InteractiveFly: GeneBrief

Tace: Biological Overview | References

Frazzled (Fra) and deleted in colorectal cancer (Dcc) are homologous receptors that promote axon attraction in response to netrin. In Drosophila, Fra also acts independently of netrin by releasing an intracellular domain (ICD) that activates gene transcription. How neurons coordinate these pathways to make accurate guidance decisions is unclear. This study shows that the ADAM metalloprotease Tace cleaves Fra, and this instructs the switch between the two pathways. Genetic manipulations that either increase or decrease Tace levels disrupt midline crossing of commissural axons. These conflicting phenotypes reflect Tace's function as a bi-directional regulator of axon guidance, a function conserved in its vertebrate homolog ADAM17: while Tace induces the formation of the Fra ICD to activate transcription, excessive Tace cleavage of Fra and Dcc suppresses the response to netrin. It is proposed that Tace and ADAM17 are key regulators of midline axon guidance by establishing the balance between netrin-dependent and netrin-independent signaling (Zang, 2022).

This study reports a conserved role for Tace and ADAM17 in coordinating different receptor signaling outputs. In the context of commissural axon guidance, strong biochemical and genetic evidence is provided demonstrating that Tace functions in both the canonical netrin-dependent Fra pathway and the non-canonical netrin-independent Fra pathway. Specifically, this study has shown that in the non-canonical pathway, Tace cleaves Fra to induce the formation of a transcriptionally active ICD fragment that regulates the expression of Fra's target gene comm. In the canonical pathway, overexpression of Tace results in excessive cleavage of Fra that inhibits the receptor's ability to respond to netrin. Because both Fra pathways are essential for proper midline attraction, such bi-directional regulation by Tace could assist the neurons in orchestrating appropriate signaling outputs. Thus, a model is proposed in which the levels and/or activity of Tace must be correctly regulated to ensure that Fra signals effectively. When the levels or activities of Tace are low, the commissural neuron favors the netrin-dependent canonical pathway to promote midline crossing. In contrast, when Tace is activated, the commissural neuron decreases its responsiveness to netrin and switches to the non-canonical Fra pathway to regulate gene transcription. Together, these two pathways are maintained in a tightly controlled balance and cooperate to facilitate commissural axon midline crossing. Importantly, this study also demonstrated a clear functional similarity between Tace and ADAM17, arguing that ADAM17-dependent cleavage of Dcc likely allows for a similar segregation of receptor signaling outputs in vertebrate systems (Zang, 2022).

Previous studies have revealed that ADAM-dependent proteolysis can impinge on downstream signaling pathways of guidance receptors in many ways. First, ADAM cleavage can terminate receptor signaling by reducing receptor surface levels. Second, ADAM cleavage can facilitate association between the receptor and its downstream effector proteins.Third, ADAM cleavage can physically separate receptor-ligand complexes to switch adhesion to repulsion. Previous studies have demonstrated that both of Fra's vertebrate homologs, Dcc and neogenin, are also ADAM substrates, yet the physiological significance and the precise mechanisms of how proteolysis affects downstream signaling of these receptors remain unclear. This study has shown that in Drosophila, Tace induces the formation of both Fra ECDs and ICDs and is required for the expression of Fra's transcriptional target gene comm. Unlike the mechanisms described above, the current data establish a distinct pathway in which an ADAM can activate an axon guidance receptor by initiating subsequent γ-secretase cleavage to induce transcriptional activity of the receptor. It remains to be seen whether the same non-canonical mechanism is conserved for ADAM17 and Fra's vertebrate homologs Dcc and neogenin. In Drosophila commissural neurons, γ-secretase cleaves Fra to generate an ICD fragment, allowing it to translocate to the nucleus to regulate the transcription of comm (Zang, 2022).

Previous studies have demonstrated that the neogenin ICD is present in the nucleus in both chick retinal ganglion cells and zebrafish embryos. In addition, in vitro evidence from cell lines suggests that both neogenin and Dcc ICDs can localize to the nucleus to modulate gene transcription, suggesting that similar non-canonical pathways exist for Dcc and neogenin as well. This study has shown that conditional removal of Adam17 disrupts midline crossing in vivo. If the only function of ADAM17 at the vertebrate midline is to downregulate netrin and Dcc signaling, it would be expected to see increased crossing when ADAM17 levels are reduced. Thus, in vertebrates, ADAM17 behaves similarly to Tace in Drosophila to positively regulate midline crossing. This observation supports a potential role for ADAM17 in activating the transcriptional activities of Dcc at the vertebrate midline (Zang, 2022).

While Dcc is arguably the most likely substrate, it should be noted that analysis in embryonic mouse spinal cords does not exclude the possibility that ADAM17 functions by regulating other substrates. One possibility is neogenin, which, in addition to netrin, also binds to its canonical ligand repulsive guidance molecule (RGM) with much higher affinity. Cleavage of neogenin by ADAM17 desensitizes axons to RGM, which abolishes its repulsive effects on neurite outgrowth. Importantly, neogenin contributes to commissure formation in the mouse spinal cord by binding and facilitating Dcc and netrin signaling through its ECDs (Zang, 2022).

Thus, it is unlikely that neogenin is the primary ADAM17 substrate, as shedding of its ECDs should have deleterious effects on midline guidance, which is contrary to what wsd observed in Adam17 cKO embryos. Another possible substrate is the repulsive receptor Robo1. While it has not been determined whether Robo1 is a substrate of ADAM17, it is possible that cleavage of Robo1 inhibits its repulsive function, which could explain the reduced commissure formation observed in Adam17 cKO embryos. One would predict that, as a result, overexpression of Tace or ADAM17 should inhibit Robo1-mediated repulsion, which is contrary to what was observed in Drosophila. This again argues against Robo1 as the primary substrate mediating the phenotypes observed in Adam17 cKO embryos (Zang, 2022).

