Introduction – Chemical Tools for Histidine Phosphorylation
Protein Histidine Phosphorylation
Biological Function of Protein Histidine Phosphorylation
- Two-component System (TCS)
- Phosphohistidine Phosphatase in TCSs
- Nucleoside Diphosphate Kinase (NDPK) in Eukaryotes
- Ca 2+ -activated K + Channel KCa3.1
- Histone H4
- Summary of Known Phosphohistidine Proteins in Biology
The best-known histidine kinase proteins so far are the two isoforms of NDPK (Nucleoside Diphosphate Kinase); NDPK-A (Nm23H1)7) and NDPK-B (NM23H2)8,9). NDPK-A has been reported to phosphorylate annexin 1 at histidine residue 10,11), and NDPK-B is responsible for His phosphorylation in the KCa3.1 potassium channel and its activation. 9,12) In addition, NDPK-B phosphorylates His266 in the β-subunit (Gβ) of trimeric G-proteins and assists in the activation of Gs by transferring the phosphoryl group to GDP and generating GTP, which binds to and activates the alpha-subunit of Gs.8 ,13). The Ca2+-activated K+ channel KCa3.1, which consists of 370 amino acids, is responsible for Ca2+ influx leading to activation of T cells and B cells.9,15) KCa3.1 has been found in blood cells. , epithelial and smooth muscle cells, and its function is to exchange cytosolic Ca2+ for K+ and thereby control the membrane potential for K+ equilibrium. This change in membrane potential can affect various physiological responses in many cell types, such as mitogenic activation of T-lymphocytes and regulation of cell volume in erythrocytes, Cl secretion in exocrine epithelial cells and proliferation of T and B cells, smooth vasculature. muscle cells and keratinocytes.20-27) Pharmacological targeting of the KCa3.1 channel is expected to have effects in proliferative diseases and autoimmune diseases such as restenosis, graft rejection, secretory diarrhea, sickle cell anemia, cystic fibrosis, etc. Compared with its homologs, KCa3.1 has a unique sequence of 14 amino acids at the C-terminus, and this sequence has a binding affinity for NDPK-B.
In fact, NDPK-B phosphorylates KCa3.1 on His358 with regulation by phosphatidylinositol 3-phosphate (PI(3)P) and the phosphorylation event activates KCa3.1.
Methods for Studying Phosphohistidine
- Preparation of Phosphohistidine
- Detection – Isotope-labeling 32 P and Amino Acid Analysis
- Detection – Mass Spectrometry
- Detection – Antibodies and Western Blots
It was then digested with trypsin or endoproteinase Asp-N, and two different pHis-containing fragments were detected. Antibodies have specific and strong interactions with their respective antigens and are used in specific detection and quantification methods such as ELISA and western blots. Generally, an immunogen is injected into animals, such as rabbits and mice, and the antibodies against the immunogen are produced.
These antibodies marked an important milestone in pHis research, as they enabled specific detection and quantification of pHis proteins.
Conclusion and Future Perspectives
For example, histone H4 was chemically phosphorylated using potassium phosphoramidate and two histidine residues were phosphorylated. In the case of small antigens, these are conjugated to carrier proteins that are immunogenic, called haptens, and the conjugated materials are injected. However, generation of pHis-specific antibodies was extremely challenging because pHis was not a stable immunization in vivo.
Katuš, H. A.; Rottbauer, W.; and Wieland, T. Interaction of nucleoside diphosphate kinase B with Gβγ dimers controls the function of heterotrimeric protein G. The solvent was evaporated and the residue was purified by column chromatography (Florisil, 100-200 mesh, 1:9 ethyl acetate/hexane). Synthesis of [13C8, 15N]tyramine (P1). The protocol for d4-tyrosine decarboxylation as described by Ntai et al18) was followed with some modifications.
Synthesis of d-desthiobiotinyl-NHS ester (P7). The protocol refers to the synthesis of d-biotinyl-NHS ester3\20).
