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Reversible phosphorylation is the most widespread posttranslational protein modification, playing regulatory role in almost every aspect of cell life. The majority of protein phosphorylation research has been focused on serine, threonine and tyrosine that form acid-stable phosphomonoesters. However, protein histidine, arginine and lysine residues also may undergo phosphorylation to yield acid-labile phosphoramidates, most often remaining undetected in conventional studies of protein phosphorylation. It has become increasingly evident that acid-labile protein phosphorylations play important roles in signal transduction and other regulatory processes. Beside acting as high-energy intermediates in the transfer of the phosphoryl group from donor to acceptor molecules, phosphohistidines have been found so far in histone H4, heterotrimeric G proteins, ion channel KCa3.1, annexin 1, P-selectin and myelin basic protein, as well as in recombinant thymidylate synthase expressed in bacterial cells. Phosphoarginines occur in histone H3, myelin basic protein and capsidic protein VP12 of granulosis virus, whereas phospholysine in histone H1. This overview of the current knowledge on phosphorylation of protein basic amino-acid residues takes into consideration its proved or possible roles in cell functioning. Specific requirements of studies on acid-labile protein phosphorylation are also indicated.
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... Protein phosphorylation, a post-translational modification, is an important process in human health and disease controlled by the coordinated activity of enzymes known as kinases and phosphatases, which add and remove phosphate groups from proteins, respectively [1]. The regulation of physiological processes including gene expression, cell proliferation and differentiation, cell cycle arrest, and apoptosis largely depends on the phosphorylation of serine, threonine, and tyrosine residues in eukaryotic proteins [2]. ...
... Fluorescence Intensity = (I MAX − I min )/ 1 + e (IC 50 −x 0 )/dx + I min (2) where I MAX means the fluorescence intensity of maximal in experiment, I min means the fluorescence intensity of minimal in experiment, and x 0 means the concentration of ethyl-3,4-dephostatin when it has I min . In the fitted curve of dPIA, R 2 value was 0.9916. ...
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We report analysis of phosphatase activity and inhibition on droplet-based microfluidic chips. Phosphatases are such attractive potential drug targets because abnormal phosphatase activity has been implicated in a variety of diseases including cancer, neurological disorders, diabetes, osteoporosis, and obesity. So far, several methods for assessing phosphatase activity have been reported. However, they require a large sample volume and additional chemical modifications such as fluorescent dye conjugation and nanomaterial conjugation, and are not cost-effective. In this study, we used an artificial phosphatase substrate 3-O-methylfluorescein phosphate as a fluorescent reporter and dual specificity phosphatase 22. Using these materials, the phosphatase assay was performed from approximately 340.4 picoliter (pL) droplets generated at a frequency of ~40 hertz (Hz) in a droplet-based microfluidic chip. To evaluate the suitability of droplet-based platform for screening phosphatase inhibitors, a dose–response inhibition study was performed with ethyl-3,4-dephostatin and the half-maximal inhibitory concentration (IC50) was calculated as 5.79 ± 1.09 μM. The droplet-based results were compared to microplate-based experiments, which showed agreement. The droplet-based phosphatase assay proposed here is simple, reproducible, and generates enormous data sets within the limited sample and reagent volumes.
... As many as 30% of eukaryotic proteins may be phosphorylated and many of these are phosphorylated at several sites [53,54]. This basic picture has, however, had to be revised following discovery of additional phosphorylated amino acids, including Arg, Lys and Asp [55][56][57][58]. It has been suggested that phosphorylation of Ser, Thr and Tyr residues may have evolved from the negatively charged Asp and Glu residues (mutations of Asp or Glu to Tyr require only one G to U substitution) and this could explain why, in some cases, phosphorylation can activate proteins, having effectively evolved from a permanently on state to a switchable state, using phosphorylation as the control mechanism [50]. ...
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Phosphate and sulfate groups are integral to energy metabolism and introduce negative charges into biological macromolecules. One purpose of such modifications is to elicit precise binding/activation of protein partners. The physico-chemical properties of the two groups, while superficially similar, differ in one important respect—the valency of the central (phosphorus or sulfur) atom. This dictates the distinct properties of their respective esters, di-esters and hence their charges, interactions with metal ions and their solubility. These, in turn, determine the contrasting roles for which each group has evolved in biological systems. Biosynthetic links exist between the two modifications; the sulfate donor 3′-phosphoadenosine-5′-phosphosulfate being formed from adenosine triphosphate (ATP) and adenosine phosphosulfate, while the latter is generated from sulfate anions and ATP. Furthermore, phosphorylation, by a xylosyl kinase (Fam20B, glycosaminoglycan xylosylkinase) of the xylose residue of the tetrasaccharide linker region that connects nascent glycosaminoglycan (GAG) chains to their parent proteoglycans, substantially accelerates their biosynthesis. Following observations that GAG chains can enter the cell nucleus, it is hypothesized that sulfated GAGs could influence events in the nucleus, which would complete a feedback loop uniting the complementary anionic modifications of phosphorylation and sulfation through complex, inter-connected signalling networks and warrants further exploration.
... e trinucleotide bloc mutation adds a lysine instead of glycine (-SRG-to -SKR-) in the 202-204 position of the motif. Together with arginine and serine, this lysine in the motif can also be a target of phosphorylation in the infected cells [18]. It has also been shown that polyphosphorylation in PASK (polyacidic serine and lysine)-rich cluster negatively changes the function of certain enzymes [19]. is information suggests that the bloc mutation might have profound impacts on the pathogenicity of SARS-CoV-2. ...