The data suggest that Tace and ADAM17 are strictly regulated to achieve a precise balance of Fra and Dcc signaling outputs. What are the mechanisms involved in commissural neurons to modulate Tace and ADAM17 expression and/or activity? In principle, this regulation could occur either at the metalloprotease level or at the substrate level. First, the surface expression or the activity of Tace and ADAM17 could be regulated. This study has demonstrated that tace and Adam17 transcripts are highly expressed in the embryonic CNS of both invertebrates and vertebrates. In Drosophila, the expression of Tace mRNA and protein does not appear to be temporally controlled, suggesting that Tace activity is likely to be regulated post-translationally. Indeed, a number of molecules have been identified as regulators of ADAM17 activity, including tissue inhibitor of metalloproteinases-3 (TIMP-3) which suppresses ADAM17 catalytic activity, and the adapter proteins iRhom1 and iRhom2, which are involved in ADAM17 maturation and stability. Thus, it would be interesting to explore the potential roles of TIMP-3 and the iRhoms and their Drosophila orthologs during axon guidance (Zang, 2022).

Alternatively, regulation of Tace and ADAM17 function could occur at the substrate level. Ligand binding could induce conformational changes in the substrate to facilitate its association with the metalloprotease, or an interacting protein could bind to the substrate to block association with the metalloprotease. Previous work has shown that netrin does not activate the transcriptional activity of Fra,sugge sting that an alternative ligand may exist for the non-canonical pathway. In mouse cortical neurons, leucine-rich repeats and immunoglobulin-like domains 2 (Lrig2) bind to neogenin to inhibit premature ADAM17 cleavage (Zang, 2022).

It remains to be seen whether similar mechanisms regulate Fra and Dcc cleavage. Tace and ADAM17 coordinate the canonical and non-canonical pathways ADAMs can facilitate a switch in responses to guidance cues. For example, ADAM10 and ADAM17 cleave Neuropilin-1 to regulate proprioceptive axon responsiveness to Sema3A. In addition, ADAM10 cleaves ephrinA5 to convert EphA3-ephrinA5-mediated adhesion to repulsion. It is proposed that Tace and ADAM17 can instruct neurons to coordinate the canonical and non-canonical pathways. It is important to point out that, instead of producing opposite signaling outcomes, the canonical and non-canonical pathways both promote midline crossing of commissural axons, which suggests that the two pathways cooperate instead of competing. This distinguishes the current model from existing mechanisms, but at the same time inevitably poses the question: how does the neuron decide between the two pathways? It is speculated that both pathways could be engaged in the same cell but are separated spatially and/or temporally. The canonical pathway is activated at the tip of the axon, where Fra responds to netrin to locally regulate the cytoskeleton (Zang, 2022).

It is possible that the non-canonical Fra pathway is activated in the soma instead, where the cleaved Fra ICDs are within close proximity to the nucleus. This is supported by the observation that both endogenous Tace and overexpressed Tace are almost exclusively detected in the cell soma but not on the axons. In addition, the two pathways could be activated by distinct ligands, so that they are controlled at different developmental time points. Given the diverse roles of ADAM family metalloproteases in development and disease, continued investigation into their regulation and mechanism of action will undoubtedly offer important biological insights (Zang, 2022).

While this study demonstrated that Tace/ADAM17 is a key regulator of Fra/Dcc signaling and controls midline axon guidance, this study has its limitations. First, tace zygotic mutants in Drosophila show midline crossing defects with low penetrance, which is likely the result of a maternal effect. Yet it was not possible to definitively test this by generating maternal zygotic tace mutants, due to developmental arrest potentially linked to its function in the Notch pathway. Second, it remains to be seen if the same non-canonical signaling mechanism is conserved for Dcc in vertebrate systems. Future transcriptomic studies comparing gene expression levels in controls and Dcc cKO embryos or cell lines, or cell lines overexpressing Dcc ICD, could help resolve its role in transcriptional regulation. Third, it is unclear if Tace/ADAM17 is required cell autonomously in commissural neurons or cell non-autonomously in neighboring cells to cleave Fra/Dcc. Generating commissural neuron-specific cKO animals in the future could provide definitive answers. Fourth, the commissure formation deficits observed in Adam17 mutants are not causatively linked to a lack of Dcc cleavage. Finally, it remains to be determined what the biological stimulus that activates the non-canonical pathway is (Zang, 2022).

ADAM17-triggered TNF signalling protects the ageing Drosophila retina from lipid droplet-mediated degeneration

Animals have evolved multiple mechanisms to protect themselves from the cumulative effects of age-related cellular damage. This study revealed an unexpected link between the TNF (tumour necrosis factor) inflammatory pathway, triggered by the metalloprotease ADAM17/TACE, and a lipid droplet (LD)-mediated mechanism of protecting retinal cells from age-related degeneration. Loss of ADAM17, TNF and the TNF receptor Grindelwald in pigmented glial cells of the Drosophila retina leads to age-related degeneration of both glia and neurons, preceded by an abnormal accumulation of glial LDs. The glial LDs initially buffer the cells against damage caused by glial and neuronally generated reactive oxygen species (ROS), but in later life the LDs dissipate, leading to the release of toxic peroxidated lipids. Finally, this study demonstrates the existence of a conserved pathway in human iPS-derived microglia-like cells, which are central players in neurodegeneration. Overall, this study has discovered a pathway mediated by TNF signalling acting not as a trigger of inflammation, but as a cytoprotective factor in the retina (Muliyil, 2020).