Sox-Based Fluorescence Sensor for Phosphohistidine
Need for Fluorescence-based Protein Phosphorylation Sensors
Intracellular phosphorylation is achieved by the enzymatic function of protein kinase against extracellular stimuli.1-4) Phosphorylation plays an important role in cellular metabolism and also in intercellular communication in eukaryotes. 5) The superfamily of protein kinases represents approximately 2% of the human genome and includes 500 kinases.2 ) Protein kinases catalyze the transfer of a γ-phosphorylation group from adenosine-5-triphosphate (ATP) to release a hydroxyl, amino, or imidazole group in a target peptide or protein10,11). Since protein kinase plays a key role and its improper function causes severe diseases12-16), the regulation of protein kinase has been of great interest and has become a major therapeutic target in recent decades17). Indeed, many pharmaceutical and biotech companies have focused on the development of protein kinase inhibitors.
A commonly used assay for protein kinase activity is a radioactivity-based assay in which phosphoryl group transfer from [γ-32P]ATP to a peptide or protein substrate is quantified by means of scintillation counting.
Design and Strategy for CHEF-based Phosphorylation Sensors
Previously, the peptide substrate was prepared using non-natural amino acid Fmoc-Sox-OH29,30) via Solid Phase Peptide Synthesis (SPPS). Instead of unnatural amino acid, we used cysteine and it was reacted with Sox halide to obtain a cysteine derivative of Sox fluorophore called CSox27,31,32).
Result and Discussion
- Preparation of Sox-Br
- Design of Model Substrates
- Preparation of Model Substrates
- Preparation of Model Kinase Sensor
- Linker Optimization of Model Kinase Sensors
- Fluorescence Assay and Phosphorylation Test of Model Kinase Sensors
- Future Direction: Preparation of the Substrates for Histidine Kinases
Using basic potassium carbonate, the yield was lower than 20%, the reason being that the commercial source of histamine was the dihydrochloride form of the salt (histamine-2HCl) and the formation of water between hydrochloric acid and potassium carbonate, which is a hindrance to the reactivity of DCC. The white solid, triphenylmethane, was dissolved in hexane and removed, but the liquid, desired product, was immiscible with the hexane and formed a layer beneath the hexane layer. Our initial synthesis was a C-terminus to N-terminus synthesis; meaning that Boc-glycine was linked to histamine, the Boc group was deprotected, and the free amine of the N-terminus on glycine was linked to 3-tritylthiopropionic acid.
Consequently, we decided to apply SPPS methods and the tetrapeptide model kinase sensor [n+3] was synthesized. Since the model kinase sensor [n+1] was ready, chemical phosphorylation was performed using potassium phosphoramidate (PPA). Fortunately, the phosphorylated sensor molecule (the sensor molecule + PPA) emitted strong intensity of fluorescence when incubated with 10 mM Mg2+.
Also, the magnesium ion concentration was varied and the fluorescence was measured to find the Kd for Mg2+ and the maximum fluorescence for the unphosphorylated sensor. Then, the sensor molecule was tested at 100 µM and the concentration of Mg2+ was 10 mM and the phosphorylation reaction was for 30 min with 10 mM PPA under pH 7.5 pH of phosphate buffer. The result was consistent with the previous data, and the fluorescence difference between the phosphorylated sensor and the non-phosphorylated sensor was definitively distinguished.
In fact, at low pH, the quinoline nitrogen of the Sox fluorophore became protonated and the fluorescence was completely quenched. Then, by comparing the phosphorylation between the sensor peptides of positions [n+α] and [n- α], we will determine which residue should be mutated to cysteine; if the enzyme recognition site is changed to a cysteine by alkylated Sox, it may not function as an enzyme recognition site and enzyme-mediated phosphorylation may be difficult.
