Lipophorin receptor 1 & Lipophorin receptor 1: Biological Overview | References
Gene name - Lipophorin receptor 1 & Lipophorin receptor 2
Cytological map positions - 96F1-96F2 & 96E10-96F1
Function - secreted receptor
Symbol - LpR1 & LpR2
Cellular location - secreted
Lipids are constantly shuttled through the body to redistribute energy and metabolites between sites of absorption, storage, and catabolism in a complex homeostatic equilibrium. In Drosophila, lipids are transported through the hemolymph in the form of lipoprotein particles, known as lipophorins. The mechanisms by which cells interact with circulating lipophorins and acquire their lipidic cargo are poorly understood. This study found that lipophorin receptor 1 and 2 (lpr1 and lpr2), two partially redundant genes belonging to the Low Density Lipoprotein Receptor (LDLR) family, are essential for the efficient uptake and accumulation of neutral lipids by oocytes and cells of the imaginal discs. Females lacking the lpr2 gene lay eggs with low lipid content and have reduced fertility, revealing a central role for lpr2 in mediating Drosophila vitellogenesis. lpr1 and lpr2 are transcribed into multiple isoforms. Interestingly, only a subset of these isoforms containing a particular LDLR type A module mediate neutral lipid uptake. Expression of these isoforms induces the extracellular stabilization of lipophorins. Furthermore, the data indicate that endocytosis of the lipophorin receptors is not required to mediate the uptake of neutral lipids. These findings suggest a model where lipophorin receptors promote the extracellular lipolysis of lipophorins. This model is reminiscent of the lipolytic processing of triglyceride-rich lipoproteins that occurs at the mammalian capillary endothelium, suggesting an ancient role for LDLR-like proteins in this process (Parra-Peralbo, 2014).
Most metazoans accumulate triacylglycerol (TAG), a strongly hydrophobic molecule with a high energy content, as the main substrate for energy storage. Large amounts of TAG are stored in fat body cells, the Drosophila equivalent of adipocytes, but most other cell types also accumulate limited amounts of it as intracellular lipid droplets. Because of their hydrophobicity, the extracellular transport of lipids requires dedicated mechanisms to increase their solubility in extracellular fluids. In mammals, lipids are packed into several types of lipoprotein particles which contain a hydrophobic core of neutral lipids (mostly TAG and esterified cholesterol) surrounded by a monolayer of phospholipids. In addition, apolipoproteins stabilize and regulate these particles. Similar lipoproteins, named lipophorins, are also found in insects. They share the same basic structure and play similar functions as mammalian lipoproteins. In Drosophila, apolipophorins are exclusively synthesized in the fat body, where they are partially lipidated and released into the hemolymph. It has been suggested that lipophorins act as a reusable shuttle in lipid transport. Lipids, primarily diacylglycerol (DAG), derived from the digestion of food in the gut or from the mobilization of lipids in the fat body, are loaded onto pre-formed, circulating lipophorins, then transported through the body via the hemolymph and unloaded upon reaching peripheral tissues for use as a source of energy and phospholipids. During this cycling process, negligible degradation of apolipophorin occurs (Parra-Peralbo, 2014).
In mammals, the Low Density Lipoprotein Receptor (LDLR) and other related proteins mediate endocytosis and the clearance of lipoproteins from plasma (Brown, 1986). Similar proteins belonging to the LDLR family, known as lipophorin receptors, were subsequently identified in insects. They can bind to lipophorins and mediate their endocytosis both in cell culture systems and in vivo (Dantuma, 1999; Van Hoof, 2003; Van Hoof, 2005). Because of these properties, it has been suggested that lipophorin receptors may play an important role in insect lipid metabolism (Rodenburg, 2005; Parra-Peralbo, 2014 and references therein).
This study examined the function of Drosophila lipophorin receptors in the uptake of neutral lipids. This organism has two lipophorin receptor genes, the lipophorin receptor 1 (lpr1) and lpr2, which are translated into multiple, functionally diverse isoforms. lpr1 and lpr2 are required for neutral lipid uptake in imaginal disc cells and oocytes. These results suggest a model where lpr1 and lpr2 promote the extracellular hydrolysis of neutral lipids contained in lipoprotein particles (Parra-Peralbo, 2014).
