Multi-omics analyses of MEN1 missense mutations identify disruption of menin-MLL and menin-JunD interactions as critical requirements for molecular pathogenicity.

BACKGROUND: Loss-of-function mutations of the multiple endocrine neoplasia type 1 (MEN1) gene are causal to the MEN1 tumor syndrome, but they are also commonly found in sporadic pancreatic neuroendocrine tumors and other types of cancers. The MEN1 gene product, menin, is involved in transcriptional and chromatin regulation, most prominently as an integral component of KMT2A/MLL1 and KMT2B/MLL2 containing COMPASS-like histone H3K4 methyltransferase complexes. In a mutually exclusive fashion, menin also interacts with the JunD subunit of the AP-1 and ATF/CREB transcription factors. RESULTS: Here, we applied and in silico screening approach for 253 disease-related MEN1 missense mutations in order to select a set of nine menin mutations in surface-exposed residues. The protein interactomes of these mutants were assessed by quantitative mass spectrometry, which indicated that seven of the nine mutants disrupt interactions with both MLL1/MLL2 and JunD complexes. Interestingly, we identified three missense mutations, R52G, E255K and E359K, which predominantly reduce the MLL1 and MLL2 interactions when compared with JunD. This observation was supported by a pronounced loss of binding of the R52G, E255K and E359K mutant proteins at unique MLL1 genomic binding sites with less effect on unique JunD sites. CONCLUSIONS: Our results underline the effects of MEN1 gene mutations in both familial and sporadic tumors of endocrine origin on the interactions of menin with the MLL1 and MLL2 histone H3K4 methyltransferase complexes and with JunD-containing transcription factors. Menin binding pocket mutants R52G, E255K and E359K have differential effects on MLL1/MLL2 and JunD interactions, which translate into differential genomic binding patterns. Our findings encourage future studies addressing the pathophysiological relevance of the separate MLL1/MLL2- and JunD-dependent functions of menin mutants in MEN1 disease model systems.

Insulin and chromium picolinate induce translocation of CD36 to the plasma membrane through different signaling pathways in 3T3-L1 adipocytes, and with a differential functionality of the CD36.

Chromium picolinate (CrPic) has been indicated to activate glucose transporter 4 (GLUT4) trafficking to the plasma membrane (PM) to enhance glucose uptake in 3T3-L1 adipocytes. In skeletal and heart muscle cells, insulin directs the intracellular trafficking of the fatty acid translocase/CD36 to induce the uptake of cellular long-chain fatty acid (LCFA). The current study describes the effects of CrPic and insulin on the translocation of CD36 from intracellular storage pools to the PM in 3T3-L1 adipocytes in comparison with that of GLUT4. Immunofluorescence microscopy and immunoblotting revealed that both CD36 and GLUT4 were expressed and primarily located intracellularly in 3T3-L1 adipocytes. Upon insulin or CrPic stimulation, PM expression of CD36 increased in a similar manner as that for GLUT4; the CrPic-stimulated PM expression was less strong than that of insulin. The increase in PM localization for these two proteins by insulin paralleled LCFA ([1-(14)C]palmitate) or [(3)H]deoxyglucose uptake in 3T3-L1 adipocytes. The induction of the PM expression of GLUT4, but not CD36, or substrate uptake by insulin and CrPic appears to be additive in adipocytes. Furthermore, wortmannin completely inhibited the insulin-stimulated translocation of GLUT4 or CD36 and prevented the increased uptake of glucose or LCFA in these cells. Taken together, for the first time, these findings suggest that both insulin and CrPic induce CD36 translocation to the PM in 3T3-L1 adipocytes and that their translocation-inducing effects are not additive. The signaling pathway inducing the translocations is different, apparently resulting in a differential activity of CD36.

The E3 ubiquitin ligase IDOL induces the degradation of the low density lipoprotein receptor family members VLDLR and ApoER2.

