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Immunological characterization of tristetraprolin as a low abundance, inducible, stable cytosolic protein.

ABSTRACT: Tristetraprolin (TTP) is a zinc finger protein that can bind to AU-rich elements within certain mRNAs, resulting in deadenylation and destabilization of those mRNAs. Its physiological targets include the mRNAs encoding the cytokines tumor necrosis factor alpha (TNF) and granulocyte-macrophage colony-stimulating factor. TTP was originally identified on the basis of its massive but transient increase in mRNA levels following mitogen stimulation of fibroblasts. It has been difficult to reconcile this transient mRNA profile with the presumed continuing "need" for TTP protein, for example, to reverse the effects of lipopolysaccharide (LPS)-stimulated TNF secretion. To investigate this and other questions concerning endogenous TTP protein in cells and tissues, we raised a high titer rabbit antiserum against full-length mouse TTP. TTP could be detected on immunoblots of mouse cytosolic tissue extracts; it was most highly expressed in spleen, but its concentration in that tissue was only about 1.5 nm. TTP could be detected readily in splenic macrophages and stromal cells from LPS-injected rats. In both LPS-treated RAW 264.7 macrophages and fetal calf serum-treated mouse embryonic fibroblasts, TTP protein was stable after induction, with minimal degradation occurring for several hours after treatment of the cells with cycloheximide. The biosynthesis of TTP was accompanied by large changes in electrophoretic mobility consistent with progressive phosphorylation. Confocal microscopy revealed that TTP accumulated in a vesicular pattern in the cytosol of the LPS-stimulated RAW 264.7 cells, and was occasionally seen in the cytosol of unstimulated dividing cells. Gel filtration of the endogenous protein suggested that its predominant structure was monomeric. TTP appears to be a low abundance, cytosolic protein in unstimulated cells and tissues, but once induced is relatively stable, in contrast to its very labile mRNA.

SUBMITTER: Cao H

PROVIDER: S-EPMC1351392 | biostudies-other | 2004 May

REPOSITORIES: biostudies-other

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Immunological characterization of tristetraprolin as a low abundance, inducible, stable cytosolic protein.

Cao Heping H Tuttle Jane S JS Blackshear Perry J PJ

The Journal of biological chemistry 20040309 20

Tristetraprolin (TTP) is a zinc finger protein that can bind to AU-rich elements within certain mRNAs, resulting in deadenylation and destabilization of those mRNAs. Its physiological targets include the mRNAs encoding the cytokines tumor necrosis factor alpha (TNF) and granulocyte-macrophage colony-stimulating factor. TTP was originally identified on the basis of its massive but transient increase in mRNA levels following mitogen stimulation of fibroblasts. It has been difficult to reconcile th ...[more]

PMID: 15010466

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Project description:Biomarker discovery produces lists of candidate markers whose presence and level must be subsequently verified in serum or plasma. Verification represents a paradigm shift from unbiased discovery approaches to targeted, hypothesis-driven methods and relies upon specific, quantitative assays optimized for the selective detection of target proteins. Many protein biomarkers of clinical currency are present at or below the nanogram/milliliter range in plasma and have been inaccessible to date by MS-based methods. Using multiple reaction monitoring coupled with stable isotope dilution mass spectrometry, we describe here the development of quantitative, multiplexed assays for six proteins in plasma that achieve limits of quantitation in the 1-10 ng/ml range with percent coefficients of variation from 3 to 15% without immunoaffinity enrichment of either proteins or peptides. Sample processing methods with sufficient throughput, recovery, and reproducibility to enable robust detection and quantitation of candidate biomarker proteins were developed and optimized by addition of exogenous proteins to immunoaffinity depleted plasma from a healthy donor. Quantitative multiple reaction monitoring assays were designed and optimized for signature peptides derived from the test proteins. Based upon calibration curves using known concentrations of spiked protein in plasma, we determined that each target protein had at least one signature peptide with a limit of quantitation in the 1-10 ng/ml range and linearity typically over 2 orders of magnitude in the measurement range of interest. Limits of detection were frequently in the high picogram/milliliter range. These levels of assay performance represent up to a 1000-fold improvement compared with direct analysis of proteins in plasma by MS and were achieved by simple, robust sample processing involving abundant protein depletion and minimal fractionation by strong cation exchange chromatography at the peptide level prior to LC-multiple reaction monitoring/MS. The methods presented here provide a solid basis for developing quantitative MS-based assays of low level proteins in blood.

Dataset Information

Immunological characterization of tristetraprolin as a low abundance, inducible, stable cytosolic protein.

Publications

Immunological characterization of tristetraprolin as a low abundance, inducible, stable cytosolic protein.

Similar Datasets

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