The Drosophila genome contains two closely related genes (lpr1 and lpr2, homologous to other described lipophorin receptors in insects like locust, mosquitoes, cockroaches, silkworm, wax moth or bees. Insect lipophorin receptors were first isolated because of their homology to the mammalian LDLR and subsequently shown to be involved in insect lipid metabolism. In particular, the locust Lipophorin Receptor, by far the best characterized member of the family, was able to induce the endocytic uptake of labeled lipophorins when expressed in mammalian cells (Dantuma, 1999). Moreover, it was required for the endocytosis of lipophorins by locust fat body cells (Van Hoof, 2003). This study generated several novel mutations in Drosophila which disrupted lpr1, lpr2 and, in view of a possible functional redundancy between the two receptors, a deficiency was generated that affects both genes simultaneously (Df(3R)lpr1/2), representing a null mutation for lipophorin receptor function. Despite the critical role lipophorins play in lipid transport in insects (Canavoso, 2001; Rodenburg, 2005) and the embryonic lethal phenotype of a null mutation in the single Drosophila apolipophorin gene (Rfabg; Callejo, 2008), complete disruption of both Drosophila lipophorin receptors does not affect the viability of flies. Moreover, no significant change were detected in total body TAG content when animals with mutations in lpr1, lpr2 or the double mutant were compared with their isogenic controls. Moreover, the fat body cells of mutant and control animals were indistinguishable, containing similar number of lipid droplets and of equivalent sizes. The rate of lipid mobilization under starvation conditions was also unaffected in lpr1 and lpr2 mutants. Taken together, these results clearly demonstrated that Drosophila lipophorin receptors are not essential for the storage of TAG in the fat body or for its mobilization. Despite this lack of a requirement, lpr1 and lpr2 expression was detected in the adult fat body, and lipophorin receptors have been identified in the fat body of other insects. Thus, the lipophorin receptors probably have functions in the fat body that are unrelated to the uptake of neutral lipids. Significantly, it has recently been reported that lpr2 is involved in immune response in Drosophila as a regulator of the serpin Necrotic metabolism (Soukup, 2009). In addition, it was shown in microarray experiments that lpr2 transcription changes upon immune challenge (De Gregorio, 2001). Since the fat body is a key immunological organ in the fly, it is possible that lpr2 expression in this tissue is related to immunity (Parra-Peralbo, 2014).
This study has shown that lpr1 and lpr2 have a key role in neutral lipid uptake in two Drosophila organs: the imaginal disc and the ovaries. In both cases, they are required to attain high levels of intracellular TAG. lpr1 and lpr2 are expressed in the wing pouch region of wing imaginal discs and mediate the uptake of neutral lipids by these cells. Another protein involved in lipid storage as a component of lipid droplets, the perilipin-like protein Lipid storage droplet-2 (Lsd-2), is also preferentially expressed and required in the wing pouch region for the accumulation of intracellular lipid droplets. Thus, lipid accumulation in this region of the disc appears to be regulated at multiple levels. Unfortunately, the functional relevance of this lipid accumulation is still unknown (Parra-Peralbo, 2014).
The results indicate that lpr1 and lpr2 genes are transcribed as multiple isoforms each with dramatically different properties. Only those isoforms transcribed from the distal promoters and containing the LA-1 module mediate lipid uptake. Similarly, the lipophorin receptor gene from the mosquito Aedes aegypti has been shown to be translated into fat body and oocyte specific isoforms from two alternative promoters (Seo, 2003). Several members of the mammalian LDLR family are similarly processed by alternative splicing. Variations in the O-glycosylation region and the LA domains in mammalian VLDLR and ApoER2 have been related to differential sensitivity to proteolytic processing by gamma-secretases, respectively. Thus, the multiple lpr1 and lpr2 isoforms might have different ligand binding and/or stability properties, allowing these receptors to be involved in processes as diverse as neutral lipid uptake, regulation of the immune system and regulation of neurite outgrowth (Parra-Peralbo, 2014).