We have previously identified the E3 ubiquitin ligase-inducible degrader of the low density lipoprotein receptor (LDLR) (Idol) as a post-translational modulator of LDLR levels. Idol is a direct target for regulation by liver X receptors (LXRs), and its expression is responsive to cellular sterol status independent of the sterol-response element-binding proteins. Here we demonstrate that Idol also targets two closely related LDLR family members, VLDLR and ApoE receptor 2 (ApoER2), proteins implicated in both neuronal development and lipid metabolism. Idol triggers ubiquitination of the VLDLR and ApoER2 on their cytoplasmic tails, leading to their degradation. We further show that the level of endogenous VLDLR is sensitive to cellular sterol content, Idol expression, and activation of the LXR pathway. Pharmacological activation of the LXR pathway in mice leads to increased Idol expression and to decreased Vldlr levels in vivo. Finally, we establish an unexpected functional link between LXR and Reelin signaling. We demonstrate that LXR activation results in decreased Reelin binding to VLDLR and reduced Dab1 phosphorylation. The identification of VLDLR and ApoER2 as Idol targets suggests potential roles for this LXR-inducible E3 ligase in the central nervous system in addition to lipid metabolism.

Locust flight activity as a model for hormonal regulation of lipid mobilization and transport.

Flight activity of insects provides a fascinating yet relatively simple model system for studying the regulation of processes involved in energy metabolism. This is particularly highlighted during long-distance flight, for which the locust constitutes a long-standing favored model insect, which as one of the most infamous agricultural pests additionally has considerable economical importance. Remarkably many aspects and processes pivotal to our understanding of (neuro)hormonal regulation of lipid mobilization and transport during insect flight activity have been discovered in the locust; among which are the peptide adipokinetic hormones (AKHs), synthesized and stored by the neurosecretory cells of the corpus cardiacum, that regulate and integrate lipid (diacylglycerol) mobilization and transport, the functioning of the reversible conversions of lipoproteins (lipophorins) in the hemolymph during flight activity, revealing novel concepts for the transport of lipids in the circulatory system, and the structure and functioning of the exchangeable apolipopotein, apolipophorin III, which exhibits a dual capacity to exist in both lipid-bound and lipid-free states that is essential to these lipophorin conversions. Besides, the lipophorin receptor (LpR) was identified and characterized in the locust. In an integrative approach, this short review aims at highlighting the locust as an unrivalled model for studying (neuro)hormonal regulation of lipid mobilization and transport during insect flight activity, that additionally has offered a broad and profound research model for integrative physiology and biochemistry, and particularly focuses on recent developments in the concept of AKH-induced changes in the lipophorin system during locust flight, that deviates fundamentally from the lipoprotein-based transport of lipids in the circulation of mammals. Current studies in this field employing the locust as a model continue to attribute to its role as a favored model organism, but also reveal some disadvantages compared to model insects with a completely sequenced genome.

Lipoprotein assembly and function in an evolutionary perspective.

Circulatory fat transport in animals relies on members of the large lipid transfer protein (LLTP) superfamily, including mammalian apolipoprotein B (apoB) and insect apolipophorin II/I (apoLp-II/I). ApoB and apoLp-II/I, constituting the structural (non-exchangeable) basis for the assembly of various lipoproteins, acquire lipids through microsomal triglyceride-transfer protein, another LLTP family member, and bind them by means of amphipathic α-helical and ß-sheet structural motifs. Comparative research reveals that LLTPs evolved from the earliest animals and highlights the structural adaptations in these lipid-binding proteins. Thus, in contrast to apoB, apoLp-II/I is cleaved post-translationally by a furin, resulting in the appearance of two non-exchangeable apolipoproteins in the single circulatory lipoprotein in insects, high-density lipophorin (HDLp). The remarkable structural similarities between mammalian and insect lipoproteins notwithstanding important functional differences relate to the mechanism of lipid delivery. Whereas in mammals, partial delipidation of apoB-containing lipoproteins eventually results in endocytic uptake of their remnants, mediated by members of the low-density lipoprotein receptor (LDLR) family, and degradation in lysosomes, insect HDLp functions as a reusable lipid shuttle capable of alternate unloading and reloading of lipid. Also, during muscular efforts (flight activity), an HDLp-based lipoprotein shuttle provides for the transport of lipid for energy generation. Although a lipophorin receptor - a homolog of LDLR - was identified that mediates endocytic uptake of HDLp during specific developmental periods, the endocytosed lipoprotein appears to be recycled in a transferrin-like manner. These data highlight that the functional adaptations in the lipoprotein lipid carriers in mammals and insects also emerge with regard to the functioning of their cognate receptors.

