A Non‐damaging Method to Analyze the Configuration and Dynamics of Nitrotyrosines in Proteins

Dı́az-Moreno, Irene; Nieto, Pedro M.; Conte, Rebecca Del; Gairì, Margarida; García-Heredia, José Manuel; Rosa, Miguel Á. De la; Díaz-Quintana, Antonio

doi:10.1002/chem.201103413

Cited by 9 publications

(9 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Notably,t he computed side-chain dipole momenti s1 6.2 D, much larger than that for p-cresol (analogoust ot he tyrosine side-chain;1 .46 D) or the e-nitro-tyrosine side-chain (5.2 D). [36] According to the data in Figure 1a nd Figure S1 in the Supporting Information, the Y48pCMFmutation induces alocalized distortion of the structure similart ot hat computed for the phosphorylated form. Our results indicatet hat pCMF mimics phosphotyrosine at position 48 better than aG lu residue.A s showni nF igure 1A,t he Y48E mutation leavesaw ide solventaccessible channel near the heme propionatest hat is not expectedt obef ormed when Tyr48 is phosphorylated.…”

Section: Tyr-by-pcmf Substitutionmentioning

confidence: 63%

See 1 more Smart Citation

Mimicking Tyrosine Phosphorylation in Human Cytochrome c by the Evolved tRNA Synthetase Technique

Guerra‐Castellano

Díaz‐Quintana

Moreno‐Beltrán

et al. 2015

Chemistry A European J

Self Cite

View full text Add to dashboard Cite

Phosphorylation of tyrosine 48 of cytochrome c is related to a wide range of human diseases due to the pleiotropic role of the heme-protein in cell life and death. However, the structural conformation and physicochemical properties of phosphorylated cytochrome c are difficult to study as its yield from cell extracts is very low and its kinase remains unknown. Herein, we report a high-yielding synthesis of a close mimic of phosphorylated cytochrome c, developed by optimization of the synthesis of the non-canonical amino acid p-carboxymethyl-L-phenylalanine (pCMF) and its efficient site-specific incorporation at position 48. It is noteworthy that the Y48pCMF mutation significantly destabilizes the Fe-Met bond in the ferric form of cytochrome c, thereby lowering the pKa value for the alkaline transition of the heme-protein. This finding reveals the differential ability of the phosphomimic protein to drive certain events. This modified cytochrome c might be an important tool to investigate the role of the natural protein following phosphorylation.

show abstract

Section: Tyr-by-pcmf Substitutionmentioning

confidence: 63%

“…The charges corresponding to p CMF and statistical data on the trajectories are given in Figure S1 in the Supporting Information. Notably, the computed side‐chain dipole moment is 16.2 D, much larger than that for p ‐cresol (analogous to the tyrosine side‐chain; 1.46 D) or the ε‐nitro‐tyrosine side‐chain (5.2 D) 36…”

Section: Resultsmentioning

confidence: 82%

Mimicking Tyrosine Phosphorylation in Human Cytochrome c by the Evolved tRNA Synthetase Technique

Guerra‐Castellano

Díaz‐Quintana

Moreno‐Beltrán

et al. 2015

Chemistry A European J

Self Cite

View full text Add to dashboard Cite

show abstract

“…Here, the delay in the insensitive nuclear enhancement by polarization transfer (INEPT) module of the pulse sequence in 1 H-15 N HSQC waso ptimized to detect 2 J(NÀH) because its duration determines the efficiency of the coherence transfer.A lthough the delay is around5ms in conventional 1 H- 15 NH SQC spectra, it was fixed to 22 ms, [40] allowing the magnetization transfer through 2 J(NÀH) weaker couplings to detect the correlations between the nitrogen atoms and the non-exchangeable protons in the imidazole ring of His96.T his approachh as been successfully appliedt oo ther kind of residues as nitrotyrosines in cytochrome c protein. [41,42] Thus, we performed series of the optimized 1 H- 15 NH SQC spectra along pH titrations( Figure 5) of TIA-1 RRM2 (either free or bound to nucleic acids) to evaluate the pH effect over the His96 side-chain, which directly interacts with 5'-UUUUU-3' (the Supporting Information, Figure S1) or 5'-TTTTT-3' oligonucleotides (the Supporting Information, Figure S2). According to the structure of the imidazole group in ah istidine (Figure 5B), the Ne 2 nucleus has two possible 2 J couplings with He 1 and Hd 2 ,w hereas the Nd 1 exhibits only one with He 1 .A ll the pH titrationsw ere performed from pH 7.0 to 4.5, monitoring the expected 1 H- 15 NH SQC region in the spectra for such correlations.…”

Section: Introductionmentioning

confidence: 99%

A Non‐Invasive NMR Method Based on Histidine Imidazoles to Analyze the pH‐Modulation of Protein–Nucleic Acid Interfaces

