A functional yeast survival screen of tumor-derived cDNA libraries designed to identify anti-apoptotic mammalian oncogenes.

Yeast cells can be killed upon expression of pro-apoptotic mammalian proteins. We have established a functional yeast survival screen that was used to isolate novel human anti-apoptotic genes overexpressed in treatment-resistant tumors. The screening of three different cDNA libraries prepared from metastatic melanoma, glioblastomas and leukemic blasts allowed for the identification of many yeast cell death-repressing cDNAs, including 28% of genes that are already known to inhibit apoptosis, 35% of genes upregulated in at least one tumor entity and 16% of genes described as both anti-apoptotic in function and upregulated in tumors. These results confirm the great potential of this screening tool to identify novel anti-apoptotic and tumor-relevant molecules. Three of the isolated candidate genes were further analyzed regarding their anti-apoptotic function in cell culture and their potential as a therapeutic target for molecular therapy. PAICS, an enzyme required for de novo purine biosynthesis, the long non-coding RNA MALAT1 and the MAST2 kinase are overexpressed in certain tumor entities and capable of suppressing apoptosis in human cells. Using a subcutaneous xenograft mouse model, we also demonstrated that glioblastoma tumor growth requires MAST2 expression. An additional advantage of the yeast survival screen is its universal applicability. By using various inducible pro-apoptotic killer proteins and screening the appropriate cDNA library prepared from normal or pathologic tissue of interest, the survival screen can be used to identify apoptosis inhibitors in many different systems.

Mapping of O-GlcNAc sites of 20 S proteasome subunits and Hsp90 by a novel biotin-cystamine tag.

The post-translational modification of proteins with O-GlcNAc is involved in various cellular processes including signal transduction, transcription, translation, and nuclear transport. This transient protein modification enables cells or tissues to adapt to nutrient conditions or stress. O-Glycosylation of the 26 S proteasome ATPase subunit Rpt2 is known to influence the stability of proteins by reducing their proteasome-dependent degradation. In contrast, knowledge of the sites of O-GlcNAcylation on the subunits of the catalytic core of the 26 S proteasome, the 20 S proteasome, and the impact on proteasome activity is very limited. This is predominantly because O-GlcNAc modifications are often substoichiometric and because 20 S proteasomes represent a complex protein mixture of different subtypes. Therefore, identification of O-GlcNAcylation sites on proteasome subunits essentially requires effective enrichment strategies. Here we describe an adapted ß-elimination-based derivatization method of O-GlcNAc peptides using a novel biotin-cystamine tag. The specificity of the reaction was increased by differential isotopic labeling with either "light" biotin-cystamine or deuterated "heavy" biotin-cystamine. The enriched peptides were analyzed by LC-MALDI-TOF/TOF-MS and relatively quantified. The method was optimized using bovine α-crystallin and then applied to murine 20 S proteasomes isolated from spleen and brain and murine Hsp90 isolated from liver. Using this approach, we identified five novel and one known O-GlcNAc sites within the murine 20 S proteasome core complex that are located on five different subunits and in addition two novel O-GlcNAc sites on murine Hsp90ß, of which one corresponds to a previously described phosphorylation site.

Immune modulation by Fas ligand reverse signaling: lymphocyte proliferation is attenuated by the intracellular Fas ligand domain.

Fas ligand (FasL) not only induces apoptosis in Fas receptor-bearing target cells, it is also able to transmit signals into the FasL-expressing cell via its intracellular domain (ICD). Recently, we described a Notch-like proteolytic processing of FasL that leads to the release of the FasL ICD into the cytoplasm and subsequent translocation into the nucleus where it may influence gene transcription. To study the molecular mechanism underlying such reverse FasL signaling in detail and to analyze its physiological importance in vivo, we established a knockout/knockin mouse model, in which wild-type FasL was replaced with a deletion mutant lacking the ICD. Our results demonstrate that FasL ICD signaling impairs activation-induced proliferation in B and T cells by diminishing phosphorylation of phospholipase C γ, protein kinase C, and extracellular signal-regulated kinase 1/2. We also demonstrate that the FasL ICD interacts with the transcription factor lymphoid-enhancer binding factor-1 and inhibits lymphoid-enhancer binding factor-1-dependent transcription. In vivo, plasma cell numbers, generation of germinal center B cells, and, consequently, production of antigen-specific immunoglobulin M antibodies in response to immunization with T cell-dependent or T cell-independent antigen are negatively affected in presence of the FasL ICD, suggesting that FasL reverse signaling participates in negative fine-tuning of certain immune responses.

The cell-penetrating peptide octa-arginine is a potent inhibitor of proteasome activities.

Oligo-arginines are cell-penetrating peptides and find use as carriers for transportation of various membrane-impermeable biopharmaceuticals into target cells. We have found that oligo-arginines of a length of 4-10 amino acids, but especially (Arg)(8), are able to inhibit the major intracellular proteolytic system, the proteasome, with mixed-type inhibition characteristics. The IC(50) values of (Arg)(8) for the proteasomal chymotrypsin-like and caspase-like activities are approximately 100 and 200 nM, respectively. The inhibition of the trypsin-like activity never exceeds 50% even at micromolar concentrations. (Arg)(8) also inhibits 20S proteasome/PA28 complexes as well as 26S proteasomes, although with a decreased efficiency. Due to its cell membrane-penetrating capability, incubation of HeLa cells in the presence of (Arg)(8) resulted in an impaired activity of proteasomes going along with an accumulation of high-molecular mass ubiquitin-conjugated proteins, the preferred substrates of 26S proteasomes. The in vivo susceptibility of the three proteasome activities resembles that found in vitro with chymotrypsin-like>caspase-like>trypsin-like activities. Since inhibition of the proteasome system might affect fundamental basic cellular processes but on the other side might also prevent the degradation of a proteinacous cargo, we suggest that this proteasome inhibitory activity should be taken into account when oligo-arginines are being considered for use as vectors for the intracellular delivery of pharmaceuticals.