YLMY Tyrosine Residue within the Cytoplasmic Tail of Newcastle Disease Virus Fusion Protein Regulates Its Surface Expression to Modulate Viral Budding and Pathogenicity

The amino-terminal cytoplasmic domains of paramyxovirus fusion glycoproteins include trafficking signals that influence protein processing and cell surface expression. This study clarified that tyrosine residues at different positions in the YLMY motif in the cytoplasmic region of the F protein regulate F protein transportation, thereby affecting viral replication and pathogenicity.

Role of PhOH and Tyrosine in Selective Oxidation of Hydrocarbons

Earlier, we established that nickel or iron heteroligand complexes, which include PhOH (nickel complexes) or tyrosine residue (nickel or iron complexes), are not only hydrocarbon oxidation catalysts (in the case of PhOH), but also simulate the active centers of enzymes (PhOH, tyrosine). The AFM method established the self-organization of nickel or iron heteroligand complexes, which included tyrosine residue or PhOH, into supramolecular structures on a modified silicon surface. Supramolecular structures were formed as a result of H-bonds and other non-covalent intermolecular interactions and, to a certain extent, reflected the structures involved in the mechanisms of reactions of homogeneous and enzymatic catalysis. Using the AFM method, we obtained evidence at the model level in favor of the involvement of the tyrosine fragment as one of the possible regulatory factors in the functioning of Ni(Fe)ARD dioxygenases or monooxygenases of the family of cytochrome P450. The principles of actions of these oxygenases were used to create highly efficient catalytic systems for the oxidation of hydrocarbons.

Functionalization of Human Serum Albumin by Tyrosine Click

Human serum albumin (HSA) is a promising drug delivery carrier. Although covalent modification of Cys34 is a well-established method, it is desirable to develop a novel covalent modification method that targets residues other than cysteine to introduce multiple functions into a single HSA molecule. We developed a tyrosine-selective modification of HSA. Three tyrosine selective modification methods, hemin-catalyzed, horseradish peroxidase (HRP)-catalyzed, and laccase-catalyzed reactions were performed, and the modification efficiencies and modification sites of the modified HSAs obtained by these methods were evaluated and compared. We found that the laccase-catalyzed method could efficiently modify the tyrosine residue of HSA under mild reaction conditions without inducing oxidative side reactions. An average of 2.2 molecules of functional groups could be introduced to a single molecule of HSA by the laccase method. Binding site analysis using mass spectrometry suggested Y84, Y138, and Y401 as the main modification sites. Furthermore, we evaluated binding to ibuprofen and found that, unlike the conventional lysine residue modification, the inhibition of drug binding was minimal. These results suggest that tyrosine-residue selective chemical modification is a promising method for covalent drug attachment to HSA.

A Novel Reagent for Radioiodine Labeling of New Chemical Entities (NCEs) and Biomolecules

Radioiodine labeling of peptides and proteins is routinely performed by using various oxidizing agents such as Chloramine T, Iodobeads, and Iodogen reagent and radioactive iodide (I−), although some other oxidizing agents were also investigated. The main objective of the present study was to develop and test a novel reagent, inorganic monochloramine (NH2Cl), for radioiodine labeling of new chemical entities and biomolecules which is cost-effective, easy to make and handle, and is selective to label amino acids, peptides, and proteins. The data presented in this report demonstrate that the yields of the non-radioactive iodine labeling reactions using monochloramine are >70% for an amino acid (tyrosine) and a cyclic peptide (cyclo Arg-Gly-Asp-d-Tyr-Lys, cRGDyK). No evidence of the formation of N-chloro derivatives in cRGDyK was observed, suggesting that the reagent is selective in iodinating the tyrosine residue in the biomolecules. The method was successfully translated into radioiodine labeling of amino acid, a peptide, and a protein, Bovine Serum Albumin (BSA).

Serine residues 726 and 780 have nonredundant roles regulating STAT5a activity in luminal breast cancer

In breast cancer, prolactin-induced activation of the transcription factor STAT5a results from the phosphorylation of STAT5a tyrosine residue 694. However, its role in mammary oncogenesis remains an unsettled debate as STAT5a exhibits functional dichotomy with both pro-differentiative and pro-proliferative target genes. Phosphorylation of STAT5a serine residues, S726 and S780, may regulate STAT5a in such a way to underlie this duality. Given hematopoiesis studies showing phospho-serine STAT5a as necessary for transformation, we hypothesized that serine phosphorylation regulates STAT5a activity to contribute to its role in mammary oncogenesis, specifically in luminal breast cancer. Here, phosphorylation of S726-, S780-, and Y694-STAT5a in response to prolactin in MCF7 luminal breast cancer cells was investigated with STAT5a knockdown and rescue with Y694F-, S726A-, or S780A-STAT5a, where the phospho-sites were mutated. RNA-sequencing and subsequent Ingenuity Pathway Analysis predicted that loss of each phospho-site differentially affected both prolactin-induced gene expression as well as functional pathways of breast cancer (e.g. cell survival, proliferation, and colony formation). In vitro studies of anchorage-independent growth and proliferation confirmed distinct phenotypes: whereas S780A-STAT5a decreased clonogenicity, S726A-STAT5a decreased proliferation in response to prolactin compared to wild type STAT5a. Collectively, these studies provide novel insights into STAT5a activation in breast cancer pathogenesis.

