The clinical benefit for patients with diverse types of metastatic cancers that has been observed upon blockade of the interaction between PD-1 and PD-L1 has highlighted the importance of this inhibitory axis in the suppression of tumour-specific T-cell responses. Notwithstanding the key role of PD-L1 expression by cells within the tumour micro-environment, our understanding of the regulation of the PD-L1 protein is limited. Here we identify, using a haploid genetic screen, CMTM6, a type-3 transmembrane protein of previously unknown function, as a regulator of the PD-L1 protein. Interference with CMTM6 expression results in impaired PD-L1 protein expression in all human tumour cell types tested and in primary human dendritic cells. Furthermore, through both a haploid genetic modifier screen in CMTM6-deficient cells and genetic complementation experiments, we demonstrate that this function is shared by its closest family member, CMTM4, but not by any of the other CMTM members tested. Notably, CMTM6 increases the PD-L1 protein pool without affecting PD-L1 (also known as CD274) transcription levels. Rather, we demonstrate that CMTM6 is present at the cell surface, associates with the PD-L1 protein, reduces its ubiquitination and increases PD-L1 protein half-life. Consistent with its role in PD-L1 protein regulation, CMTM6 enhances the ability of PD-L1-expressing tumour cells to inhibit T cells. Collectively, our data reveal that PD-L1 relies on CMTM6/4 to efficiently carry out its inhibitory function, and suggest potential new avenues to block this pathway.

Please be advised that this information was generated on 2018-05-11 and may be subject to change. letters to natureSequences (MIPS) databases were reconciled. Ty elements and dubious open reading frames (ORFs) were excluded. The data set (5,790 proteins) and search results can be viewed at the URL http://acer.gen.tcd.ie/~khwolfe/yeast. Repetitive regions within proteins were masked using the SEG filter in BLAST.Statistical analysis. Chi-square tests (data not shown) indicate that duplicated genes in yeast are distributed in a highly non-random manner with regard to both the order in which homologous genes occur on pairs of chromosomes and the transcriptional orientations of those genes. A simultaneous origin of duplicate regions, as opposed to 55 independent duplications, is supported by a chi-square test on block orientations and by the lack of triplicated regions. The Poisson expectation if blocks were duplicated sequentially is for approximately 40 duplicated blocks, and 7 blocks that are replicated more than once (mainly triplicated). There is only one possible candidate for a triplicated region: the genes YDR474Q YDR492W10 inferred substitutions on branch A. One gene pair, ORC1/SÏR3, was omitted because one of the yeast genes appeared more similar to its human homologue than to its duplicate.

Following caspase-8 mediated cleavage, a carboxylterminal fragment of the BH3 domain-only Bcl-2 family member Bid transmits the apoptotic signal from death receptors to mitochondria. In a screen for possible regulators of Bid, we defined Bfl-1/A1 as a potent Bid interacting protein. Bfl-1 is an anti-apoptotic Bcl-2 family member, whose preferential expression in hematopoietic cells and endothelium is controlled by inflammatory stimuli. Its mechanism of action is unknown. We find that Bfl-1 associates with both full-length Bid and truncated (t)Bid, via the Bid BH3 domain. Cellular expression of Bfl-1 confers protection against CD95-and Trail receptor-induced cytochrome c release. In vitro assays, using purified mitochondria and recombinant proteins, demonstrate that Bfl-1 binds full-length Bid, but does not interfere with its processing by caspase-8, or with its mitochondrial association. Confocal microscopy supports that Bfl-1, which at least in part constitutively localizes to mitochondria, does not impede tBid translocation. However, Bfl-1 remains tightly and selectively bound to tBid and blocks collaboration between tBid and Bax or Bak in the plane of the mitochondrial membrane, thereby preventing mitochondrial apoptotic activation. Lack of demonstrable interaction between Bfl-1 and Bak or Bax in the mitochondrial membrane suggests that Bfl-1 generally prevents the formation of a pro-apoptotic complex by sequestering BH3 domainonly proteins.

Figure 4CD95 induces cyt c release in a Bcl-2-and FLIPL-inhibitable manner. Cytosolic extracts (10 µg of protein) of J neo and J Bcl-2 (a), and J16 and JFL2 (b), prepared after the indicated times of treatment with anti-CD95 mAb (200 ng/ml), were separated by SDS-PAGE, and cyt c and actin content were evaluated by immunoblotting.