During vitellogenesis, the nurse cells and the oocyte grow rapidly accumulating large amounts of yolk proteins and lipids from the hemolymph over approximately 18 hours. Work from the Mahowald lab has shown that Yolkless, an LDLR family protein, mediates the endocytic uptake of yolk proteins in Drosophila. This study demonstrates that a different receptor type, the lipophorin receptor, is essential for the uptake of neutral lipids during vitellogenesis. This is clearly shown in Df(3R)lpr2 females and in double mutant lpr1-, lpr2-germ-line clones. In both cases, the mutant egg chambers accumulate low levels of neutral lipids. In addition to impaired lipid uptake during vitellogenesis, a second phenotype in was observed Df(3R)lpr1/2 double mutant females, where most of the egg chambers degenerated at mid-oogenesis. A simple explanation for this phenotype would be that degeneration was triggered by the low lipid content of Df(3R)lpr1/2 egg chambers. In fact, it is known that multiple challenges like starvation, extreme temperatures or chemical treatments, trigger a mid-oogenesis checkpoint and induce apoptosis at this stage. Significantly, flies with a mutation in the gene midway, which encodes an acyl coenzyme A: diacylglycerol acyltransferase required for the synthesis of TAG, were described to have severely reduced levels of neutral lipids in the germ-line and displayed apoptosis at mid oogenesis, thus paralleling the Df(3R)lpr1/2 phenotype. However, it was difficult to fully attribute degeneration to low lipid levels, as some experimental conditions were observed that resulted in egg chambers with very low levels of neutral lipids but that did not undergo degeneration. In particular, in Df(3R)lpr1/2 germ-line clones degeneration was absent even though the neutral lipid content of the egg chambers was low. Similarly, expression of UAS-lpr1J exclusively in the follicle cells of Df(3R)lpr1/2 females abolished egg chamber degeneration even though neutral lipid accumulation in the nurse cells and oocytes was low. These experiments suggest that the lipophorin receptors might have an additional function in the follicle cells which is necessary to avoid egg chamber degeneration. Accordingly, lpr1 expression was detected in the follicular epithelium. In this direction, it has recently been described that blocking the nutrient sensing TOR pathway in follicle cells induced apoptosis at mid oogenesis. Thus, Lpr1 could be required to maintain elevated levels of TOR activity in follicle cells. In interpreting these results, the non-autonomous effects of lipophorin receptors should also be considered. This study has shown that expression of UAS-lpr2E exclusively in the oocyte and nurse cells increases lipid uptake in the follicle cells, which could potentially impact on their nutritional status and restore their putative anti-apoptotic activity. Conversely, expression of the transgene in the follicle cells might slightly increase lipid uptake by the oocyte and nurse cells, even though it has not been possible to detect this effect, and thus provide enough lipids to bypass the mid-oogenesis checkpoint. More studies will be required to assess the role of the lipophorin receptors in the follicular epithelium (Parra-Peralbo, 2014).
Drosophila Lpr1 and Lpr2 are bona fide members of the LDLR family, sharing a similar organization of proteins domains with the human LDLR, ApoER2 and VLDLR. The human LDLR is the archetypical endocytic receptor. It is expressed in the liver where it mediates the endocytosis of cholesterol-rich LDL, regulating LDL concentration in serum. Endocytosis of LDL results in the catabolic processing of both, the lipidic and proteic moieties of LDL in lysosomes. Other members of the LDLR family are also well known endocytic receptors with a broad variety of ligands. Drosophila lipophorin receptors can also mediate endocytosis of their ligands. It has recently been reported that Lpr1 is expressed in garland cells and pericardial athrocytes where it is critical for the endocytic clearance of serpin/protease complexes from the hemolymph, thus regulating the innate immune response (Soukup, 2009). Overexpression of Lpr1 and Lpr2 in imaginal discs also induced the endocytosis of lipophorins, which colocalized with endocytic markers (Callejo, 2008; Khaliullina1, 2009). Similarly, the locust lipophorin receptor mediated lipophorin endocytosis in the fat body and in cell culture (Dantuma, 1999; Van Hoof, 2003). Despite this well documented endocytic activity of LDLRs, the current data demonstrates that neutral lipid uptake mediated by Drosophila lipophorin receptors does not require the endocytosis of lipophorin particles. Three lines of evidence support this conclusion: (1) Blocking endocytosis did not affect lipid uptake in the egg chambers; (2) overexpression of Lpr2E in groups of imaginal disc cells induced lipid uptake both