MEKK1-Dependent Activation of the CRL4 Complex Is Important for DNA Damage-Induced Degradation of p21 and DDB2 and Cell Survival.

Cullin-4 ubiquitin ligase (CRL4) complexes are differentially composed and highly dynamic protein assemblies that control many biological processes, including the global genome nucleotide excision repair (GG-NER) pathway. Here, we identified the kinase mitogen-activated protein kinase kinase kinase 1 (MEKK1) as a novel constitutive interactor of a cytosolic CRL4 complex that disassembles after DNA damage due to the caspase-mediated cleavage of MEKK1. The kinase activity of MEKK1 was important to trigger autoubiquitination of the CRL4 complex by K48- and K63-linked ubiquitin chains. MEKK1 knockdown prohibited DNA damage-induced degradation of the CRL4 component DNA-damage binding protein 2 (DDB2) and the CRL4 substrate p21 and also cell recovery and survival. A ubiquitin replacement strategy revealed a contribution of K63-branched ubiquitin chains for DNA damage-induced DDB2/p21 decay, cell cycle regulation, and cell survival. These data might also have implications for cancer, as frequently occurring mutations of MEKK1 might have an impact on genome stability and the therapeutic efficacy of CRL4-dependent immunomodulatory drugs such as thalidomide derivatives.

Transcriptional control of regulatory T cells.

Regulatory T cells (Tregs) constitute unique T cell lineage that plays a key role for immunological tolerance. Tregs are characterized by the expression of the forkhead box transcription factor Foxp3, which acts as a lineage-specifying factor by determining the unique suppression profile of these immune cells. Here, we summarize the recent progress in understanding how Foxp3 expression itself is epigenetically and transcriptionally controlled, how the Treg-specific signature is achieved and how unique properties of Treg subsets are defined by other transcription factors. Finally, we will discuss recent studies focusing on the molecular targeting of Tregs to utilize the specific properties of this unique cell type in therapeutic settings.

The Treg-specific demethylated region stabilizes Foxp3 expression independently of NF-κB signaling.

Regulatory T cells (Tregs) obtain immunosuppressive capacity by the upregulation of forkhead box protein 3 (Foxp3), and persistent expression of this transcription factor is required to maintain their immune regulatory function and ensure immune homeostasis. Stable Foxp3 expression is achieved through epigenetic modification of the Treg-specific demethylated region (TSDR), an evolutionarily conserved non-coding element within the Foxp3 gene locus. Here, we present molecular data suggesting that TSDR enhancer activity is restricted to T cells and cannot be induced in other immune cells such as macrophages or B cells. Since NF-κB signaling has been reported to be instrumental to induce Foxp3 expression during Treg development, we analyzed how NF-κB factors are involved in the molecular regulation of the TSDR. Unexpectedly, we neither observed transcriptional activity of a previously postulated NF-κB binding site within the TSDR nor did the entire TSDR show any transcriptional responsiveness to NF-κB activation at all. Finally, the NF-κB subunit c-Rel revealed to be dispensable for epigenetic imprinting of sustained Foxp3 expression by TSDR demethylation. In conclusion, we show that NF-κB signaling is not substantially involved in TSDR-mediated stabilization of Foxp3 expression in Tregs.

The WD40-repeat protein Han11 functions as a scaffold protein to control HIPK2 and MEKK1 kinase functions.

Protein kinases are organized in hierarchical networks that are assembled and regulated by scaffold proteins. Here, we identify the evolutionary conserved WD40-repeat protein Han11 as an interactor of the kinase homeodomain-interacting protein kinase 2 (HIPK2). In vitro experiments showed the direct binding of Han11 to HIPK2, but also to the kinases DYRK1a, DYRK1b and mitogen-activated protein kinase kinase kinase 1 (MEKK1). Han11 was required to allow coupling of MEKK1 to DYRK1 and HIPK2. Knockdown experiments in Caenorhabditis elegans showed the relevance of the Han11 orthologs Swan-1 and Swan-2 for the osmotic stress response. Downregulation of Han11 in human cells lowered the threshold and amplitude of HIPK2- and MEKK1-triggered signalling events and changed the kinetics of kinase induction. Han11 knockdown changed the amplitude and time dependence of HIPK2-driven transcription in response to DNA damage and also interfered with MEKK1-triggered gene expression and stress signalling. Impaired signal transmission also occurred upon interference with stoichiometrically assembled signalling complexes by Han11 overexpression. Collectively, these experiments identify Han11 as a novel scaffold protein regulating kinase signalling by HIPK2 and MEKK1.

Ajoene inhibits both primary tumor growth and metastasis of B16/BL6 melanoma cells in C57BL/6 mice.

Ajoene is an organosulphur compound derived from garlic with important effects on several membrane-associated processes such as platelet aggregation, as well as being cytotoxic for tumor cell lines in vitro. In the present study, we investigated the effect of ajoene on different cell types in vitro, as well as its inhibitory effects on both primary tumors and metastasis in a mouse model. We found ajoene to inhibit tumor cell growth in vitro, but also to inhibit strongly metastasis to lung in the B16/BL6 melanoma tumor model in C57BL/6 mice. As far as we are aware, this is the first report of the anti-metastatic effect of ajoene. Ajoene also inhibited tumor-endothelial cell adhesion, as well as the in vivo TNF-alpha response to lipopolysaccharide. Possible mechanisms of its antitumoral activity are discussed in the light of these results.