoprotein synthesis. Specifically, we synthesized the sfGFP protein and MEK1 protein with site-specific phosphoserine (O-pSer) or its nonhydrolyzable analog, 2-amino-4-phosphonobutyric acid (C-pSer). This study successfully realized building cell-free systems for site-specific incorporation of phosphonate mimics into the target protein. Our work lays the foundation for developing a highly expansible CFPS platform and the streamlined production of user-defined phosphoproteins, which can facilitate research on the physiological mechanism and potential interference tools toward protein phosphorylation.
PubMed: 36514487
DOI: 10.1016/j.synbio.2022.11.004