in cells expressing the receptor and in a 1-2 cell diameter region of adjacent cells and (3) expression of Lpr2E in the oocyte and nurse cells promoted lipid uptake in the adjacent, somatic follicular epithelium. These results also indicate that Lpr2E is able to locally increase the concentration of lipophorins in the extracellular space. Taking into account this data, the following model is proposed for lipophorin receptor-mediated neutral lipid uptake: lipophorin receptors interact with lipophorins at the cell surface and promote the extracellular hydrolysis of their DAG core by facilitating the activity of an as-yet-unidentified lipase, associated with the extracellular matrix. The free fatty acids generated during DAG hydrolysis could diffuse a few cell diameters away before being captured by cells, explaining why lipophorin receptors can promote lipid uptake non-autonomously. Significantly, physiological data obtained from studies of flight muscles and oocytes in insects indicated that lipid uptake mostly occurs without the concomitant degradation of the apolipophorin, which is consistent with the current hypothesis. Moreover, a lipophorin-specific lipase activity associated with muscle and oocyte cell membranes has been detected. The model offers a possible explanation to understand why only a subset of lpr1 and lpr2 isoforms mediates lipid uptake, whereby only the lipid-uptake promoting isoforms can stabilize lipophorins in the extracellular matrix. Alternatively, if lipophorin receptors must interact with both, a lipophorin particle and a lipase to generate a ternary complex and facilitate lipolysis, then the lipid uptake-defective isoforms might lack the ability to interact with the lipase. Identification of such putative lipase(s) will be necessary to test this hypothesis (Parra-Peralbo, 2014).
The proposed model displays a number of resemblances to the lipolytic processing of triglyceride-rich lipoproteins in the microvascular endothelium of adipose tissue, heart and striated muscles in mammals. Circulating triglyceride-rich lipoproteins, chylomicrons from the intestine and VLDL synthesized by the liver, reach the capillary endothelium where they interact with lipoprotein lipase at the luminal surface. Lipoprotein lipase is essential for the lipolytic processing of chylomicrons and VLDL, generating non-esterified fatty acids from the TAG fraction of lipoproteins. The free fatty acids are then transported to the underlying adipocytes and myocytes by specific transporters such as CD-36. Once inside these cells they are re-esterified into newly synthesized TAG stores or enter the β-oxidation cycle. Recent data indicated that the extracellular lipolysis of TAG-rich lipoproteins is strongly potentiated by the endothelial protein GPIHBP1. This protein is essential for the transcytosis of lipoprotein lipase from the basolateral to the apical capillary endothelial surface. In addition, it has been suggested that it may facilitate lipolysis by simultaneously interacting with lipoprotein lipase and chylomicrons in the luminal surface of capillaries, providing a molecular platform for lipolysis to occur. In agreement with this essential functions, Gpihbp1-deficient mice manifested severe hyperchylomicronemia. The VLDLR, which is also expressed at the capillary endothelium, seems to participate in the lipolytic processing of TAG-rich lipoproteins in similar ways. The VLDLR can mediate the transcytosis of lipoprotein lipase across cultured endothelial cells and interacts with both, lipoprotein lipase and ApoE containing TAG-rich lipoproteins, potentially tethering them to the endothelium surface and thus promoting the action of lipoprotein lipase. These potential functions were supported by the phenotype of vldlr- mice, which showed delayed clearance of TAG-rich lipoproteins after a meal and increased plasma TAG levels under a high fat diet but normal lipoprotein profiles under regular feeding conditions. Unfortunately, these weak phenotypes have hampered the elucidation of the precise roles that VLDLR plays during the processing of TAG-rich lipoproteins in vivo. It is proposed that in Drosophila, lipophorin receptors have an activity similar to the bridging role proposed for GPIHBP1 and VLDLR in mammals, bringing lipophorins and a putative lipophorin-specific lipase into close contact on the cell surface and promoting in this way the lipolysis of lipophorins. It is speculated that during evolution, a protein related to VLDLR had a critical role in promoting the extracellular hydrolysis of lipoproteins. In insects, this function is carried out by the lipophorin receptors whereas in mammals, GPIHBP1 appears to have taken most of this function, with VLDLR retaining a minor role. The data supports an ancient function for the LDLR family in promoting the extracellular lipolytic processing of lipoproteins (Parra-Peralbo, 2014).
