Tumour-associated ligands of the activating NKG2D (natural killer group 2, member D; also called KLRK1) receptor-which are induced by genotoxic or cellular stress-trigger activation of natural killer cells and co-stimulation of effector T cells, and may thus promote resistance to cancer. However, many progressing tumours in humans counter this anti-tumour activity by shedding the soluble major histocompatibility complex class-I-related ligand MICA, which induces internalization and degradation of NKG2D and stimulates population expansions of normally rare NKG2D+CD4+ T cells with negative regulatory functions. Here we show that on the surface of tumour cells, MICA associates with endoplasmic reticulum protein 5 (ERp5; also called PDIA6 or P5), which, similar to protein disulphide isomerase, usually assists in the folding of nascent proteins inside cells. Pharmacological inhibition of thioreductase activity and ERp5 gene silencing revealed that cell-surface ERp5 function is required for MICA shedding. ERp5 and membrane-anchored MICA form transitory mixed disulphide complexes from which soluble MICA is released after proteolytic cleavage near the cell membrane. Reduction of the seemingly inaccessible disulphide bond in the membrane-proximal alpha3 domain of MICA must involve a large conformational change that enables proteolytic cleavage. These results uncover a molecular mechanism whereby domain-specific deconstruction regulates MICA protein shedding, thereby promoting tumour immune evasion, and identify surface ERp5 as a strategic target for therapeutic intervention.

powers erythrocyte invasion (6), while PfCDPK5 was shown to be critical for egress of P. falciparum merozoites from erythrocytes (7). In this report, we describe bumped kinase inhibitors (BKIs) that block infection of mosquitoes by malaria parasites. These compounds selectively and potently inhibit CDPK4, which is required for exflagellation of Plasmodium berghei microgametes (8) and has recently been shown to be connected with induction of exflagellation in P. falciparum microgametes (9), before fusion with the macrogamete, to form a zygote. The zygote undergoes transitional ookinete and oocyst stages to mature into infective sporozoites that are injected into a mammalian host during the female mosquito blood meal. Blocking exflagellation through the selective inhibition of Plasmodium CDPK4 would be expected to interrupt malaria transmission without being toxic to humans (10). Results and DiscussionWe have previously demonstrated that the ATP-binding pockets of Toxoplasma gondii and Cryptosporidium parvum CDPK1 can be selectively targeted by BKIs with large aromatic moieties displayed from the 3 position of the pyrazolopyrimidine scaffold due to the anomalously small gatekeeper residues (glycine) present in these kinases. Selective inhibition of Tg/CpCDPK1 with BKIs leads to blockage of mammalian-host cell invasion (11,12). PfCDPK4 has a serine at the gatekeeper position (Figure 1), smaller than the gatekeeper in almost all mammalian kinases, and an overall binding pocket that is very similar to those of TgCDPK1 and CpCDPK1. A number of compounds in our Tg/CpCDPK1 BKI library were found to inhibit recombinant PfCDPK4 (rPfCDPK4), the most potent being BKI-1 with an IC 50 (concentration to inhibit 50% of enzyme activity) of 4 nM (Table 1). However, not all compounds that are potent inhibitors of Tg/CpCDPK1 have comparable activity against rPfCDPK4. Despite the overall structural similarities in the ATP-binding pockets of CDPKs, small differences in the size of the gatekeeper residue may have a large effect on inhibitor potency.Effective control and eradication of malaria will require new tools to prevent transmission. Current antimalarial therapies targeting the asexual stage of Plasmodium do not prevent transmission of circulating gametocytes from infected humans to mosquitoes. Here, we describe a new class of transmission-blocking compounds, bumped kinase inhibitors (BKIs), which inhibit microgametocyte exflagellation. Oocyst formation and sporozoite production, necessary for transmission to mammals, were inhibited in mosquitoes fed on either BKI-1-treated human blood or mice treated with BKI-1. BKIs are hypothesized to act via inhibition of Plasmodium calcium-dependent protein kinase 4 and predicted to have little activity against mammalian kinases. Our data show that BKIs do not inhibit proliferation of mammalian cell lines and are well tolerated in mice. Used in combination with drugs active against asexual stages of Plasmodium, BKIs could prove an important tool for malaria control and eradication.

The matrilins form a four-member family of modular, multisubunit matrix proteins, which are expressed in cartilage but also in many other forms of extracellular matrix. They participate in the formation of fibrillar or filamentous structures and are often associated with collagens. It appears that they mediate interactions between collagen-containing fibrils and other matrix constituents, such as aggrecan. This adaptor function may be modulated by physiological proteolysis that causes the loss of single subunits and thereby a decrease in binding avidity. Attempts to study matrilin function by gene inactivation in mouse have been frustrating and so far not yielded pronounced phenotypes, presumably because of the extensive redundancy within the family allowing compensation by one family member for another. However, mutations in matrilin-3 in humans cause different forms of chondrodysplasias and perhaps also hand osteoarthritis. As loss of matrilin-3 is not critical in mouse, these phenotypes are likely to be caused by dominant negative effects.

The stimulatory natural killer group 2 member D (NKG2D) lymphocyte receptor and its tumor-associated ligands are important mediators in the immune surveillance of cancer. With advanced human tumors, however, persistent NKG2D ligand expression may favor tumor progression. We have found that cancer cells themselves express NKG2D in complex with the DNAX-activating protein 10 (DAP10) signaling adaptor. Triggering of NKG2D on ex vivo cancer cells or on tumor lines which express only few receptor complexes activates the oncogenic PI3K-protein kinase B (PKB/ AKT)-mammalian target of rapamycin (mTOR) signaling axis and downstream effectors, the ribosomal protein S6 kinase 1 (S6K1) and the translation initiation factor 4E-binding protein 1 (4E-BP1). In addition, as in lymphocytes, NKG2D ligand engagement stimulates phosphorylation of JNK and ERK in MAP kinase cascades. Consistent with these signaling activities, above-threshold expression of NKG2D-DAP10 in a ligand-bearing tumor line increases its bioenergetic metabolism and proliferation, thus suggesting functional similarity between this immunoreceptor and tumor growth factor receptors. This relationship is supported by significant correlations between percentages of cancer cells that are positive for surface NKG2D and criteria of tumor progression. Hence, in a conceptual twist, these results suggest that tumor co-option of NKG2D immunoreceptor expression may complement the presence of its ligands for stimulation of tumor growth.growth stimulation | signal transduction