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Identification of S-nitrosylated targets of thioredoxin using a quantitative proteomic approach.

ABSTRACT: Reversible protein cysteine nitrosylation (S-nitrosylation) is a common mechanism utilized in signal transduction and other diverse cellular processes. Protein denitrosylation is largely mediated by cysteine denitrosylases, but the functional scope and significance of these enzymes are incompletely defined, in part due to limited information on their cognate substrates. Here, using Jurkat cells, we employed stable isotope labeling by amino acids in cell culture (SILAC), coupled to the biotin switch technique and mass spectrometry, to identify 46 new substrates of one denitrosylase, thioredoxin 1. These substrates are involved in a wide range of cellular functions including cytoskeletal organization, cellular metabolism, signal transduction, and redox homeostasis. We also identified multiple S-nitrosylated proteins that are not substrates of thioredoxin 1. A verification of our principal findings was made in a second cell type (RAW264.7 cells). Our results point to thioredoxin 1 as a major protein denitrosylase in mammalian cells and demonstrate the utility of quantitative proteomics for large-scale identification of denitrosylase substrates.

SUBMITTER: Benhar M

PROVIDER: S-EPMC3008576 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Identification of S-nitrosylated targets of thioredoxin using a quantitative proteomic approach.

Benhar Moran M Thompson J Will JW Moseley M Arthur MA Stamler Jonathan S JS

Biochemistry 20100801 32

Reversible protein cysteine nitrosylation (S-nitrosylation) is a common mechanism utilized in signal transduction and other diverse cellular processes. Protein denitrosylation is largely mediated by cysteine denitrosylases, but the functional scope and significance of these enzymes are incompletely defined, in part due to limited information on their cognate substrates. Here, using Jurkat cells, we employed stable isotope labeling by amino acids in cell culture (SILAC), coupled to the biotin swi ...[more]

PMID: 20695533

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Project description:Acute kidney injury (AKI) is a common and increasingly encountered complication in hospitalized patients with critical illness in intensive care units (ICU). According to the etiology, Sepsis-induced AKI (SAKI) is a leading contributor to AKI and significantly has very poor prognosis, which might be related to the late detection when the elevation of BUN and serum creatinine (SCr) is used. Many genes are up-regulated in the damaged kidney with the corresponding protein products appearing in plasma and urine. Some of these are candidate biomarkers for more timely diagnosis of SAKI. Therefore, extensive research efforts over this past decade have been directed at the discovery and validation of novel SAKI biomarkers to detect injury prior to changes in kidney function, a number of serum and urinary proteins, including NGAL, KIM-1, cystatin-C, IL-18, and L-FABP, have been identified for predicting SAKI before a rise in BUN and serum creatinine in several experimental and clinical trainings. Unfortunately, an ideal biomarker of SAKI with highly sensitivity and specificity has not been identified yet. Recent progresses in quantitative proteomics have offered opportunities to discover biomarkers for SAKI. In the present study, kidney tissue samples from SAKI mice were analyzed by two-dimensional differential gel electrophoresis (2D-DIGE), and 4 up-regulated proteins, which were actin (ACTB), myosin regulatory light chain 12B (MYL12B), myosin regulatory light polypeptide 9 (MYL9), and myosin regulatory light chain 12A (MYL12A) were identified by matrix assisted laser desorption ionization-time of flight/time of flight mass spectrometry (MALDI-TOF/TOF MS). Among all the varied proteins, MYL12B was validated by western blot. Interestingly, there was no change between the SAKI and control kidney tissues, however, phosphorylated MYL12B was detected to be consistent with the proteomics data. Furthermore, phosphorylated MYL12B was found similarly to be increased in SAKI plasma, while MYL12B was changeless in plasma of control group. Taking together, phosphorylated MYL12B may be employed as a potential plasma biomarker for the early diagnosis of SAKI.

Dataset Information

Identification of S-nitrosylated targets of thioredoxin using a quantitative proteomic approach.

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Identification of S-nitrosylated targets of thioredoxin using a quantitative proteomic approach.

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