Apoptosis inhibitor 5 is an endogenous inhibitor of caspase-2.

Caspases are key enzymes responsible for mediating apoptotic cell death. Across species, caspase-2 is the most conserved caspase and stands out due to unique features. Apart from cell death, caspase-2 also regulates autophagy, genomic stability and ageing. Caspase-2 requires dimerization for its activation which is primarily accomplished by recruitment to high molecular weight protein complexes in cells. Here, we demonstrate that apoptosis inhibitor 5 (API5/AAC11) is an endogenous and direct inhibitor of caspase-2. API5 protein directly binds to the caspase recruitment domain (CARD) of caspase-2 and impedes dimerization and activation of caspase-2. Interestingly, recombinant API5 directly inhibits full length but not processed caspase-2. Depletion of endogenous API5 leads to an increase in caspase-2 dimerization and activation. Consistently, loss of API5 sensitizes cells to caspase-2-dependent apoptotic cell death. These results establish API5/AAC-11 as a direct inhibitor of caspase-2 and shed further light onto mechanisms driving the activation of this poorly understood caspase.

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.

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.

LmxMPK4, an essential mitogen-activated protein kinase of Leishmania mexicana is phosphorylated and activated by the STE7-like protein kinase LmxMKK5.

The essential mitogen-activated protein kinase (MAP kinase), LmxMPK4, of Leishmania mexicana is minimally active when purified following recombinant expression in Escherichia coli and was therefore unsuitable for drug screening until now. Using an E. coli protein co-expression system we identified LmxMKK5, a STE7-like protein kinase from L. mexicana, which phosphorylates and activates recombinant LmxMPK4 in vitro. LmxMKK5 is comprised of 525 amino acids and has a calculated molecular mass of 55.9kDa. The co-expressed, purified LmxMPK4 showed strong phosphotransferase activity in radiometric kinase assays and was confirmed by immunoblot and tandem mass spectrometry analyses to be phosphorylated on threonine 190 and tyrosine 192 of the typical TXY MAP kinase activation motif. The universal protein kinase inhibitor staurosporine reduced the phosphotransferase activity of co-expressed and activated LmxMPK4 in a dose-dependent manner. To our knowledge this is the first time that an in vitro activator of an essential Leishmania MAP kinase was identified and our findings form the basis for the development of drug screening assays to identify small molecule inhibitors of LmxMPK4 in the search for new therapeutic drugs against leishmaniasis.