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Analysis of S-nitrosylated Proteins

Author and Curator: Larry H Bernstein, MD, FCAP

 

The biotin switch method for the detection of S-nitrosylated proteins.

Jaffrey S. R. and Snyder S. H.
Sci STKE. 2001, pl1.

 

• apoptosis thymocytes induced by GSNO.

Meanwhile, the level of the protein S-nitrosylation

• was inhibited by the NOS inhibitor L-NMMA, which is consistent with
• the protection of L-NMMA from apoptosis.

We also found that proteins with

• moderate molecular weight (20-60 kDa)
• are more sensitive to GSNO S-nitrosylation.

Thymocytes of 3-4 week-old inbred male/female Balb/c mice (11.5-13.0 g), were obtained by gently pressing the thymus against a nylon net  submerged in  SWIM’S S-77 medium without fetal calf serum (FCS) and sulphydryl from cysteine. The suspension was put in Ficoll and prepared by density gradient centrifugation  at 410 g.

Thymocytes were resuspended in S-77 medium (1×10  cells/ml) and cultured at 37°C, 5% CO2. The viability of untreated thymocytes were identified by trypan blue exclusion assay or/and MoFlo high performance cell sorter (MoFlo cell sorter) to be always greater than about 96% during the 3-hour real-time process (data not shown).

GSNO is the nitroso derivative of glutathione (GSH) and must be freshly synthesized right before the experiments. To prepare 30 mM GSNO, GSH was dissolved in 0.1 N HCl to the final concentration of 60 mM, and then mixed with equal molar amount of sodium nitrite. During the preparation, the mixture was protected from light. The final GSNO was characterized using ultraviolet-spectroscopy [7].

Thymocytes were treated with GSNO for 3 hrs in S-77 medium and then washed with PBS. The washed cells were stained by double labeled Hoechst 33342 (final concentration 10 µM) and PI (5 µg/ml) for 20 min, and subjected to MoFlo high-performance cell sorter (Dako, USA) using excitation /emission equal to 351/460 nm for Hoechst 33342 and 488/630 nm for PI respectively.

Biotin-switch method and western blotting-detected S-nitrosated proteins

The analysis of S-nitrosylated proteins was described previously [8, 9]. After the exposure to 0.3 mM GSNO for 3 h, cells were washed three times with ice-cold PBS, and lysed in HEN buffer (250 mM Hepes-NaOH pH 7.7, 1 mM EDTA, 0.1 mM Neocupeoine) containing 0.5% NP-40 for 30 min on ice and centrifuged at 10,000 g for 10 min. One volume of the supernatant was incubated with four volumes of blocking buffer (9 volumes of HEN buffer plus one volume 25% SDS, 20 mM MMTS) at 50 °C for 20 min with frequent vortexing. MMTS was then removed by protein precipitation with ten volumes of pre-chilled acetone. After SDS-PAGE sample buffer was added, the samples were resolved by SDS-PAGE and transferred for immunoblotting with streptravidin-HRP. S-nitrosated bovine serum albumin (BSA) was used as a positive control.

In order to gain reasonable data of PCR amplification, two controls were set up in parallel. One of them sets water as the template, the other of them sets un-reversed transcribed total RNA as the template. Every samples were serially diluted 10 times for a calibration curve. The housekeeping gene beta-actin was set in this study. The primers were used :

beta-actin-FP: 5’-GAG ACC TTC AAC ACC CCA GCC-3’,
beta-actin-RP: 5’-AAT GTC ACG CAC GAT TTC CC-3’;
p53-FP: 5’ACG TGC CCT GTG CAG TTG T-3’,
p53-RP: 5’GGA TAG GTC GGC GGT TCA T-3’;
ING1-FP: 5’-CTC CAG GGC TTT GTC CAT-3’，
ING1-RP 5’-GCA ACC AGG TCT CCT ACG-3’;
bax-FP: 5’-GTA GAA GAG GGC AAC CAC G-3’,
bax-RP :5’CCA GGA TGC GTC CAC CAA-3’,
All raw data of Real time PCR in this study were obtained from software of Gene Amp 5700 Sequence Detection System.

 

Reference

Nitric oxide: NO apoptosis or turning it ON? Cell Death Differ 2003, 10:864-869.

Hess DT, Matsumoto A, Kim SO, Marshall HE, Stamler JS: Protein S-nitrosylation: purview and parameters. Nat Rev Mol Cell Biol 2005,  6:150-166.

Messmer UK, Reed UK, Brune B: Bcl-2 protects macrophages from nitric oxide-induced apoptosis. J Biol Chem 1996, 271:20192-20197.

Brune B, Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C: A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 1991, 139:271-279.

Lin DY, Ma WY, Duan SJ, Zhang Y, Du LY: Real-time imaging of viable-apoptotic switch in GSNO-induced mouse thymocyte apoptosis. Apoptosis 2006, 11:1289-1298.

Fehsel K, Kroncke KD, Meyer KL, Huber H, Wahn V, Kolb-Bachofen V: Nitric oxide induces apoptosis in mouse thymocytes. J Immunol 1995, 155:2858-2865.

Gabor G, Allon N: Spectrofluorometric method for NO determination. Anal Biochem 1994, 220:16-19.

Jaffrey SR, Snyder SH: The biotin switch method for the detection of S-nitrosylated proteins. Sci STKE 2001, 2001:PL1.

Sumbayev VV, Budde A, Zhou J, Brune B: HIF-1 alpha protein as a target for S-nitrosation. FEBS Lett 2003, 535:106-112.

 

SJ Williams:

There are two very good volumes of Methods in Enzymology (Volumes 528 (2013) and Volume 437 (2008) which deal with methods to quantitate nitric oxide modifications in cells,(including whole cell imaging). These methods usually have delt with the reversible nitrosylation reaction however the irreversible covalent modification (highlighted in our Nitric oxide ebook) is quite difficult to measure yet is a very biologically relevant modification.