This paper reports a previously unrecognised role of ADAM17 and TNF in protecting Drosophila retinal cells from age- and activity-related degeneration. Loss of ADAM17 and TNF signalling in retinal glial cells causes an abnormal accumulation of LDs in young glial cells. These LDs disperse by about 2 weeks after eclosion (middle age for flies), and their loss coincides with the onset of severe glial and neuronal cell death. By 4 weeks of age, no intact glia or neurons remain. Cell death depends on neuronal activity: retinal degeneration and, to a lesser extent, LD accumulation are rescued in flies reared fully in the dark. LD accumulation does not merely precede, but is actually responsible for subsequent degeneration, because preventing the accumulation of LDs fully rescues cell death. The data indicate that Eiger/TNF released by ADAM17 acts specifically through the Grindelwald TNF receptor. Loss of ADAM17-mediated TNF signalling also leads to elevated production of mitochondrial ROS in glial cells, causing activation of the JNK pathway and elevated lipogenic gene expression. Together, these changes trigger cell death through the production of toxic peroxidated lipids. Importantly, toxicity is also contributed to by ROS generated by normal activity of neighbouring neurons. Finally, this study shows that a similar signalling module is conserved in mammalian cells: when ADAM17 is inhibited in human iPSC-derived microglial-like cells, the same series of events is seen: LD accumulation, elevated mitochondrial ROS and high levels of toxic peroxidated lipids (Muliyil, 2020).

It is proposed that TNF is an autocrine trophic factor that protects retinal pigmented glial cells from age-related cumulative damage caused by the ROS that are normal by-products of neuronal activity. This ADAM17/TNF protection system is located specifically in retinal glial cells, but its role is to protect both glia and neighbouring neurons. In the absence of this TNF cytoprotective pathway, severe early-onset retinal neurodegeneration is seen. The data imply that cells die by being overwhelmed by toxic peroxidated lipids when abnormal accumulations of LDs disperse. This occurs in Drosophila middle age, when LDs stop accumulating and begin to disperse, triggering the cytotoxic phase of the ADAM17-/- phenotype. It is important to emphasise that despite the ADAM17/TNF protection system being located specifically in retinal glial cells, there is neuronal involvement. Not only does TNF indirectly protect against neurodegeneration, but photoreceptor neurons are also significant sources of the ROS that generate the toxic peroxidated lipids in glia. More generally, this work provides a model for investigating more widely the functional links between ageing, cellular stress, lipid droplet accumulation and neurodegeneration. Indeed, in the light of the discovery that the pathway discovered in Drosophila is conserved in human microglia-like cells, it is significant that lipid droplets have been reported to accumulate in human microglia, cells that are increasingly prominent in the pathology of Alzheimer's disease and other neurodegenerative conditions (Muliyil, 2020).

In a Drosophila model of neuronal mitochondrionopathies, abnormal neuronal ROS production led to elevated neuronal lipid production, followed by transfer of the lipids to the PGCs, where LDs accumulated. In that work, lipase expression in neurons suppressed LD accumulation; in contrast, suppression was only observed when lipase was expressed in PGCs, not neurons, suggesting that in the case of ADAM17 mutants, the primary source of accumulating lipids is the glial cells. Despite not being able to detect a role for neuronal lipid production in ADAM17-/- mutants, it was found that photoreceptor neurons are significant sources of the ROS that generate the toxic peroxidated lipids in glia. Despite these differences between this work and what has been previously reported, a growing body of work points to a close coupling between ROS, the JNK pathway, lipid droplets and cellular degeneration, a relationship conserved in mammals. This study did not investigate the involvement of SREBP in mediating LD accumulation caused by ADAM17 loss, but its well-established connection with stress-induced and JNK-mediated lipid synthesis suggests that it is a likely additional shared component of this conserved regulatory axis (Muliyil, 2020).

It has become clear that LDs are much more than simply passive storage vessels for cellular lipids; they have multiple regulatory functions. Indeed, although this study highlighted a developing picture of an LD/ROS-dependent trigger of cell death, in other contexts LDs have protective functions against oxidative damage, both in flies and mammals. This may occur by providing an environment that shields fatty acids from peroxidation by ROS and/or by sequestering toxic peroxidated lipids. Although this superficially appears to contradict the theme of LD/ROS toxicity, it is important to recall that in LD-related cell death is not simultaneous with LD accumulation. In fact, degeneration temporally correlates with the dispersal of LDs in middle age, rather than their earlier accumulation. Together, the strands of evidence from several studies suggest that it is the combination of elevated ROS and the dispersal of abnormally high quantities of lipids from previously accumulated LDs that trigger death. This suggests that cells die by being overwhelmed by toxic peroxidated lipids when abnormal accumulations of LDs break down in the presence of high levels of ROS. Experiments with Brummer lipase are consistent with this idea: the Brummer lipase was expressed from early in development, thereby preventing abnormal LD accumulation, and this protected against cell death. This sequence of events implies the existence of a metabolic switch, when LDs stop accumulating and begin to disperse, triggering the toxic phase of ADAM17 loss. It will be interesting in the future and may provide insights into the normal ageing process, to understand the molecular mechanism of this age- and/or activity-dependent change (Muliyil, 2020).