Activity-dependent modifications strongly influence neural development. However, molecular programs underlying their context and circuit-specific effects are not well understood. To study global transcriptional changes associated with chronic elevation of synaptic activity, cell-type-specific transcriptome profiling was performed of Drosophila ventral lateral neurons (LNvs) in the developing visual circuit, and activity-modified transcripts were identified that are enriched in neuron morphogenesis, circadian regulation, and lipid metabolism and trafficking. Using bioinformatics and genetic analyses, activity-induced isoform-specific upregulation was validated of Drosophila lipophorin receptors LpR1 and LpR2, the homologs of mammalian low-density lipoprotein receptor (LDLR) family proteins. Furthermore, these morphological and physiological studies uncovered critical functions of neuronal lipophorin receptors (LpRs) in maintaining the structural and functional integrities in neurons challenged by chronic elevations of activity. Together, these findings identify LpRs as molecular targets for activity-dependent transcriptional regulation and reveal the functional significance of cell-type-specific regulation of neuronal lipid uptake in experience-dependent plasticity and adaptive responses (Yin, 2018).
This study investigated experience-dependent regulation of dendrite plasticity using cell-type-specific transcriptome profiling followed by transgenic RNAi screens. Candidate genes were identified that are subject to activity-dependent transcriptional regulation and function in regulating dendrite development in the Drosophila CNS. Additionally, combined bioinformatics and genetic analyses revealed isoform-specific expression of lipoprotein receptors LpR1 and LpR2 in LNvs and uncovered their roles in supporting dendrite morphogenesis and synaptic functions in the CNS. Together, these findings provide in vivo evidence for neuronal lipoprotein receptors serving as targets of activity-dependent transcriptional regulation, a previously unrecognized component of the neuronal homeostatic mechanism that maintains structural and functional integrity in response to chronic elevation of input activity (Yin, 2018).
Activity-dependent transcriptional factors serve important functions in synapse development, maturation, and elimination as well as dendritic and axonal outgrowth. However, a molecular understanding of circuit and context-specific transcriptional events induced by sensory experience is just starting to emerge. This cell-type-specific RNA-seq analyses identified over 200 experience-modified transcripts, among which are previously identified activity-dependent genes, including CrebB, Hr38, dnc, and Irk1, as well as many candidate genes that have not been previously linked to activity-dependent regulation or been functionally characterized in neurons. Therefore, this study generated a large number of potential targets for future molecular studies on experience-dependent dendrite plasticity. Besides the list of activity-modified transcripts, this analyses also revealed isoform-specific regulation of LpR genes, demonstrating the possibility of studying alternative splicing events associated with chronic alterations of activity using the RNA-seq dataset (Yin, 2018).
Both insect LpRs and mammalian LDLR family proteins have multiple isoforms generated by alternative splicing events and the usage of alternative promoters. In the fly imaginal disc and oocyte, long isoforms of LpRs acquire their lipoprotein cargo by interacting with lipid transfer particles (LTP), which stabilize the receptor-lipoprotein complex on the cell surface and possibly facilitate lipolysis. In these non-neuronal tissues, long isoforms of LpRs are also solely responsible for endocytosis-independent neutral lipid uptake. The function of the short isoforms, however, is unclear. When overexpressed in imaginal discs, the short isoform of LpR2 localizes in early endosomes and mediates uptake of lipoproteins through endocytic activities similar to those observed in mammalian LDLRs. These observations suggest that isoform-specific transcriptional regulation generates LpRs with diverse properties and functionalities (Yin, 2018).
These studies in LNvs indicate that the short-isoform of LpR2 has an endosomal localization in neurons and the ability to reconstitute LpR2 function in supporting dendrite growth. In conjunction with previous studies, these results suggest that isoform-specific transcriptional regulation may lead to distinct modes of lipoprotein intake in cells expressing different isoforms. It is proposed that the short isoforms of LpR1 and LpR2 expressed in neurons are endocytic lipoprotein receptors responsible for neuronal lipid uptake. In the CNS, lipid trafficking and homeostasis involve neural-glia interactions, which likely alter local concentrations of lipoprotein complexes and generate unique demands for lipoprotein uptake compared to non-neuronal tissues. Future in vivo imaging studies on CNS lipoprotein trafficking combined with functional studies of different LpR isoforms will contribute to understanding of the molecular regulation of neuronal lipid uptake (Yin, 2018).
