Dynamic Proteomics: a database for dynamics and localizations of endogenous fluorescently-tagged proteins in living human cells.

Recent advances allow tracking the levels and locations of a thousand proteins in individual living human cells over time using a library of annotated reporter cell clones (LARC). This library was created by Cohen et al. to study the proteome dynamics of a human lung carcinoma cell-line treated with an anti-cancer drug. Here, we report the Dynamic Proteomics database for the proteins studied by Cohen et al. Each cell-line clone in LARC has a protein tagged with yellow fluorescent protein, expressed from its endogenous chromosomal location, under its natural regulation. The Dynamic Proteomics interface facilitates searches for genes of interest, downloads of protein fluorescent movies and alignments of dynamics following drug addition. Each protein in the database is displayed with its annotation, cDNA sequence, fluorescent images and movies obtained by the time-lapse microscopy. The protein dynamics in the database represents a quantitative trace of the protein fluorescence levels in nucleus and cytoplasm produced by image analysis of movies over time. Furthermore, a sequence analysis provides a search and comparison of up to 50 input DNA sequences with all cDNAs in the library. The raw movies may be useful as a benchmark for developing image analysis tools for individual-cell dynamic-proteomics. The database is available at http://www.dynamicproteomics.net/.

Antiapoptotic function of Bcl-2 in mast cells is dependent on its association with heat shock protein 90beta.

In the present study, we demonstrated that the antiapoptotic function of Bcl-2 in mast cells is significantly dependent on its association with the heat shock protein 90beta (Hsp90beta). Dissociation of these 2 proteins inhibits the antiapoptotic activity of Bcl-2 by initiating the release of cytochrome c from mitochondria into cytosol and increasing the activity of caspase 3 and caspase 7, resulting in mast-cell apoptosis. The antiapoptotic activity of Bcl-2 was greatly affected by knocking-out specifically Hsp90beta using the RNA interference approach. Thus, for the first time, it has been shown that Hsp90beta might modulate the antiapoptotic activity of Bcl-2 at least in mast cells. These findings could have implications for a novel strategy of regulating apoptosis in patients with mastocytosis and other mast cell-associated diseases.

The involvement of Bcl-2 in mast cell apoptosis.

Apoptosis or programmed cell death plays an important role in a wide variety of physiological processes. Apoptosis is regulated by proteins of the Bcl-2 family consisting of both anti-apoptotic and pro-apoptotic factors. The direct involvement of the Bcl-2 protein family in the process of mast cell apoptosis has not been clarified. We have used a single-chain antibody (scFv) raised against Bcl-2 derived from human phage-display antibody library. The addition of TAT sequence, which is responsible for translocation through the membrane, endows the anti-Bcl-2-scFv with the ability to penetrate living cells. The association of anti-Bcl-2-scFv-TAT with intracellular Bcl-2 leads to neutralization of Bcl-2 and eradication of its anti-apoptotic activity in two types of mast cells and in a human breast cancer cell line. Moreover, we found by mass spectrometry and co-immunoprecipitation assay that heat shock protein 90b (Hsp90b) forms a complex with Bcl-2 in mast cells. Thus, understanding the network of interactions between Bcl-2 and non-Bcl-2 family members might help in development of more specific drugs and cancer therapy.

A novel strategy using single-chain antibody to show the importance of Bcl-2 in mast cell survival.

Apoptosis or programmed cell death plays an important role in a wide variety of physiologic processes and is regulated by proteins of the Bcl-2 family consisting of both antiapoptotic and proapoptotic factors. The direct involvement of the Bcl-2 protein family in the process of mast cell apoptosis has not been clarified. In the present work we have used a single-chain antibody (scFv) raised against Bcl-2 derived from a semisynthetic human phage-display antibody library. The addition of TAT sequence, which is responsible for translocation through the membrane, endows the anti-Bcl-2-scFv with the ability to penetrate living cells. Moreover, it specifically neutralizes Bcl-2 intracellularly by binding to the BH1 domain and eradicates its antiapoptotic activity in 2 types of mast cells and in a human breast cancer cell line.