Substance P Serves as a Balanced Agonist for MRGPRX2 and a Single Tyrosine Residue Is Required for β-arrestin Recruitment and Receptor Internalization

The neuropeptide substance P (SP) mediates neurogenic inflammation and pain and contributes to atopic dermatitis in mice through the activation of mast cells (MCs) via Mas-related G protein-coupled receptor (GPCR)-B2 (MrgprB2, human ortholog MRGPRX2). In addition to G proteins, certain MRGPRX2 agonists activate an additional signaling pathway that involves the recruitment of β-arrestins, which contributes to receptor internalization and desensitization (balanced agonists). We found that SP caused β-arrestin recruitment, MRGPRX2 internalization, and desensitization. These responses were independent of G proteins, indicating that SP serves as a balanced agonist for MRGPRX2. A tyrosine residue in the highly conserved NPxxY motif contributes to the activation and internalization of many GPCRs. We have previously shown that Tyr279 of MRGPRX2 is essential for G protein-mediated signaling and degranulation. To assess its role in β-arrestin-mediated MRGPRX2 regulation, we replaced Tyr279 in the NPxxY motif of MRGPRX2 with Ala (Y279A). Surprisingly, we found that, unlike the wild-type receptor, Y279A mutant of MRGPRX2 was resistant to SP-induced β-arrestin recruitment and internalization. This study reveals the novel findings that activation of MRGPRX2 by SP is regulated by β-arrestins and that a highly conserved tyrosine residue within MRGPRX2’s NPxxY motif contributes to both G protein- and β-arrestin-mediated responses.

An Azo Coupling-Based Chemoproteomic Approach to Systematically Profile the Tyrosine Reactivity in the Human Proteome

<p>The tyrosine residue of proteins participates in a wide range of activities including enzymatic catalysis, protein-protein interaction, and protein-ligand binding. However, the functional annotation of the tyrosine residues on a large scale is still very challenging. Here, we report a novel method integrating azo coupling, bioorthogonal chemistry, and multiplexed proteomics to globally investigate the tyrosine reactivity in the human proteome. Based on the azo-coupling reaction between aryl diazonium salt and the tyrosine residue, the two different probes were evaluated, and the probe with the best performance was employed to specifically target the tyrosine residues. After the reaction, tagged tyrosine containing peptides were selectively enriched using bioorthogonal chemistry, and a small tag on the peptides from the cleavage perfectly fits for site-specific analysis by MS. Coupling with multiplexed proteomics, we quantified over 5,000 tyrosine sites in MCF7 cells and these quantified sites displayed a wide range of reactivity. The tyrosine residues with high reactivity were found on functionally and structurally diverse proteins, including those with the catalytic activity and binding property. This method can be extensively applied to advance our understanding of protein functions and facilitate the development of covalent drugs to regulate protein activity.</p>

An Azo Coupling-Based Chemoproteomic Approach to Systematically Profile the Tyrosine Reactivity in the Human Proteome

<p>The tyrosine residue of proteins participates in a wide range of activities including enzymatic catalysis, protein-protein interaction, and protein-ligand binding. However, the functional annotation of the tyrosine residues on a large scale is still very challenging. Here, we report a novel method integrating azo coupling, bioorthogonal chemistry, and multiplexed proteomics to globally investigate the tyrosine reactivity in the human proteome. Based on the azo-coupling reaction between aryl diazonium salt and the tyrosine residue, the two different probes were evaluated, and the probe with the best performance was employed to specifically target the tyrosine residues. After the reaction, tagged tyrosine containing peptides were selectively enriched using bioorthogonal chemistry, and a small tag on the peptides from the cleavage perfectly fits for site-specific analysis by MS. Coupling with multiplexed proteomics, we quantified over 5,000 tyrosine sites in MCF7 cells and these quantified sites displayed a wide range of reactivity. The tyrosine residues with high reactivity were found on functionally and structurally diverse proteins, including those with the catalytic activity and binding property. This method can be extensively applied to advance our understanding of protein functions and facilitate the development of covalent drugs to regulate protein activity.</p>