Lipids are essential building blocks for plasma membranes and vesicles. They also have versatile roles in regulating cellular metabolism and mediating signaling transductions. In the nervous system, neuronal lipid uptake and recycling involve complex neuron-glia interactions that are critical for lipid homeostasis in the brain. Although neurons have the intrinsic ability to synthesize lipids, glia-derived cholesterol and phospholipids are essential for the formation and maintenance of the synapse in mammalian CNS neurons. Recent studies in the Drosophila system also demonstrated that glial lipid droplet formation protects neural stem cells from systematic stress in the developing Drosophila larval brain and that the inability of neurons to transport lipids for glial lipid droplet formation leads to accelerated neurodegeneration. However, whether and how neuronal lipid uptake contributes to normal development and activity-induced plasticity events remain unknown (Yin, 2018).
These studies demonstrate the function of LpRs in supporting dendrite growth. Reducing or eliminating LpRs in LNvs leads to a significant decrease in dendrite volume. Counterintuitively, LpRs are upregulated in LL conditions, which also generate reductions in LNv dendrite volume. Although an increased LpR level in the LL condition does not increase dendrite size, it is critical for preventing a further reduction in dendrites and a loss of physiological functions, suggesting that LpRs counteract activity-induced morphological and functional alterations. Based on these observations and current knowledge of lipid homeostasis in the larval CNS, it is proposed that activity-dependent regulation of LpRs serves as a homeostatic compensatory mechanism to augment the capacity for neuronal lipoprotein uptake in response to chronic elevation of input activity. Lipoprotein complexes recruited by LpRs are either released by glia or captured from the circulating hemolymph. This model is supported by RNA-seq studies, which revealed that a number of lipid-metabolism-related genes are downregulated in the constant light condition, including FASN1, Dgk, CG31140, GlcAT-S, fwd, and retm. LNv transcriptome analysis indicated a strong impact of elevated input activity on lipid metabolism, which may lead to altered lipid homeostasis and increased demand for lipid uptake. In vivo studies on activity-induced modifications of lipid homeostasis and trafficking will improve understanding of the compensatory role of LpR upregulation in neuronal adaptive responses (Yin, 2018).
Taken together, these studies strongly suggest that neuronal lipoprotein receptors are important components of activity-regulated neural plasticity and adaptive responses. In addition, transcriptome studies on neuronal-specific responses induced by excessive input activity provide molecular insights for studies related to human epilepsy and seizure disorders and potentially reveal the previously unappreciated role of altered neuronal lipoprotein uptake in neurological disorders associated with the dysregulation of lipid homeostasis (Yin, 2018).
The humoral response to fungal and Gram-positive infections is regulated by the serpin-family inhibitor, Necrotic. Following immune-challenge, a proteolytic cascade is activated which signals through the Toll receptor. Toll activation results in a range of antibiotic peptides being synthesised in the fat-body and exported to the haemolymph. As with mammalian serpins, Necrotic turnover in Drosophila is rapid. This serpin is synthesised in the fat-body, but its site of degradation has been unclear. By 'freezing' endocytosis with a temperature sensitive Dynamin mutation, this study has demonstrated that Necrotic is removed from the haemolymph in two groups of giant cells: the garland and pericardial athrocytes. Necrotic uptake responds rapidly to infection, being visibly increased after 30 mins and peaking at 6-8 hours. Co-localisation of anti-Nec with anti-AP50, Rab5, and Rab7 antibodies establishes that the serpin is processed through multi-vesicular bodies and delivered to the lysosome, where it co-localises with the ubiquitin-binding protein, HRS. Nec does not co-localise with Rab11, indicating that the serpin is not re-exported from athrocytes. Instead, mutations which block late endosome/lysosome fusion (dor, hk, and car) cause accumulation of Necrotic-positive endosomes, even in the absence of infection. Knockdown of the 6 Drosophila orthologues of the mammalian LDL receptor family with dsRNA identifies LpR1 as an enhancer of the immune response. Uptake of Necrotic from the haemolymph is blocked by a chromosomal deletion of LpR1. In conclusion, this study identified the cells and the receptor molecule responsible for the uptake and degradation of the Necrotic serpin in Drosophila melanogaster. The scavenging of serpin/proteinase complexes may be a critical step in the regulation of proteolytic cascades (Soukup, 2009).