Cruz-Gallardo

Conte

Velázquez‐Campoy

et al. 2015

Chemistry A European J

Self Cite

View full text Add to dashboard Cite

A useful (2) J(N-H) coupling-based NMR spectroscopic approach is proposed to unveil, at the molecular level, the contribution of the imidazole groups of histidines from RNA/DNA-binding proteins on the modulation of binding to nucleic acids by pH. Such protonation/deprotonation events have been monitored on the single His96 located at the second RNA/DNA recognition motif (RRM2) of T-cell intracellular antigen-1 (TIA-1) protein. The pKa values of the His96 ionizable groups were substantially higher in the complexes with short U-rich RNA and T-rich DNA oligonucleotides than those of the isolated TIA-1 RRM2. Herein, the methodology applied to determine changes in pKa of histidine side chains upon DNA/RNA binding, gives valuable information to understand the pH effect on multidomain DNA/RNA-binding proteins that shuttle among different cellular compartments.

show abstract

“…While flow cytometry can be quantitative for cell-surface proteins or some select intracellular proteins, these methods are largely incapable of determining accurate quantities of intracellular proteins or protein modifications present in biological specimens. Strategies to measure nitration without damaging the protein include: nuclear magnetic resonance, electron paramagnetic resonance, and electronic absorption spectra 18 , 19 . Proteomic approaches using older mass spectrometry methods capable of high mass accuracy have made significant advancements in characterizing the various protein and protein modification biomarkers present within complex biological samples 20 .…”

Section: Introductionmentioning

confidence: 99%

Nitric oxide mediated inhibition of antigen presentation from DCs to CD4+ T cells in cancer and measurement of STAT1 nitration

Markowitz

Wang

Vangundy

et al. 2017

Sci Rep

View full text Add to dashboard Cite

Myeloid derived suppressor cells (MDSC) produce nitric oxide (NO) and inhibit dendritic cell (DC) immune responses in cancer. DCs present cancer cell antigens to CD4 + T cells through Jak-STAT signaltransduction. In this study, NO donors (SNAP and DETA-NONOate) inhibited DC antigen presentation. As expected, MDSC isolated from peripheral blood mononuclear cells (PBMC) from cancer patients produced high NO levels. We hypothesized that NO producing MDSC in tumor-bearing hosts would inhibit DC antigen presentation. Antigen presentation from DCs to CD4 + T cells (T cell receptor transgenic OT-II) was measured via a [ 3 H]-thymidine incorporation proliferation assay. MDSC from melanoma tumor models decreased the levels of proliferation more than pancreatic cancer derived MDSC. T cell proliferation was restored when MDSC were treated with inhibitors of inducible nitric oxide synthase (L-NAME and NCX-4016). A NO donor inhibited OT II T cell receptor recognition of OT II specific tetramers, thus serving as a direct measure of NO inhibition of antigen presentation. Our group has previously demonstrated that STAT1 nitration also mediates MDSC inhibitory effects on immune cells. Therefore, a novel liquid chromatography-tandem mass spectrometry assay demonstrated that nitration of the STAT1-Tyr701 occurs in PBMC derived from both pancreatic cancer and melanoma patients.Melanoma cells are recognized by the immune system, but the anti-tumor activity of T cells and natural killer (NK) cells is inhibited by multiple mechanisms mediated by immune suppressor cells including depletion of nutrients from the tumor microenvironment, production of reactive oxygen and nitrogen species, secretion of immune-suppressive cytokines and induction of additional inhibitory immune cells 1 . Presentation of antigens to T cells by dendritic cells (DCs) is defective in the setting of melanoma 2 . Recently, it has been shown that stimulation of DCs with type I interferons (IFN-α and β) and down-stream signal transduction via the Janus kinase-signal transducer and activator of transcription (Jak-STAT) pathway is critically important to immune surveillance and the generation of effective host T cell immune responses to cancer 3,4 . Furthermore, in dendritic cells, IFN-α signaling is responsible for up-regulation of class I and class II MHC molecules for the presentation of antigens by dendritic cells [5][6][7] . It has been demonstrated that the anti-tumor effects of IFN-α were dependent on STAT1 signal transduction in immune cells via phosphorylation of tyrosine 701 8 . Jak-STAT signaling was markedly inhibited in human peripheral blood immune cells from tumor bearing patients 9 , More recently, we

show abstract

A Non‐damaging Method to Analyze the Configuration and Dynamics of Nitrotyrosines in Proteins

Cited by 9 publications

References 36 publications

Mimicking Tyrosine Phosphorylation in Human Cytochrome c by the Evolved tRNA Synthetase Technique

Mimicking Tyrosine Phosphorylation in Human Cytochrome c by the Evolved tRNA Synthetase Technique

A Non‐Invasive NMR Method Based on Histidine Imidazoles to Analyze the pH‐Modulation of Protein–Nucleic Acid Interfaces

Nitric oxide mediated inhibition of antigen presentation from DCs to CD4+ T cells in cancer and measurement of STAT1 nitration

Contact Info

Product

Resources

About