Effects of AMPK activators on the sub-cellular distribution of fatty acid transporters CD36 and FABPpm.

In heart and skeletal muscle, enhanced contractile activity induces an increase in the uptake of glucose and long-chain fatty acids (LCFA) via an AMP-activated protein kinase (AMPK)-regulated mechanism. AMPK activation induces glucose uptake through translocation of glucose transporter 4 (GLUT4) from intracellular pools to the plasma membrane (PM). AMPK-mediated LCFA uptake has been suggested to be regulated by a similar translocation of the LCFA transporters CD36 and plasma membrane-associated fatty acid binding protein (FABPpm). In contrast to the well-characterized GLUT4 translocation, documentation of the proposed translocation of both LCFA transporters is rudimentary. Therefore, we adopted a cell culture system to investigate the localization of CD36 and FABPpm compared with GLUT4, in the absence and presence of AMPK activators oligomycin and AICAR. To this end, intact Chinese hamster ovary (CHO) cells stably expressing CD36 or myc-tagged GLUT4 (GLUT4myc) were used; FABPpm is endogenously expressed in CHO cells. Immuno-fluorescence microscopy revealed that CD36 PM localization resembled that of GLUT4, while FABPpm localized to other PM domains. Upon stimulation with oligomycin or AICAR, CD36 translocated (1.5-fold increase) to a PM location similar to that of GLUT4myc. In contrast, the PM FABPpm content did not change upon AMPK activation. Thus, for the first time in intact cells, we present evidence for AMPK-mediated translocation of CD36 from intracellular pools to the PM, similar to GLUT4, whereas FABPpm is not relocated.

Circulatory lipid transport: lipoprotein assembly and function from an evolutionary perspective.

Circulatory transport of neutral lipids (fat) in animals relies on members of the large lipid transfer protein (LLTP) superfamily, including mammalian apolipoprotein B (apoB) and insect apolipophorin II/I (apoLp-II/I). Latter proteins, which constitute the structural basis for the assembly of various lipoproteins, acquire lipids through microsomal triglyceride transfer protein (MTP)--another LLTP family member--and bind them by means of amphipathic structures. Comparative research reveals that LLTPs have evolved from the earliest animals and additionally highlights the structural and functional adaptations in these lipid carriers. For instance, in contrast to mammalian apoB, the insect apoB homologue, apoLp-II/I, is post-translationally cleaved by a furin, resulting in their appearance of two non-exchangeable apolipoproteins in the insect low-density lipoprotein (LDL) homologue, high-density lipophorin (HDLp). An important difference between mammalian and insect lipoproteins relates to the mechanism of lipid delivery. Whereas in mammals, endocytic uptake of lipoprotein particles, mediated via members of the LDL receptor (LDLR) family, results in their degradation in lysosomes, the insect HDLp was shown to act as a reusable lipid shuttle which is capable of reloading lipid. Although the recent identification of a lipophorin receptor (LpR), a homologue of LDLR, reveals that endocytic uptake of HDLp may constitute an additional mechanism of lipid delivery, the endocytosed lipoprotein appears to be recycled in a transferrin-like manner. Binding studies indicate that the HDLp-LpR complex, in contrast to the LDL-LDLR complex, is resistant to dissociation at endosomal pH as well as by treatment with EDTA mimicking the drop in Ca(2+) concentration in the endosome. This remarkable stability of the ligand-receptor complex may provide a crucial key to the recycling mechanism. Based on the binding and dissociation capacities of mutant and hybrid receptors, the specific binding interaction of the ligand-binding domain of the receptor with HDLp was characterized. These structural similarities and functional adaptations of the lipid transport systems operative in mammals and insects are discussed from an evolutionary perspective.