The Necrotic serpin controls activation of the Toll-mediated immune-response in Drosophila, which represents the best-studied example of serpin-regulated proteolytic cascade in insects. The Drosophila melanogaster genome encodes 15 putative inhibitory serpin transcripts that carry secretion-signal peptides. In addition to nec, the Spn27A serpin controls Toll-mediated morphogenesis in the embryo and the phenol-oxidase cascade in adults. Spn28D (CG7219) also regulates the phenol-oxidase cascade, while Spn77Ba regulates tracheal melanization, which also can trigger systemic expression of Drosomycin via the Toll pathway. In addition, the Spn42Da transcript inhibits furin, which is involved in the maturation of secreted proteins (Soukup, 2009).
In mammals, serpins are removed from circulation by endocytosis and degraded in the liver as inactive serpin/proteinase complexes. This aspect of serpin metabolism, however, has not been studied previously in Drosophila. This study identified the mechanism of serpin clearance by endocytosis in the garland and pericardial cells. These cells are known to take up ferritin and have been suggested to be homologous to mammalian reticulo-endothelial cells or nephrocytes. As in mammals, serpin turnover in Drosophila is extremely rapid, so it was not possible to detect the immune-response serpins Nec and Spn27A under normal conditions. However, freezing the pinching-off of endocytotic vesicles, using the shits1 mutation, allows detection of serpin uptake. Endocytosed Nec is sorted, first to Rab5-positive early-endosomes and then to Rab7-positive late-endosomes. Disrupting these steps by expression of dominant negative UAS-Rab5S43 or UAS-Rab7Q67L, leads to accumulation of Nec-positive vesicles. Similarly, co-localisation of anti-Nec and anti-HRS antibody staining indicates that the serpin is present in early endosomes in the ubiquitin-dependent sorting pathway; while anti-Nec and anti-Fab1 confirms that the serpin is destined for lysosomal degradation. Blocking late-endosome/lysosome fusion using HOPS-complex mutants causes accumulation of Nec-positive endosomes/MVB, indicating that Nec sorting to MVB is required for lysosomal delivery. Co-localization of anti-Nec and anti-Lamp1 antibody staining confirms that Nec is delivered to lysosomes for degradation, while the absence of Nec staining in Rab11-positive vesicles indicates that none of the serpin is recycled from MVB to the haemolymph. In summary, Nec is cleared from the haemolymph and sorted through MVB, via the ubiquitin-dependent pathway, to lysosomes for degradation (Soukup, 2009).
In vitro studies in mammals have shown that different members of LDLR family have different binding specificities to different native serpins and serpin/proteinase complexes. This study has shown that LpR1 is the Nec trafficking receptor in vivo, but that neither LpR1 nor LpR2 traffics Spn27A. By analogy to mammalian systems, Nec is probably taken up by LpR1 as a complex with its target proteinase. In addition, pre-digestion of Nec with PPE increases Nec uptake in garland cells that are not deficient for the LpR1 receptor. The results establish that active trafficking of Nec from the haemolymph can modulate the immune response. Nec clearance is extremely rapid, but deletion of the LpR1 gene sensitises the immune response: nec transcript levels decrease and Drs transcript levels increase. These results imply a regulatory feedback loop at the transcriptional level. In this context, it is significant that LpR1 appears to bind the non-inhibitory serpin/proteinase complex, in preference to the native Nec serpin. Clearance of the serpin/protease complex through the athrocytes appears to compete with a regulatory feedback loop affecting nec transcription (Soukup, 2009).
In summary, this study has established that the Nec serpin is taken-up via LpR1 from the haemolymph and degraded in the garland and pericardial athrocytes (Soukup, 2009).
