Apoptotic signalling targets the post-endocytic sorting machinery of the death receptor Fas/CD95.

Fas plays a major role in regulating ligand-induced apoptosis in many cell types. It is well known that several cancers demonstrate reduced cell surface levels of Fas and thus escape a potential control system via ligand-induced apoptosis, although underlying mechanisms are unclear. Here we report that the endosome associated trafficking regulator 1 (ENTR1), controls cell surface levels of Fas and Fas-mediated apoptotic signalling. ENTR1 regulates, via binding to the coiled coil domain protein Dysbindin, the delivery of Fas from endosomes to lysosomes thereby controlling termination of Fas signal transduction. We demonstrate that ENTR1 is cleaved during Fas-induced apoptosis in a caspase-dependent manner revealing an unexpected interplay of apoptotic signalling and regulation of endolysosomal trafficking resulting in a positive feedback signalling-loop. Our data provide insights into the molecular mechanism of Fas post-endocytic trafficking and signalling, opening possible explanations on how cancer cells regulate cell surface levels of death receptors.

Proteins interacting with cloning scars: a source of false positive protein-protein interactions.

A common approach for exploring the interactome, the network of protein-protein interactions in cells, uses a commercially available ORF library to express affinity tagged bait proteins; these can be expressed in cells and endogenous cellular proteins that copurify with the bait can be identified as putative interacting proteins using mass spectrometry. Control experiments can be used to limit false-positive results, but in many cases, there are still a surprising number of prey proteins that appear to copurify specifically with the bait. Here, we have identified one source of false-positive interactions in such studies. We have found that a combination of: 1) the variable sequence of the C-terminus of the bait with 2) a C-terminal valine "cloning scar" present in a commercially available ORF library, can in some cases create a peptide motif that results in the aberrant co-purification of endogenous cellular proteins. Control experiments may not identify false positives resulting from such artificial motifs, as aberrant binding depends on sequences that vary from one bait to another. It is possible that such cryptic protein binding might occur in other systems using affinity tagged proteins; this study highlights the importance of conducting careful follow-up studies where novel protein-protein interactions are suspected.

Expression of the major fas family and Bcl-2 family of proteins in epithelial ovarian cancer (EOC) and their correlation to chemotherapeutic response and outcome.

Platinum-based chemotherapeutic drugs trigger apoptosis, and deregulation of apoptotic pathways may contribute to chemoresistance. We investigated the role of major Fas and Bcl-2 family members as predictors of response to platinum-based chemotherapy in epithelial ovarian cancer (EOC). The expression of Fas, FasL, FAP-1, Bax, Bcl-2, and Bcl-X(L) was analyzed in 35 women with EOC at the transcript level by semiquantitative RT-PCR and at the protein level by immunohistochemistry. The apoptotic index was determined by TUNEL assay. The response to chemotherapy was documented and at the end of six cycles of chemotherapy. Based on their response, two groups were identified: primary chemosensitive (n = 20) and primary chemoresistant (n = 15). Further, after a follow-up of 12-46 months, two groups were identified: no evidence of disease (n = 10) and evidence of disease (n = 25). The primary chemoresistant tumors in comparison to the chemosensitive tumors had significantly lower levels of Fas transcript (p = 0.026), Bax transcript (p = 0.042) and Bcl-2 protein (p = 0.038) and higher levels of Bcl-X(L) (p = 0.040). The apoptotic index revealed a significant inverse correlation only with Bcl-X(L) protein levels (p = 0.003). Patients with evidence of disease at last follow-up in comparison to those with no evidence of disease showed lower Bax transcript (p = 0.012), Bcl-2 protein (p = 0.014) and lower apoptotic index (p = 0.005) and higher Bcl-X(L) protein levels (p = 0.023). In conclusion, Bcl-2 family members and apoptotic index are useful in prediction of response to chemotherapy in EOC. These initial observations need to be validated in large-scale studies.