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... Hydroxylated amino acids such as serine (75%-80%), threonine (15%-20%), and tyrosine (1%-5%) majorly participate in phosphorylation of proteins (Champion et al., 2004). Apart from these hydroxylated amino acids, histidine and aspartic acid can also phosphorylate (Ciesla, Frączyk, & Rode, 2011). The protein kinases and phosphatases are the regulators of phosphorylation and these gene families are abundant in plants. ...
... Methylation is a signi cant PTM because lysine residues in proteins are methylated, in uencing their interaction with DNA and gene expression. e molecular switch is another protein regulatory mechanism that adjusts the protein to perform such as protein structure conformational changes, protein activation and deactivation, and signal transduction pathways [59][60][61][62]. ...
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... Because lysine residues in certain proteins are methylated, this changes their interaction with DNA and regulates gene expression, methylation is a key PTM. Another essential method for protein regulation is the molecular switch, which adapts the protein to execute functions such as protein structure conformational changes, protein activation and deactivation, and signal transduction pathways (Deutscher and Saier, 2005;Puttick et al., 2008;Cieśla et al., 2011;Sawicka and Seiser, 2014). Among these predictions, the ConSurf Conservation profile shows that rs137 6162684 is highly conserved, exposed, and functionally relevant, indicating its relevance. ...
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... For eukaryotic protein kinases, the main phosphorylatable sites are serine (Ser), threonine (Thr) and tyrosine (Tyr); whereas in prokaryotes, the main sites are aspartic acid (Asp), glutamic acid (Glu) and histidine (His) [1]. Other phosphorylatable amino acid residues include arginine (Arg), lysine (Lys) and cysteine (Cys) [2]. The human genome encodes~518 kinase genes which Abbreviations ALK, anaplastic lymphoma kinase; ALL, acute lymphocytic leukaemia; BCR, B-cell receptor; BTK, Bruton's tyrosine kinase; CDK, cyclindependent kinase; CETSA, cellular thermal shift assay; CML, chronic myelogenous leukaemia; CSF1R, colony stimulating factor 1 receptor; DIA, data-independent acquisition; EGFR, epidermal growth factor receptor; EMT, epithelial-to-mesenchymal transition; FDA, Food and Drug Administration; FGFR, fibroblast growth factor receptor; FLT3, FMS-like tyrosine kinase 3 receptor; HCC, hepatocellular carcinoma; HGF, hepatocyte growth factor; JAK, Janus kinase; KI, kinase inhibitor; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase kinase; MS, mass spectrometry; mTOR, mechanistic target of rapamycin; NRY, non-receptor protein-tyrosine kinase; NSCLC, nonsmall cell lung cancer; PDGFR, platelet-derived growth factor beta-receptor; PI3K, phosphoinositide 3-kinases; PTM, post-translational modification; RCC, renal cell carcinoma; RET, rearranged during transfection; RTK, receptor protein-tyrosine kinase; S/T, protein-serine/ threonine kinase; Syk, spleen tyrosine kinase; T/Y, dual specificity protein kinase; TKI, tyrosine kinase inhibitor; TPP, thermal proteome profiling; TRK, tropomyosin receptor kinases; VEGFR, vascular endothelial growth factor receptor. ...
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Chapter
Post-translational modifications (PTMs) regulate complex biological processes through the modulation of protein activity, stability, and localization. Insights into the specific modification type and localization within a protein sequence can help ascertain functional significance. Computational models are increasingly demonstrated to offer a low-cost, high-throughput method for comprehensive PTM predictions. Algorithms are optimized using existing experimental PTM data, thus accurate prediction performance relies on the creation of robust datasets. Herein, advancements in mass spectrometry-based proteomics technologies to maximize PTM coverage are reviewed. Further, requisite experimental validation approaches for PTM predictions are explored to ensure that follow-up mechanistic studies are focused on accurate modification sites.
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Novel N-15-isotope enriched potassium and diammonium thiophosphoramidates were synthesized and their spectroscopic properties, along with reactivity towards several compounds, including histidine, thymidine, glucose and 2-deoxyribose are presented. The application of quantum mechanical DFT calculations for estimation of P-31 NMR chemical shifts for several thiophosphoramidate ions and its derivatives are also discussed.
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Article
This chapter discusses protein histidine phosphorylation, protein histidine kinases (HKs), and protein histidine phosphatases (PHPs) and describes their roles in bacteria, fungi, plants, and mammalian cells. Current methods of detection of phosphohistidine in proteins are also described, including HK assays, phosphoamino acid analysis, and approaches involving mass spectrometric (MS) methods. The HKs in bacteria, fungi, and plants are two-component protein systems composed of two major functional parts: the HK and the response regulator protein. The receptor or sensor protein that has the HK activity exists in the cell membrane as a preformed dimer or in some cases, may dimerize in response to the extracellular signal. One of the simplest ways to confirm that the site of phosphorylation in a phosphoprotein is a particular amino acid is to perform phosphoamino acid analysis. In this process, the protein substrate is phosphorylated using a nucleotide in which the γ-phosphate is radiolabelled, commonly with 32P, resulting in the formation of a [32P] phosphoprotein product. Phosphohistidine in proteins is directly detected using 31P and 1H nuclear magnetic resonance (NMR).
Article
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