The Hedgehog (Hh) family of secreted signaling proteins has a broad variety of functions during metazoan development and implications in human disease. Despite Hh being modified by two lipophilic adducts, Hh migrates far from its site of synthesis and programs cellular outcomes depending on its local concentrations. Recently, lipoproteins were suggested to act as carriers to mediate Hh transport in Drosophila. This study examined the role of lipophorins (Lp), the Drosophila lipoproteins, in Hh signaling in the wing imaginal disk, a tissue that does not express Lp but obtains it through the hemolymph. The up-regulation of the Lp receptor 2 (LpR2), the main Lp receptor expressed in the imaginal disk cells, was used to increase Lp endocytosis and locally reduce the amount of available free extracellular Lp in the wing disk epithelium. Under this condition, secreted Hh is not stabilized in the extracellular matrix. Similar results were obtained after a generalized knock-down of hemolymph Lp levels. These data suggest that Hh must be packaged with Lp in the producing cells for proper spreading. Interestingly, it was also shown that Patched (Ptc), the Hh receptor, is a lipoprotein receptor; Ptc actively internalizes Lp into the endocytic compartment in a Hh-independent manner and physically interacts with Lp. Ptc, as a lipoprotein receptor, can affect intracellular lipid homeostasis in imaginal disk cells. However, by using different Ptc mutants, it was shown that Lp internalization does not play a major role in Hh signal transduction but does in Hh gradient formation (Callejo, 2008).
At least two models have been proposed to explain how the lipophilic Hh can spread through an aqueous tissue. Fractionation studies of the supernatant of Hh-expressing cells showed that Hh participates in high molecular weight structures that probably represent multimeric complexes, and cholesterol and palmitic acid seems to mediate this multimerization. The lipid moieties are thought to be embedded in the core of these complexes, in analogy to micelles. Recently, a second model was proposed: it suggests that lipoprotein particles could carry lipid-modified ligands such as Hh and Wingless, acting as vehicles for long-range transport. Vertebrate lipoprotein particles are scaffolded by apolipoproteins and consist of a phospholipid monolayer surrounding a core of esterified cholesterol and triglycerides. Insects form similar particles that are called Lipophorins (Lp) and contain Apolipophorins I and II (ApoLI and ApoLII). These proteins are produced in the fat body by cleavage of the precursor pro-Apolipophorin, and are not synthesized by imaginal disk cells but receive them through the hemolymph. Panakova (2005) described that a systemic reduction of lipoprotein levels in the hemolymph, by expression of Lp (ApoLI-II) RNAi in the fat body, affects long-range but not short-range Hh signaling. That study also found that Wnt and Hh proteins copurify with lipoproteins from tissue homogenates and colocalize with lipoprotein particles in the developing wing epithelium. More recently, an interaction between Lp and the glypicans, Dally and Dally-like, has been found (Callejo, 2008).
This study has tested the role of lipoproteins in Hh signaling. To this aim, the lipoprotein gene was knocked down by RNA interference, reducing Lp supply in the hemolymph. In addition, the amount of extracellular Lp was locally reduced in the wing imaginal disk cells by overexpressing Lipophorin receptor 2 (LpR2), which increases Lp endocytosis. Under both experimental conditions a decrease was observed in extracellular Hh. These results suggest an important role of lipoproteins in Hh anchoring and spreading through the extracellular matrix. Moreover, this study has observed that Ptc actively internalizes Lipophorins, effectively acting as a Lipoprotein receptor, and that its over-expression can alter intracellular lipid homeostasis. Collectively, these results are consistent with the model of lipoprotein particles acting as vehicles for Hh transport (Callejo, 2008).
Search PubMed for articles about Drosophila Lipophorin receptor
Brown, M. S. and Goldstein, J. L. (1986). A receptor-mediated pathway for cholesterol homeostasis. Science 232: 34-47. PubMed ID: 3513311
Callejo, A., Culi, J. and Guerrero, I. (2008). Patched, the receptor of Hedgehog, is a lipoprotein receptor. Proc Natl Acad Sci U S A 105: 912-917. PubMed ID: 18198278
Canavoso, L. E., Jouni, Z. E., Karnas, K. J., Pennington, J. E. and Wells, M. A. (2001). Fat metabolism in insects. Annu Rev Nutr 21: 23-46. PubMed ID: 11375428
Dantuma, N. P., Potters, M., De Winther, M. P., Tensen, C. P., Kooiman, F. P., Bogerd, J. and Van der Horst, D. J. (1999). An insect homolog of the vertebrate very low density lipoprotein receptor mediates endocytosis of lipophorins. J Lipid Res 40: 973-978. PubMed ID: 10224168
De Gregorio, E., Spellman, P. T., Rubin, G. M. and Lemaitre, B. (2001). Genome-wide analysis of the Drosophila immune response by using oligonucleotide microarrays. Proc Natl Acad Sci U S A 98: 12590-12595. PubMed ID: 11606746
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date revised: 13 December 2018
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