ADAM17 is one of the most important shedding enzymes in humans, responsible for the proteolytic release of a vast array of cell surface signals, receptors and other proteins. Because of its role in signalling by both TNF and ligands of the EGF receptor, it has been the focus of major pharmaceutical efforts, with a view to treating inflammatory diseases and cancer. It is therefore surprising that it has been very little studied in Drosophila. This is the first report of Drosophila ADAM17 mutants. This study also confirmed for the first time that Drosophila ADAM17 is indeed an active metalloprotease, able to shed cell surface proteins including the Drosophila TNF homologue Eiger. The only other description of Drosophila ADAM17 function is mechanistically consistent with the data, despite relating to a different physiological context. In that case, ADAM17 was shown to cause the release of soluble TNF from the fat body so that it can act as a long range adipokine (Agrawal, 2016). Other ADAM17 substrates in different developmental or physiological contexts cannot be ruled out, although the relatively subtle phenotype of null mutant flies implies that ADAM17 does not have essential functions that lead to obvious defects when mutated. Moreover, wdevelopmental defects or LD accumulation were not observed in any neuronal or non-neuronal ADAM17-/- larval tissues, suggesting that the mechanism reported in this study is both age and tissue specific (Muliyil, 2020).

Although TNF is sometimes viewed as a specific cell death-promoting signal, and the pathways by which it activates caspase-induced apoptosis have been studied extensively in flies and mammals, the response to TNF is in fact very diverse, depending on the biological context. Indeed, its most well-studied role in mammals is as the primary inflammatory cytokine, released by macrophages and other immune cells, and triggering the release of other cytokines, acting as a chemoattractant, stimulating phagocytosis, and promoting other inflammatory responses. However, TNF has not previously been shown to have trophic activity, protecting cells in the nervous system from stress-induced damage, although a link with the Nrf2/Keap1 redox pathway in cardiomyocytes provides an interesting parallel (Muliyil, 2020).

In conclusion, this work highlights three important biological concepts. The first is to identify a new function for the ADAM17/TNF pathway in a cytoprotective role that protects Drosophila retinal cells against age- and activity-dependent degeneration. This contrasts with its well-established roles in inflammation and cell death. Secondly, the existence is highlighted of a glia-centric cellular pathway by which the breakdown of accumulated LDs and ROS together participate in promoting stress-induced and age-related cell death. Finally, this study has shown that the core phenomenon of the ADAM17 protease, acting to regulate the homeostatic relationship between ROS and LD biosynthesis, is conserved in human microglial cells, which themselves are intimately involved in neuroprotection (Muliyil, 2020).

Proteolytic maturation of Drosophila Neuroligin 3 by tumor necrosis factor alpha-converting enzyme in the nervous system.

The functions of autism-associated Neuroligins (Nlgs) are modulated by their post-translational modifications, such as proteolytic cleavage. A previous study has shown that there are different endogenous forms of DNlg3 in Drosophila, indicating it may undergo proteolytic processing. However, the molecular mechanism underlying DNlg3 proteolytic processing is unknown. This study reports a novel proteolytic mechanism that is essential for DNlg3 maturation and function in the nervous system. Molecular cloning, cell culture, immunohistochemistry, western blotting and genetic studies were employed to map the DNlg3 cleavage region, identify the protease and characterize the cleavage manner. Behavior analysis, immunohistochemistry and genetic manipulations were employed to study the functions of different DNlg3 forms in the nervous system and neuromuscular junction (NMJs). Tumor necrosis factor alpha-converting enzyme (TACE) cleaved DNlg3 exclusively at its extracellular acetylcholinesterase-like domain to generate the N-terminal fragment and the short membrane-anchored fragment (sDNlg3). DNlg3 was constitutively processed in an activity-independent manner. Interestingly, DNlg3 was cleaved intracellularly in the Golgi apparatus before it arrived at the cell surface, a unique cleavage mechanism that is distinct from 'conventional' ectodomain shedding of membrane proteins, including rodent Nlg1. Genetic studies showed that sDNlg3 was essential for maintaining proper locomotor activity in Drosophila. These results revealed a unique cleavage mechanism of DNlg3 and a neuron-specific role for DNlg3 maturation which is important in locomotor activity. This study provides a new insight into a cleavage mechanism of Nlgs maturation in the nervous system (Wu, 2018).

The Drosophila TNF Eiger is an adipokine that acts on insulin-producing cells to mediate nutrient response

Adaptation of organisms to ever-changing nutritional environments relies on sensor tissues and systemic signals. Identification of these signals would help understand the physiological crosstalk between organs contributing to growth and metabolic homeostasis. This study shows that Eiger, the Drosophila TNF-alpha, is a metabolic hormone that mediates nutrient response by remotely acting on insulin-producing cells (IPCs). In the condition of nutrient shortage, a metalloprotease of the TNF-alpha converting enzyme (TACE) family protein CG7908 is active in fat body (adipose-like) cells, allowing the cleavage and release of adipose Eiger in the hemolymph. In the brain IPCs, Eiger activates its receptor Grindelwald, leading to JNK-dependent inhibition of insulin production. Therefore, this study has identified a humoral connexion between the fat body and the brain insulin-producing cells relying on TNF-alpha that mediates adaptive response to nutrient deprivation (Agrawal, 2016).