Delipidation of insect lipoprotein, lipophorin, affects its binding to the lipophorin receptor, LpR: implications for the role of LpR-mediated endocytosis.

The insect lipophorin receptor (LpR), an LDL receptor (LDLR) homologue that is expressed during restricted periods of insect development, binds and endocytoses high-density lipophorin (HDLp). However, in contrast to LDL, HDLp is not lysosomally degraded, but recycled in a transferrin-like manner, leaving a function of receptor-mediated uptake of HDLp to be uncovered. Since a hallmark of circulatory HDLp is its ability to function as a reusable shuttle that selectively loads and unloads lipids at target tissues without being endocytosed or degraded, circulatory HDLp can exist in several forms with respect to lipid loading. To investigate whether lipid content of the lipoprotein affects binding and subsequent endocytosis by LpR, HDLp was partially delipidated in vitro by incubation with alpha-cyclodextrin, yielding a particle of buoyant density 1.17g/mL (HDLp-1.17). Binding experiments demonstrated that LpR bound HDLp-1.17 with a substantially higher affinity than HDLp both in LpR-transfected Chinese hamster ovary (CHO) cells and isolated insect fat body tissue endogenously expressing LpR. Similar to HDLp, HDLp-1.17 was targeted to the endocytic recycling compartment after endocytosis in CHO(LpR) cells. The complex of HDLp-1.17 and LpR appeared to be resistant to endosomal pH, as was recently demonstrated for the LpR-HDLp complex, corroborating that HDLp-1.17 is recycled similar to HDLp. This conclusion was further supported by the observation of a significant decrease with time of HDLp-1.17-containing vesicles after endocytosis of HDLp-1.17 in LpR-expressing insect fat body tissue. Collectively, our results indicate that LpR favors the binding and subsequent endocytosis of HDLp-1.17 over HDLp, suggesting a physiological role for LpR in selective endocytosis of relatively lipid-unloaded HDLp particles, while lipid reloading during their intracellular itinerary might result in decreased affinity for LpR and thus allows recycling.

The complex of the insect LDL receptor homolog, lipophorin receptor, LpR, and its lipoprotein ligand does not dissociate under endosomal conditions.

The insect low-density lipoprotein (LDL) receptor (LDLR) homolog, lipophorin receptor (LpR), mediates endocytic uptake of the single insect lipoprotein, high-density lipophorin (HDLp), which is structurally related to LDL. However, in contrast to the fate of LDL, which is endocytosed by LDLR, we previously demonstrated that after endocytosis, HDLp is sorted to the endocytic recycling compartment and recycled for re-secretion in a transferrin-like manner. This means that the integrity of the complex between HDLp and LpR is retained under endosomal conditions. Therefore, in this study, the ligand-binding and ligand-dissociation capacities of LpR were investigated by employing a new flow cytometric assay, using LDLR as a control. At pH 5.4, the LpR-HDLp complex remained stable, whereas that of LDLR and LDL dissociated. Hybrid HDLp-binding receptors, containing either the beta-propeller or both the beta-propeller and the hinge region of LDLR, appeared to be unable to release ligand at endosomal pH, revealing that the stability of the complex is imparted by the ligand-binding domain of LpR. The LpR-HDLp complex additionally appeared to be EDTA-resistant, excluding a low Ca(2+) concentration in the endosome as an alternative trigger for complex dissociation. From binding of HDLp to the above hybrid receptors, it was inferred that the stability upon EDTA treatment is confined to LDLR type A (LA) ligand-binding repeats 1-7. Additional (competition) binding experiments indicated that the binding site of LpR for HDLp most likely involves LA-2-7. It is therefore proposed that the remarkable stability of the LpR-HDLp complex is attributable to this binding site. Together, these data indicate that LpR and HDLp travel in complex to the endocytic recycling compartment, which constitutes a key determinant for ligand recycling by LpR.

Insulin-induced translocation of CD36 to the plasma membrane is reversible and shows similarity to that of GLUT4.

