Human CD137 (4-1BB), a member of the TNF receptor family, and its ligand CD137L (4-1BBL), are expressed on immune cells and tumor cells. CD137/CD137L interaction mediates bidirectional cellular responses of potential relevance in inflammatory diseases, autoimmunity and oncology. A soluble form of CD137 exists, elevated levels of which have been reported in patients with rheumatoid arthritis and various malignancies. Soluble CD137 (sCD137) is considered to represent a splice variant of CD137. In this report, however, evidence is presented that A Disintegrin and Metalloproteinase (ADAM)10 and potentially also ADAM17 are centrally involved in its generation. Release of sCD137 by transfected cell lines and primary T cells was uniformly inhibitable by ADAM10 inhibition. The shedding function of ADAM10 can be blocked through inhibition of its interaction with surface exposed phosphatidylserine (PS), and this effectively inhibited sCD137 generation. The phospholipid scramblase Anoctamin-6 (ANO6) traffics PS to the outer membrane and thus modifies ADAM10 function. Overexpression of ANO6 increased stimulated shedding, and hyperactive ANO6 led to maximal constitutive shedding of CD137. sCD137 was functionally active and augmented T cell proliferation. Our findings shed new light on the regulation of CD137/CD137L immune responses with potential impact on immunotherapeutic approaches targeting CD137.

Ca2+-activated Cl− channels (TMEM16, also known as anoctamins) perform important functions in cell physiology, including modulation of cell proliferation and cancer growth. Many members, including TMEM16F/ANO6, additionally act as Ca2+-activated phospholipid scramblases. We recently presented evidence that ANO6-dependent surface exposure of phosphatidylserine (PS) is pivotal for the disintegrin-like metalloproteases ADAM10 and ADAM17 to exert their sheddase function. Here, we compared the influence of seven ANO family members (ANO1, 4, 5, 6, 7, 9, and 10) on ADAM sheddase activity. Similar to ANO6, overexpression of ANO4 and ANO9 led to increased release of ADAM10 and ADAM17 substrates, such as betacellulin, TGFα, and amphiregulin (AREG), upon ionophore stimulation in HEK cells. Inhibitor experiments indicated that ANO4/ANO9-mediated enhancement of TGFα-cleavage broadened the spectrum of participating metalloproteinases. Annexin V-staining demonstrated increased externalisation of PS in ANO4/ANO9-overexpressing cells. Competition experiments with the soluble PS-headgroup phosphorylserine indicated that the ANO4/ANO9 effects were due to increased PS exposure. Overexpression of ANO4 or ANO9 in human cervical cancer cells (HeLa), enhanced constitutive shedding of the growth factor AREG and increased cell proliferation. We conclude that ANO4 and ANO9, by virtue of their scramblase activity, may play a role as important regulators of ADAM-dependent cellular functions.

Proteolytic ectodomain release is a key mechanism for regulating the function of many cell surface proteins. The sheddases ADAM10 and ADAM17 are the best-characterized members of the family of transmembrane disintegrin-like metalloproteinase. Constitutive proteolytic activities are low but can be abruptly upregulated via inside-out signaling triggered by diverse activating events. Emerging evidence indicates that the plasma membrane itself must be assigned a dominant role in upregulation of sheddase function. Data are discussed that tentatively identify phospholipid scramblases as central players during these events. We propose that scramblase-dependent externalization of the negatively charged phospholipid phosphatidylserine (PS) plays an important role in the final activation step of ADAM10 and ADAM17. In this manuscript, we summarize the current knowledge on the interplay of cell membrane changes, PS exposure, and proteolytic activity of transmembrane proteases as well as the potential consequences in the context of immune response, infection, and cancer. The novel concept that scramblases regulate the action of ADAM-proteases may be extendable to other functional proteins that act at the cell surface.

The cyanobacterium Synechocystis sp.PCC 6803 possesses a bidirectional NiFe-hydrogenase, HoxEFUYH. It functions to produce hydrogen under dark, fermentative conditions and photoproduces hydrogen when dark-adapted cells are illuminated. Unexpectedly, we found that the deletion of the large subunit of the hydrogenase (HoxH) in Synechocystis leads to an inability to grow on arginine and glucose under continuous light in the presence of oxygen. This is surprising, as the hydrogenase is an oxygen-sensitive enzyme. In wild-type (WT) cells, thylakoid membranes largely disappeared, cyanophycin accumulated, and the plastoquinone (PQ) pool was highly reduced, whereas ΔhoxH cells entered a dormant-like state and neither consumed glucose nor arginine at comparable rates to the WT. Hydrogen production was not traceable in the WT under these conditions. We tested and could show that the hydrogenase does not work as an oxidase on arginine and glucose but has an impact on the redox states of photosynthetic complexes in the presence of oxygen. It acts as an electron valve as an immediate response to the supply of arginine and glucose but supports the input of electrons from arginine and glucose oxidation into the photosynthetic electron chain in the long run, possibly via the NDH-1 complex. Despite the data presented in this study, the latter scenario requires further proof. The exact role of the hydrogenase in the presence of arginine and glucose remains unresolved. In addition, a unique feature of the hydrogenase is its ability to shift electrons between NAD(H), NADP(H), ferredoxin, and flavodoxin, which was recently shown in vitro and might be required for fine-tuning. Taken together, our data show that Synechocystis depends on the hydrogenase to metabolize organic carbon and nitrogen in the presence of oxygen, which might be an explanation for its prevalence in aerobic cyanobacteria.

ADAM17, a prominent member of the “Disintegrin and Metalloproteinase” (ADAM) family, controls vital cellular functions through the cleavage of transmembrane substrates, including epidermal growth factor receptor (EGFR) ligands such as transforming growth factor (TGF)-alpha and Epiregulin (EREG). Several ADAM17 substrates are relevant to oncogenesis and tumor growth. We have presented evidence that surface exposure of phosphatidylserine (PS) is pivotal for ADAM17 to exert sheddase activity. The scramblase Xkr8 is instrumental for calcium-independent exposure of PS in apoptotic cells. Xkr8 can be dually activated by caspase-3 and by kinases. In this investigation, we examined whether Xkr8 would modulate ADAM17 activity under apoptotic and non-apoptotic conditions. Overexpression of Xkr8 in HEK293T cells led to significantly increased caspase-dependent as well as PMA-induced release of EREG and TGF-alpha. Conversely, siRNA-mediated downregulation of Xkr8 in colorectal Caco-2 cancer cells led to decreased PS externalization upon induction of apoptosis, which was accompanied by reduced shedding of endogenously expressed EREG and reduced cell survival. We conclude that Xkr8 shares with conventional scramblases the propensity to upmodulate the ADAM-sheddase function. Liberation of growth factors could serve a rescue function in cells on the pathway to apoptotic death.