This study shows that the fly TNF Eiger functions as a metabolic hormone produced by the fat body in response to chronic protein deprivation. Elevated circulating levels of human TNF-α and TNFR are also observed in malnutrition conditions and in catabolic states associated with cachexia induced by sepsis and cancer. Moreover, this study presents evidence that the molecular mechanism leading to decreased insulin expression by TNF-α is conserved in mammalian β cells. Therefore, activation of TNF signaling could be an evolutionary conserved response to undernutrient imbalance, recently highjacked as an adaptive response to continuous nutritional surplus. Drosophila Eiger has so far mostly been implicated in local, autonomous responses, activating JNK signaling in the cells or tissues that express it. In recent studies, however, it has been postulated that Egr can diffuse outside of its expression domain in a paracrine manner. The current data now demonstrate that Egr circulates in the hemolymph and acts remotely, allowing crossorgan communication. This raises questions relative to the mode of transport of Eiger in the hemolymph and its specificity of action on remote target tissues. Indeed, although overexpression of Egr in fat cells leads to a strong increase in Egr levels in the hemolymph, flies show no obvious defects, suggesting that secreted Egr has limited access to peripheral tissues while in the hemolymph. Previous studies have shown that human and mouse TNF-α efficiently cross the blood-brain barrier (BBB) after i.v. injection and are detected in the cerebrospinal fluid in a process requiring the presence of TNF receptors in glial cells. This study shows that hemolymph Eiger can penetrate the brain and access to the insulin-producing cells. It will be important to evaluate in future experiments the mechanisms by which Egr travels in the hemolymph and across the larval BBB (Agrawal, 2016).

This study shows that an important aspect of Egr secretion relies on its shedding from the membrane by the convertase enzyme TACE. Drosophila TACE was recently shown to be required for Egr function in a tumor model using activated ras (Chabu, 2014). This study shows that transcription of TACE, but not egr, is induced in fat cells under low-protein diet (LPD) and that adipose TACE activity is critical for metabolic adaptation to low protein. Moreover, a genetic link was identified between TACE expression and TORC1, the main amino acid sensor in fat cells. Interestingly, REPTOR and REPTOR-BP, two transcription factors that are responsible for most of the transcription response to TORC1 inhibition, are not required for TACE expression, suggesting that an alternative mechanism is required for TACE induction in response to TORC1 inhibition following exposure to LPD. Vertebrate TACE/ADAM17 acts on a small number of cytokines and growth factors including TNF-α and several membrane receptors (Menghini, 2013). Mice deficient in TACE function are lean and resistant to high-fat-diet-induced obesity and diabetes type 2, a range of phenotypes that could be linked to TNF-α shedding defects. In mammals, TACE activity is controlled through balanced expression of the Tissue Inhibitor of Metalloprotease 3 (TIMP3). Although a TIMP3 homolog exists in Drosophila, there is no indication that it participates in modulating Drosophila TACE activity in addition to TACE transcriptional activation observed in LPD (Agrawal, 2016).

Circulating Eiger produced by adipose cells remotely acts on the brain neurosecretory cells that produce insulin (IPCs), leading to general body growth inhibition. This correlates with specific expression of the TNF receptor Grindelwald in the IPCs. Indeed, knocking down Grnd in these neurons mimics the effect of knocking down Egr in the fat body. No effect is observed upon knocking down the other fly TNFR Wengen in IPCs, indicating that Grnd mediates Egr metabolic action and that specific targeting of TNF signaling to the IPCs is the consequence of localized Grnd expression. As a consequence of Grnd activation, JNK signaling is elevated in the IPCs of animals raised on a LPD. TNF signaling is not required in the IPCs for the retention of Dilp2 observed upon acute protein starvation. By contrast, activation of JNK in larval IPCs leads to reduced expression of Dilp2 and Dilp5, two major circulating Dilps. Strikingly, this is reminiscent of the role described for JNK in vertebrate pancreatic β cells. Indeed, JNK inhibitors increase insulin expression in Langerhans islets from obese mice, suggesting that JNK represses expression of the insulin gene. These results are also in line with the present finding that TNF-α inhibits INS1 and INS2 gene transcription from Min6 cells and mouse islets (Agrawal, 2016).

In conclusion, this work unravels an anciently conserved mechanism by which TNF signaling mediates direct response to low nutrient through a fat-brain crossorgan communication leading to the modulation of growth. Other signals contributing to reduction of insulin signaling in condition of nutrient shortage have recently been identified in Drosophila. Systemic Hedgehog is produced by gut cells in response to low nutrients and targets both fat cells and ecdysone-producing cells to adapt larval growth to nutrient shortage. A hormone called Limostatin produced by the corpora cardiaca in low-glucose condition acts directly on adult insulin-producing cells to block insulin secretion. These findings together with the present work indicate that a variety of signals allow the integration of different physiological contexts for the proper control of insulin production (Agrawal, 2016).

Oncogenic Ras stimulates Eiger/TNF exocytosis to promote growth

Oncogenic mutations in Ras deregulate cell death and proliferation to cause cancer in a significant number of patients. Although normal Ras signaling during development has been well elucidated in multiple organisms, it is less clear how oncogenic Ras exerts its effects. Furthermore, cancers with oncogenic Ras mutations are aggressive and generally resistant to targeted therapies or chemotherapy. This study identified the exocytosis component Sec15 as a synthetic suppressor of oncogenic Ras in an in vivo Drosophila mosaic screen. Oncogenic Ras elevates exocytosis and promotes the export of the pro-apoptotic ligand Eiger (Drosophila TNF). This blocks tumor cell death and stimulates overgrowth by activating the JNK-JAK-STAT non-autonomous proliferation signal from the neighboring wild-type cells. Inhibition of Eiger/TNF exocytosis or interfering with the JNK-JAK-STAT non-autonomous proliferation signaling at various steps suppresses oncogenic Ras-mediated overgrowth. These findings highlight important cell-intrinsic and cell-extrinsic roles of exocytosis during oncogenic growth and provide a new class of synthetic suppressors for targeted therapy approaches (Chabu, 2014).