In cardiac and skeletal muscles, insulin regulates the uptake of long-chain fatty acid (LCFA) via the putative LCFA transporter CD36. Biochemical studies propose an insulin-induced translocation of CD36 from intracellular pools to the plasma membrane (PM), similar to glucose transporter 4 (GLUT4) translocation. To characterize insulin-induced CD36 translocation in intact cells, Chinese hamster ovary (CHO) cells stably expressing CD36 or myc-tagged GLUT4 (GLUT4myc) were created. Immuno-fluorescence microscopy revealed CD36 to be located both intracellularly (in--at least partially--different compartments than GLUT4myc) and at the PM. Upon stimulation with insulin, CD36 translocated to a PM localization similar to that of GLUT4myc; the increase in PM CD36 content, as quantified by surface-protein biotinylation, amounted to 1.7-fold. The insulin-induced CD36 translocation was shown to be phosphatidylinositol-3 kinase-dependent, and reversible (as evidenced by insulin wash-out) in a similar time frame as that for GLUT4. The expression of GLUT4myc in non-stimulated cells, and the insulin-induced increase in PM GLUT4myc correlated with increased deoxyglucose uptake. By contrast, CD36 expression in non-stimulated cells and the insulin-induced increase in PM CD36 were not paralleled by a rise in LCFA uptake, suggesting that in these cells, such increase requires additional proteins, or a protein activation step. Taken together, this study is the first to present morphological evidence for CD36 translocation, and shows this process to resemble GLUT4 translocation.

Insect lipoprotein biogenesis depends on an amphipathic beta cluster in apolipophorin II/I and is stimulated by microsomal triglyceride transfer protein.

Lipoproteins transport lipids in the circulation of an evolutionally wide diversity of animals. The pathway for lipoprotein biogenesis has been revealed to a large extent in mammals only, in which apolipoprotein B (apoB) acquires lipids via the assistance of microsomal triglyceride transfer protein (MTP) and binds them by means of amphipathic protein structures. To investigate whether this is a common mechanism for lipoprotein biogenesis in animals, we studied the structural elements involved in the assembly of the insect lipoprotein, lipophorin. LOCATE sequence analysis predicted that the insect lipoprotein precursor, apolipophorin II/I (apoLp-II/I), contains clusters of amphipathic alpha-helices and beta-strands, organized along the protein as N-alpha(1)-beta-alpha(2)-C, reminiscent of a truncated form of apoB. Recombinant expression of a series of C-terminal truncation variants of Locusta migratoria apoLp-II/I in an insect cell (Sf9) expression system revealed that the formation of a buoyant high density lipoprotein requires the amphipathic beta cluster. Coexpression of apoLp-II/I with the MTP homolog of Drosophila melanogaster affected insect lipoprotein biogenesis quantitatively as well as qualitatively, as the secretion of apoLp-II/I proteins was increased several-fold and the buoyant density of the secreted lipoprotein decreased concomitantly, indicative of augmented lipidation. Based on these findings, we propose that, despite specific modifications, the assembly of lipoproteins involves MTP as well as amphipathic structures in the apolipoprotein carrier, both in mammals and insects. Thus, lipoprotein biogenesis in animals appears to rely on structural elements that are of early metazoan origin.

The Spn4 gene from Drosophila melanogaster is a multipurpose defence tool directed against proteases from three different peptidase families.

By alternative use of four RSL (reactive site loop) coding exon cassettes, the serpin (serine protease inhibitor) gene Spn4 from Drosophila melanogaster was proposed to enable the synthesis of multiple protease inhibitor isoforms, one of which has been shown to be a potent inhibitor of human furin. Here, we have investigated the inhibitory spectrum of all Spn4 RSL variants. The analyses indicate that the Spn4 gene encodes inhibitors that may inhibit serine proteases of the subtilase family (S8), the chymotrypsin family (S1), and the papain-like cysteine protease family (C1), most of them at high rates. Thus a cohort of different protease inhibitors is generated simply by grafting enzyme-adapted RSL sequences on to a single serpin scaffold, even though the target proteases contain different types and/or a varying order of catalytic residues and are descendents of different phylogenetic lineages. Since all of the Spn4 RSL isoforms are produced as intracellular residents and additionally as variants destined for export or associated with the secretory pathway, the Spn4 gene represents a versatile defence tool kit that may provide multiple antiproteolytic functions.