Conclusion
Methods
Dioxane was removed by rotary evaporation and the remaining yellowish oil was redissolved in ethyl acetate (200 ml), washed with brine (20 ml), water (20 ml), saturated potassium carbonate solution (20 ml) and dried over MgSO 4 . Then the reaction solution was diluted with diethyl ether (200 ml), washed with saturated ammonium chloride (40 ml), water (2 x 40 ml) and brine (40 ml) and dried over MgSO 4 . The reaction mixture was diluted with 1200 ml diethyl ether, washed with water (150 ml) and brine (100 ml) and dried over MgSO4.
Histamine dihydrochloride (200 mg, 1.1 mmol) was added and the reaction solution was stirred overnight at 40 °C. The organic layer was evaporated and the residue was purified by column chromatography on silica gel. The reaction solution was evaporated under reduced pressure and the product was extracted with ethyl acetate and washed with distilled water.
The temperature was then raised to 35°C and the reaction solution was stirred for 2.5 hours. The reaction mixture was poured into hexane and the product was extracted with an aqueous layer (25 mL H2O x2 and 25 mL Na2CO3 solution x2). The collected organic layer was dried over Na2SO4 and the solvent was removed by rotary evaporation to leave the product.
The DMF was removed by rotary evaporation and the residue was diluted in ethyl acetate, washed with water and brine and dried over Na2SO4. After the reaction was complete, the solvent was evaporated and the residue was diluted with DCM, washed with water and brine and dried over MgSO4.
Experimental Data
After reaction was completed, the reaction solution was removed by a rotary evaporator and the residue was redissolved in water and passed through cation exchange column. Synthesis of d-desthiobiotin-NHS ester was referred to the synthesis of biotin-NHS ester using DCC as the coupling agent. When the reaction was complete, the precipitated solid (DCU) was removed by filtration and the solvent was evaporated.
Since NHS-activated ester is stable in mild acid, d-desthiobiotin-NHS ester remained in the organic phase and other mixtures were extracted in the aqueous phase. As a test, unlabeled with d-desthiobiotin-NHS ester was reacted in a mixture of methanol and water. Reaction was monitored by TLC, and when the reaction was complete, solvent was removed by rotary evaporation.
Isotope-labeled tyramines were also conjugated with desthiobiotin-NHS ester to produce three types of isotope-labeled DBT products. A series of isotopically labeled tyrosine was converted to tyramine and subsequent dethiobiotin conjugation was successful. The reaction mixture was concentrated in vacuo and passed through Dowex 50WX cation exchange resin (hydrogen form).
The protocol was identical to the synthesis of [13C8,15N]tyramine (P1), except for the use of [ring-13C6]tyrosine as starting material. The reaction mixture was concentrated in vacuo and the residue was extracted with ethyl acetate (50 mL x 8 times) from water (50 mL).
Proteomic Labeling Strategy through Desthiobiotin with Conjugation of Isotope-Labeled
Design of Isotope-labeled Desthiobiotinyl Tyramide
To prepare the desthiobiotin-phenol derivative, a labeled substrate for APEX, desthiobiotin was previously coupled with tyramine to form desthiobiotinyl tyramide (DBT). This time, it was necessary to prepare tyramine, which contains non-radioactive stable isotopes, i.e. 13-carbon and 15-nitrogen.
Results and Discussion
We bought three types of tyrosine containing isotopes and converted them to tyramines containing isotopes, which is what we wanted. In the case of biotin-NHS ester, it was not soluble in isopropanol, while other compounds such as DCC, DCU, NHS (unreacted and remaining), TEA (base), and DMF solvent were dissolved in isopropanol. The residue was dissolved in ethyl acetate and treated with ethyl acetate and acidic NH 4 Cl solution.
C18 column, 100% water to 75% water and 25% acetonitrile in 5 min, and 75% water and 25% acetonitrile to 60% water and 40% acetonitrile in 40 min.
Conclusion
The tyramine product was eluted from the resin with approximately 50 mL of 1 mM HCl and the solvent was evaporated to give [13C8,15N]tyramine as a brown powder (74 mg, 97% yield). 5 mL of methanol was added and the resulting solution was stirred at room temperature for 16 h.
