A small-molecule ARTS mimetic promotes apoptosis through degradation of both XIAP and Bcl-2.

Many human cancers over-express B cell lymphoma 2 (Bcl-2) or X-linked inhibitor of apoptosis (IAP) proteins to evade cell death. The pro-apoptotic ARTS (Sept4_i2) protein binds directly to both Bcl-2 and XIAP and promotes apoptosis by stimulating their degradation via the ubiquitin-proteasome system (UPS). Here we describe a small molecule, A4, that mimics the function of ARTS. Microscale thermophoresis assays showed that A4 binds XIAP, but not cellular inhibitor of apoptosis protein 1 (cIAP1). A4 binds to a distinct ARTS binding pocket in the XIAP-BIR3 (baculoviral IAP repeat 3) domain. Like ARTS, A4 stimulated poly-ubiquitylation and UPS-mediated degradation of XIAP and Bcl-2, but not cIAP1, resulting in caspase-9 and -3 activation and apoptosis. In addition, over-expression of XIAP rescued HeLa cells from A4-induced apoptosis, consistent with the idea that A4 kills by antagonizing XIAP. On the other hand, treatment with the SMAC-mimetic Birinapant induced secretion of tumour necrosis factor-α (TNFα) and killed ~50% of SKOV-3 cells, and addition of A4 to Birinapant-treated cells significantly reduced secretion of TNFα and blocked Birinapant-induced apoptosis. This suggests that A4 acts by specifically targeting XIAP. The effect of A4 was selective as peripheral blood mononuclear cells and normal human breast epithelial cells were unaffected. Furthermore, proteome analysis revealed that cancer cell lines with high levels of XIAP were particularly sensitive to the killing effect of A4. These results provide proof of concept that the ARTS binding site in XIAP is "druggable". A4 represents a novel class of dual-targeting compounds stimulating apoptosis by UPS-mediated degradation of important anti-apoptotic oncogenes.

PAX8 activates a p53-p21-dependent pro-proliferative effect in high grade serous ovarian carcinoma.

High grade serous carcinoma (HGSC) is the most common subtype of ovarian cancer and it is now widely accepted that this disease often originates from the fallopian tube epithelium. PAX8 is a fallopian tube lineage marker with an essential role in embryonal female genital tract development. In the adult fallopian tube, PAX8 is expressed in the fallopian tube secretory epithelial cell (FTSEC) and its expression is maintained through the process of FTSEC transformation to HGSC. We now report that PAX8 has a pro-proliferative and anti-apoptotic role in HGSC. The tumor suppressor gene TP53 is mutated in close to 100% of HGSC; in the majority of cases, these are missense mutations that endow the mutant p53 protein with potential gain of function (GOF) oncogenic activities. We show that PAX8 positively regulates the expression of TP53 in HGSC and the pro-proliferative role of PAX8 is mediated by the GOF activity of mutant p53. Surprisingly, mutant p53 transcriptionally activates the expression of p21, which localizes to the cytoplasm of HGSC cells where it plays a non-canonical, pro-proliferative role. Together, our findings illustrate how TP53 mutations in HGSC subvert a normal regulatory pathway into a driver of tumor progression.

Degradation of Bcl-2 by XIAP and ARTS Promotes Apoptosis.

We describe a mechanism by which the anti-apoptotic B cell lymphoma 2 (Bcl-2) protein is downregulated to induce apoptosis. ARTS (Sept4_i2) is a tumor suppressor protein that promotes cell death through specifically antagonizing XIAP (X-linked inhibitor of apoptosis). ARTS and Bcl-2 reside at the outer mitochondrial membrane in living cells. Upon apoptotic induction, ARTS brings XIAP and Bcl-2 into a ternary complex, allowing XIAP to promote ubiquitylation and degradation of Bcl-2. ARTS binding to Bcl-2 involves the BH3 domain of Bcl-2. Lysine 17 in Bcl-2 serves as the main acceptor for ubiquitylation, and a Bcl-2 K17A mutant has increased stability and is more potent in protection against apoptosis. Bcl-2 ubiquitylation is reduced in both XIAP- and Sept4/ARTS-deficient MEFs, demonstrating that XIAP serves as an E3 ligase for Bcl-2 and that ARTS is essential for this process. Collectively, these results suggest a distinct model for the regulation of Bcl-2 by ARTS-mediated degradation.

The tryptophan pathway genes of the Sargasso Sea metagenome: new operon structures and the prevalence of non-operon organization.

BACKGROUND: The enormous database of microbial DNA generated from the Sargasso Sea metagenome provides a unique opportunity to locate genes participating in different biosynthetic pathways and to attempt to understand the relationship and evolution of those genes. In this article, an analysis of the Sargasso Sea metagenome is made with respect to the seven genes of the tryptophan pathway. RESULTS: At least 5% of all the genes that are related to amino acid biosynthesis are tryptophan (trp) genes. Many contigs and scaffolds contain whole or split operons that are similar to previously analyzed trp gene organizations. Only two scaffolds discovered in this analysis possess a different operon organization of tryptophan pathway genes than those previously known. Many marine organisms lack an operon-type organization of these genes or have mini-operons containing only two trp genes. In addition, the trpB genes from this search reveal that the dichotomous division between trpB_1 and trpB_2 also occurs in organisms from the Sargasso Sea. One cluster was found to contain trpB sequences that were closely related to each other but distinct from most known trpB sequences. CONCLUSION: The data show that trp genes are widely dispersed within this metagenome. The novel organization of these genes and an unusual group of trpB_1 sequences that were found among some of these Sargasso Sea bacteria indicate that there is much to be discovered about both the reason for certain gene orders and the regulation of tryptophan biosynthesis in marine bacteria.