The cellular release of soluble TNF from its precursor transmembrane form and its resulting activity are mediated by the TNF-converting enzyme, Tace (Black, 1997; Blobel, 1997; Moss, 1997). Eiger/TNF contains a Tace cleavage site equivalent to TNFα cleavage site in vertebrates. This study first verified that Drosophila Tace plays a similar role in Eiger/TNF signaling. First, it was found that RNAi knockdown of the sole Drosophila Tace in the developing eye suppressed the Eiger/TNF-mediated small-eye phenotype. Similar results were obtained in wing imaginal discs. Subsequently, tests were performed for a role for Tace in Eiger/TNF non-autonomous signaling using the hanging-eye phenotype model. It was found that coexpression of eiger and Tace in cells rescued by expression of the dominant-negative JNK allele bsk-DN in the developing eye (GMR>egr,Tace,bsk-DN) produced a hanging-eye phenotype. The majority of GMR>egr,Tace,bsk-DN animals die in late pupal stages (73%, N=157) and show a ring of necrotic tissue around the eyes. Interestingly, the GMR>egr,Tace,bsk-DN animals did not have a dramatic hanging-eye phenotype. These data indicate that Tace plays an important role in the secretion and signaling of Eiger. Finally, Tace was knocked down by RNAi in the RasV12 cells, and this was found to abolished Eiger/TNF accumulation around the RasV12 clones. Collectively, these data indicate that the pool of Eiger/TNF seen around RasV12 clones originates from RasV12 cells, and Sec15 plays an important role in Eiger/TNF protein export (Chabu, 2014).

Kuz and TACE can activate Notch independent of ligand

A central mechanism in activation of the Notch signaling pathway is cleavage of the Notch receptor by ADAM metalloproteases. ADAMs also cleave Delta, the ligand for Notch, thereby downregulating Notch signals. Two ADAMs, Kuzbanian (Kuz) and TNF-alpha converting enzyme (TACE), are known to process both Delta and Notch, yet the role of these cleavages in signal propagation has remained controversial. Using an in vitro model, this study shows that Kuz regulates Notch signaling primarily by activating the receptor and has little overall effect on signaling via disabling Delta. It was confirmed that Kuz-dependent activation of Notch requires stimulation of Notch by Delta. However, over-expression of Kuz gives ligand-independent Notch activation. In contrast, TACE, which is elevated in expression in the developing Drosophila nervous system, can efficiently activate Notch in a ligand-independent manner. Altogether, these data demonstrate the potential for Kuz and TACE to participate in context- and mechanism-specific modes of Notch activation (Delwig, 2008).

Drosophila TIMP is a potent inhibitor of MMPs and TACE: similarities in structure and function to TIMP-3

The four tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors that regulate the activity of matrix metalloproteinases (MMPs) and certain disintegrin and metalloproteinase (ADAM) family proteases in mammals. The protease inhibitory activity is present in the N-terminal domains of TIMPs (N-TIMPs). In this work, the N-terminal inhibitory domain of the only TIMP produced by Drosophila (dN-TIMP) was expressed in Escherichia coli and folded in vitro. The purified recombinant protein is a potent inhibitor of human MMPs, including membrane-type 1-MMP, although it lacks a disulfide bond that is conserved in all other known N-TIMPs. Titration with the catalytic domain of human MMP-3 [MMP-3(DeltaC)] showed that dN-TIMP prepared by this method is correctly folded and fully active. dN-TIMP also inhibits, in vitro, the activity of the only two MMPs of Drosophila, dm1- and dm2-MMPs, indicating that the Drosophila TIMP is an endogenous inhibitor of the Drosophila MMPs. dN-TIMP resembles mammalian N-TIMP-3 in strongly inhibiting human tumor necrosis factor-alpha-converting enzyme (TACE/ADAM17) but is a weak inhibitor of human ADAM10. Models of the structures of dN-TIMP and N-TIMP-3 are strikingly similar in surface charge distribution, which may explain their functional similarity. Although the gene duplication events that led to the evolutionary development of the four mammalian TIMPs might be expected to be associated with functional specialization, Timp-3 appears to have conserved most of the functions of the ancestral TIMP gene (Wei, 2003).

kuzbanian-mediated cleavage of Drosophila Notch

Loss of Kuzbanian, a member of the ADAM family of metalloproteases, produces neurogenic phenotypes in Drosophila. It has been suggested that this results from a requirement for Kuzbanian-mediated cleavage of the Notch ligand Delta. Using transgenic Drosophila expressing transmembrane Notch proteins, this study shows that Kuzbanian, independent of any role in Delta processing, is required for the cleavage of Notch. Kuzbanian can physically associate with Notch and removal of kuzbanian activity by RNA-mediated interference in Drosophila tissue culture cells eliminates processing of ligand-independent transmembrane Notch molecules. These data suggest that in Drosophila, kuzbanian can mediate S2 cleavage of Notch (Lieber, 2002).

