Post-translational regulation of lipophorin receptors

Resumen del póster presentado al IX Meeting of the Spanish Society for Developmental Biology celebrado en Granada del 12 al 14 de noviembre de 2012.Peer Reviewe

Uptake of the Necrotic Serpin in Drosophila melanogaster via the Lipophorin Receptor-1

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, we demonstrate 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, we identify 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

Drosophila Lipophorin Receptors Mediate the Uptake of Neutral Lipids in Oocytes and Imaginal Disc Cells by an Endocytosis-Independent Mechanism

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. We have 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, our 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

Molecular mechanisms of neutral lipid uptake in Drosophila

Resumen del trabajo presentado al 1st Spanish Conference on the Molecular, Cellular and Developmental Biology of Drosophila, celebrado en Girona (España) del 25 al 28 de septiembre de 2012.The lipophorin receptors are required in tissues such as the imaginal discs, the oenocytes and the egg chambers, for the cellular uptake of neutral lipids from hemolymph. In lipophorin receptor mutants, these tissues are severely deprived on intracellular lipid droplets. Our previous research suggested a model in which the lipophorin receptors interact with circulating lipophorin particles -insect diacylglycerol-rich lipoproteinsstabilizing them on the cell surface. This interaction is required for the transfer of neutral lipids to the cell by a still not completely understood mechanism that does not require the endocytosis of the lipophorin particle and that is reminiscent of the extracellular lipolysis of mammalian lipoproteins in the microcapillaries of peripheral tissues. To better understand the molecular mechanisms mediating lipid uptake, we searched for molecular partners of the lipophorin receptors by tandem affinity purification. A key cofactor identified was a large protein with homology to human ApoB, suggesting it was a novel circulating lipoprotein. Isopicnic gradient ultracentrifugation showed it floated at a density of 1.23 g/ml. We generated null mutations that are larval lethal. Silencing of this novel lipoprotein in adults resulted in a strong accumulation of neutral lipids in the gut and a reduction in the ovaries, a phenotype very similar to that of lipophorin knockdown. This suggests that both lipoproteins are essential for the transfer of neutral lipids from the gut to peripheral tissues. Studies in other insects had similarly identified two circulating lipoproteins: lipophorin, which is the main lipid carrier, and Lipid Transfer Particle (LTP), a high density lipoprotein that catalyzes the transfer of lipids between lipophorin and tissues by an unknown mechanism. Our data suggests that the novel high density lipoprotein we identified corresponds to Drosophila LTP. We are currently examining the functional relevance of the interaction between Drosophila LTP and the lipophorin receptors.Peer reviewe

A sensor mechanism involving the lipophorin receptors couples the metabolic status of the female fly with oogenesis progression

Resumen del trabajo presentado al 1st Spanish Conference on the Molecular, Cellular and Developmental Biology of Drosophila, celebrado en Girona (España) del 25 al 28 de septiembre de 2012.It has been known for a long time that females adjust the number of eggs laid to nutrient availability. Work mostly from Drummond-Barbosa's lab indicated that this regulation was mediated in part by two systemic signals, insulin-like peptides and ecdyson. Both signals impact on germ-line stem cells proliferation and maintenance in the germarium. In addition, under poor nutrition there is a strong increase in the loss of egg chambers at stage 8 by apoptosis. How this cell death is triggered is not currently known. We have observed that disruption of lipid metabolism causes a similar effect, the abrupt death of most egg chambers by apoptosis at stage 9-10a. In particular, knockdown of lipophorin, of LTP or a mutation in the lipophorin receptors all cause this phenotype. Expression of the lipophorin receptors exclusively in the somatic follicle cells that surround the oocyte and nurse cells rescues egg chambers death under these conditions. This suggests that follicle cells sense the availability of lipids and trigger the death of the egg chamber when they are low. We have identified the TOR pathway as a central component of this sensor mechanism. Our data suggests that in the absence of lipid uptake in the follicular epithelia, the energysensing kinase AMPK represses the TOR pathway, triggering the destruction of the egg chamber. In contrast, the insulin pathway is not involved. We are currently analyzing the link between the lipophorin receptors and AMPK.Peer reviewe

Post-transcriptional regulation of lipophorin receptors

Resumen del trabajo presentado al 1st Spanish Conference on the Molecular, Cellular and Developmental Biology of Drosophila, celebrado en Girona (España) del 25 al 28 de septiembre de 2012.The lipophorin receptors genes generate multiple isoforms with astonishing different functional properties, since only a subset of them is able to mediate neutral lipids uptake. We have previously demonstrated that a 47 amino acids long, differentially spliced complement-type domain (LA-1) is critical for this activity. However, all the isoforms that contain LA-1 also have an atypically long signal peptide (>60 amino acids) of unknown function. Our preliminary data suggests that this long signal peptide allows the translation of some lipophorin receptors isoforms under conditions of cellular stress and generalized inhibition of translation by a novel mechanism that we are currently characterizing. Other Drosophila proteins such as Crumbs and the LDLR family protein Megalin also contain long signal peptides. However, this is the first time a cellular function has been established for them.Peer Reviewe

Especificacion de las celulas precursoras de los organos sensoriales en 'Drosophila'

Centro de Informacion y Documentacion Cientifica (CINDOC). C/Joaquin Costa, 22. 28002 Madrid. SPAIN / CINDOC - Centro de Informaciòn y Documentaciòn CientìficaSIGLEESSpai

An atypical long signal peptide controls the translation efficiency and distribution of lipophorin receptors isoforms

Resumen del póster presentado al 23rd European Drosophila Research Congress, celebrado en Barcelona (España) del 16 al 19 de octubre de 2013.Drosophila Lipophorin receptor 1 and 2 (Lpr1, Lpr2) are homologous to the human LDLR and they are involved in multiple processes, most notably lipid metabolism. Each receptor has two alternative promoters (P and D) that give rise to multiple isoforms (P- and D-isoforms) having strikingly different functions and tissue distributions. We found that these isoforms are subjected to differential post-transcriptional regulation when expressed in imaginal discs resulting in different translational efficiency, subcellular distribution and activity. We have mapped the protein domains required for this translational regulation to the signal peptide characteristic of D-isoforms. This signal peptide is predicted to be atypically long and thus, it may have additional functions besides targeting to the endoplasmic reticulum. We are currently studying how the signal peptide affects protein translation efficiency and function in multiple tissues where the lipophorin receptors have a critical role during lipid uptake.Peer Reviewe

Drosophila lipophorin receptors mediate the uptake of neutral lipids in oocytes and imaginal disc cells by an endocytosis-independent mechanism

Resumen del póster presentado al 52nd Annual Drosophila Research Conference, celebrado en San Diego, California (US) del 30 de marzo al 3 de abril de 2011.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. We have 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, our data indicates that endocytosis 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.Peer Reviewe