TACE, another member of the ADAM family of metalloproteases, mediates S2 cleavage of mammalian N in vitro. Drosophila S2 cells do not contain any detectable TACE RNA, and exogenous TACE only poorly complements the RNAi-mediated loss of kuz activity. The restoration of some S3 product upon expression of TACE is in accord with the residual activity of LNR^LexA in kuz embryos, and in vitro TACE and N do interact, albeit less well than do Kuz and N. In the absence of a TACE mutant, it is not possible to say for certain whether kuz and TACE have redundant functions, if another member of the ADAM family is responsible for the residual S3 cleavage, or if the residual in vivo activity is caused by the expression of LNR^LexA from a heterologous promoter. Hardly any S3 product was generated from Delta1-18 LNR^LexA by exogenous TACE in S2 cells that had been treated with kuz double-stranded RNA, accounting for the in vivo Kuz-dependence. It is not clear why the ability of exogenous TACE to produce S3-cleaved N differs between LNR^LexA and Delta1-18 LNRLexA; however, in S2 cells, even in the presence of endogenous Kuz, Delta1-18 LNR^LexA is not cleaved as well as is LNR^LexA, suggesting that perhaps differences in the secondary structure of the molecules account for their differing responses to TACE (Lieber, 2002).

The pattern of cleavage products generated by expression of TACE in kuz^- S2 cells also provides an explanation for the in vivo biochemical data, which had seemed to suggest that kuz is responsible for S3 cleavage. It is intriguing that both in kuz embryos and in TACE-complemented kuz^- S2 cells there is an accumulation of a protein the size of S2-cleaved N, which is not efficiently cleaved further to produce S3-cleaved N. It is proposed that although TACE can cleave N at juxtamembrane sites, a large fraction of this cleavage is occurring at a site close to but distinct from the S2 site that allows for efficient S3 cleavage. This suggests that cleavage of N at any juxtamembrane site is not immediately followed by efficient S3 cleavage (Lieber, 2002).

Cloning of a disintegrin metalloproteinase that processes precursor tumour-necrosis factor-alpha

Tumour-necrosis factor-alpha (TNF-alpha) is a cytokine that contributes to a variety of inflammatory disease states. The protein exists as a membrane-bound precursor of relative molecular mass 26K which can be processed by a TNF-alpha-converting enzyme (TACE), to generate secreted 17K mature TNF-alpha. This study has purified TACE and cloned its complementary DNA. TACE is a membrane-bound disintegrin metalloproteinase. Structural comparisons with other disintegrin-containing enzymes indicate that TACE is unique, with noteable sequence identity to MADM, an enzyme implicated in myelin degradation, and to KUZ, a Drosophila homologue of MADM important for neuronal development. The expression of recombinant TACE (rTACE) results in the production of functional enzyme that correctly processes precursor TNF-alpha to the mature form. The rTACE provides a readily available source of enzyme to help in the search for new anti-inflammatory agents that target the final processing stage of TNF-alpha production (Moss, 1997).

A structural model of the iRhom-ADAM17 sheddase complex reveals functional insights into its trafficking and activity

Several membrane-anchored signal mediators such as cytokines (e.g. TNFalpha) and growth factors are proteolytically shed from the cell surface by the metalloproteinase ADAM17, which, thus, has an essential role in inflammatory and developmental processes. The membrane proteins iRhom1 and iRhom2 are instrumental for the transport of ADAM17 to the cell surface and its regulation. However, the structure-function determinants of the iRhom-ADAM17 complex are poorly understood. This study used AI-based modelling to gain insights into the structure-function relationship of this complex. Different regions were identified in the iRhom homology domain (IRHD) that are differentially responsible for iRhom functions. This study has supported the validity of the predicted structure-function determinants with several in vitro, ex vivo and in vivo approaches and demonstrated the regulatory role of the IRHD for iRhom-ADAM17 complex cohesion and forward trafficking. Overall, mechanistic insights are provided into the iRhom-ADAM17-mediated shedding event, which is at the centre of several important cytokine and growth factor pathways (Kahveci-Turkoz, 2023).

EGFR signaling leads to downregulation of PTP-LAR via TACE-mediated proteolytic processing

Proteolytic processing and ectodomain shedding have been described for a broad spectrum of transmembrane proteins under both normal and pathophysiological conditions and has been suggested as one mechanism to regulate a protein's function. It has also been documented for the receptor-like protein tyrosine phosphatase PTP-LAR, induced by treating cells with the tumor promoter TPA or the calcium ionophor A23187. The epidermal growth factor receptor (EGFR) has been identified as both an association partner of PTP-LAR, that mediates phosphorylation of the latter, as well as an inducer of LAR-cleavage. Both overexpression of this kinase and stimulation of endogenous EGFR in various tumor cell lines have been shown to induce proteolytic processing of the catalytic LAR-P-subunit. In contrast to TPA-induced shedding of PTP-LAR, EGFR-mediated cleavage does not require PKC-activity. For both stimuli, however, processing of the P-subunit turns out to be dependent on the activation of the MAP kinases ERK1 and ERK2, and is completely abrogated upon pre-treating cells with Batimastat, indicating the involvement of a metalloproteinase in this pathway. Being strongly impaired in fibroblasts derived from ADAM-17/TACE-knockout-mice or tumor cells that express a dominant negative mutant of ADAM-17/TACE, cleavage of PTP-LAR is suggested to be mediated by this metalloproteinase. Paralleled by rapid reduction of cell surface-localized LAR-E-subunit, EGFR-induced cleavage could be shown to lead to degradation of the catalytic LAR-P-subunit, thereby resulting in a significantly reduced overall cellular phosphatase activity of PTP-LAR. These results for the first time identify a protein tyrosine phosphatase as a potential substrate of TACE and describe proteolytic processing of PTP-LAR as a means of regulating phosphatase activity downstream and thus under the control of EGFR-mediated signaling pathways (Ruhe, 2006).

A metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells

Mammalian cells proteolytically release (shed) the extracellular domains of many cell-surface proteins. Modification of the cell surface in this way can alter the cell's responsiveness to its environment and release potent soluble regulatory factors. The release of soluble tumour-necrosis factor-alpha (TNF-alpha) from its membrane-bound precursor is one of the most intensively studied shedding events because this inflammatory cytokine is so physiologically important. The inhibition of TNF-alpha release (and many other shedding phenomena) by hydroxamic acid-based inhibitors indicates that one or more metalloproteinases is involved. This study has purified and cloned a metalloproteinase that specifically cleaves precursor TNF-alpha. Inactivation of the gene in mouse cells caused a marked decrease in soluble TNF-alpha production. This enzyme (called the TNF-alpha-converting enzyme, or TACE) is a new member of the family of mammalian adamalysins (or ADAMs), for which no physiological catalytic function has previously been identified. These results should facilitate the development of therapeutically useful inhibitors of TNF-alpha release, and they indicate that an important function of adamalysins may be to shed cell-surface proteins (Black, 1997).

Search PubMed for articles about Drosophila Tace

Agrawal, N., Delanoue, R., Mauri, A., Basco, D., Pasco, M., Thorens, B. and Leopold, P. (2016). The Drosophila TNF Eiger is an adipokine that acts on insulin-producing cells to mediate nutrient response. Cell Metab 23: 675-684. PubMed ID: 27076079

Blobel, C. P. (1997). Metalloprotease-disintegrins: links to cell adhesion and cleavage of TNF alpha and Notch. Cell 90(4): 589-592. PubMed ID: 9288739

Chabu, C. and Xu, T. (2014). Oncogenic Ras stimulates Eiger/TNF exocytosis to promote growth. Development 141(24): 4729-4739. PubMed ID: 25411211

Menghini, R., Fiorentino, L., Casagrande, V., Lauro, R. and Federici, M. (2013). The role of ADAM17 in metabolic inflammation. Atherosclerosis 228(1): 12-17. PubMed ID: 23384719

Moss, M. L., Jin, S. L., Milla, M. E., Bickett, D. M., Burkhart, W., Carter, H. L., Chen, W. J., Clay, W. C., Didsbury, J. R., Hassler, D., Hoffman, C. R., Kost, T. A., Lambert, M. H., Leesnitzer, M. A., McCauley, P., McGeehan, G., Mitchell, J., Moyer, M., Pahel, G., Rocque, W., Overton, L. K., Schoenen, F., Seaton, T., Su, J. L., Becherer, J. D. and et al. (1997). Cloning of a disintegrin metalloproteinase that processes precursor tumour-necrosis factor-alpha. Nature 385(6618): 733-736. PubMed ID: 9034191

Kahveci-Turkoz, S., Blasius, K., Wozniak, J., Rinkens, C., Seifert, A., Kasparek, P., Ohm, H., Oltzen, S., Nieszporek, M., Schwarz, N., Babendreyer, A., Preisinger, C., Sedlacek, R., Ludwig, A. and Dusterhoft, S. (2023). A structural model of the iRhom-ADAM17 sheddase complex reveals functional insights into its trafficking and activity. Cell Mol Life Sci 80(5): 135. PubMed ID: 37119365

Lieber, T., Kidd, S. and Young, M. W. (2002). kuzbanian-mediated cleavage of Drosophila Notch. Genes Dev. 16: 209-221. 11799064

Delwig, A. and Rand, M. D. (2008). Kuz and TACE can activate Notch independent of ligand. Cell Mol Life Sci 65(14): 2232-2243. PubMed ID: 18535782

Muliyil, S., Levet, C., Dusterhoft, S., Dulloo, I., Cowley, S. A. and Freeman, M. (2020). ADAM17-triggered TNF signalling protects the ageing Drosophila retina from lipid droplet-mediated degeneration. Embo J: e104415. PubMed ID: 32715522

Ruhe, J. E., Streit, S., Hart, S. and Ullrich, A. (2006). EGFR signaling leads to downregulation of PTP-LAR via TACE-mediated proteolytic processing. Cell Signal. [Epub ahead of print]. 16478662

Black, R. A., Rauch, C. T., Kozlosky, C. J., Peschon, J. J., Slack, J. L., Wolfson, M. F., Castner, B. J., Stocking, K. L., Reddy, P., Srinivasan, S., Nelson, N., Boiani, N., Schooley, K. A., Gerhart, M., Davis, R., Fitzner, J. N., Johnson, R. S., Paxton, R. J., March, C. J. and Cerretti, D. P. (1997). A metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells. Nature 385(6618): 729-733. PubMed ID: 9034190

Wei, S., Xie, Z., Filenova, E. and Brew, K. (2003). Drosophila TIMP is a potent inhibitor of MMPs and TACE: similarities in structure and function to TIMP-3. Biochemistry 42(42): 12200-12207. PubMed ID: 14567681

Wu, J., Tao, N., Tian, Y., Xing, G., Lv, H., Han, J., Lin, C. and Xie, W. (2018). Proteolytic maturation of Drosophila Neuroligin 3 by tumor necrosis factor alpha-converting enzyme in the nervous system. Biochim Biophys Acta Gen Subj 1862(3): 440-450. PubMed ID: 29107812

Zang, Y., Chaudhari, K. and Bashaw, G. J. (2022). Tace/ADAM17 is a bi-directional regulator of axon guidance that coordinates distinct Frazzled and Dcc receptor signaling outputs. Cell Rep 41(10): 111785. PubMed ID: 36476876

date revised: 2 August 2023

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