Molecular diversity and evolution of the large lipid transfer protein superfamily.

Circulatory lipid transport in animals is mediated to a substantial extent by members of the large lipid transfer (LLT) protein (LLTP) superfamily. These proteins, including apolipoprotein B (apoB), bind lipids and constitute the structural basis for the assembly of lipoproteins. The current analyses of sequence data indicate that LLTPs are unique to animals and that these lipid binding proteins evolved in the earliest multicellular animals. In addition, two novel LLTPs were recognized in insects. Structural and phylogenetic analyses reveal three major families of LLTPs: the apoB-like LLTPs, the vitellogenin-like LLTPs, and the microsomal triglyceride transfer protein (MTP)-like LLTPs, or MTPs. The latter are ubiquitous, whereas the two other families are distributed differentially between animal groups. Besides similarities, remarkable variations are also found among LLTPs in their major lipid-binding sites (i.e., the LLT module as well as the predicted clusters of amphipathic secondary structure): variations such as protein modification and number, size, or occurrence of the clusters. Strikingly, comparative research has also highlighted a multitude of functions for LLTPs in addition to circulatory lipid transport. The integration of LLTP structure, function, and evolution reveals multiple adaptations, which have come about in part upon neofunctionalization of duplicated genes. Moreover, the change, exchange, and expansion of functions illustrate the opportune application of lipid-binding proteins in nature. Accordingly, comparative research exposes the structural and functional adaptations in animal lipid carriers and brings up novel possibilities for the manipulation of lipid transport.

Sequence analysis of the non-recurring C-terminal domains shows that insect lipoprotein receptors constitute a distinct group of LDL receptor family members.

Lipoprotein-mediated delivery of lipids in mammals involves endocytic receptors of the low density lipoprotein (LDL) receptor (LDLR) family. In contrast, in insects, the lipoprotein, lipophorin (Lp), functions as a reusable lipid shuttle in lipid delivery, and these animals, therefore, were not supposed to use endocytic receptors. However, recent data indicate additional endocytic uptake of Lp, mediated by a Lp receptor (LpR) of the LDLR family. The two N-terminal domains of LDLR family members are involved in ligand binding and dissociation, respectively, and are composed of a mosaic of multiple repeats. The three C-terminal domains, viz., the optional O-linked glycosylation domain, the transmembrane domain, and the intracellular domain, are of a non-repetitive sequence. The present classification of newly discovered LDLR family members, including the LpRs, bears no relevance to physiological function. Therefore, as a novel approach, the C-terminal domains of LDLR family members across the entire animal kingdom were used to perform a sequence comparison analysis in combination with a phylogenetic tree analysis. The LpRs appeared to segregate into a specific group distinct from the groups encompassing the other family members, and each of the three C-terminal domains of the insect receptors is composed of unique set of sequence motifs. Based on conservation of sequence motifs and organization of these motifs in the domains, LpR resembles most the groups of the LDLRs, very low density lipoprotein (VLDL) receptors, and vitellogenin receptors. However, in sequence aspects in which LpR deviates from these three receptor groups, it most notably resembles LDLR-related protein-2, or megalin. These features might explain the functional differences disclosed between insect and mammalian lipoprotein receptors.

Lipoprotein-mediated lipid transport in insects: analogy to the mammalian lipid carrier system and novel concepts for the functioning of LDL receptor family members.

In all animals, lipoproteins are used to transport lipids through the aqueous circulation. Lipids are delivered to mammalian cells by two different mechanisms: via endocytic uptake of the complete lipoprotein particle mediated by members of the low density lipoprotein (LDL) receptor (LDLR) family, or by selective delivery of lipoprotein-carried lipids at the cell surface, such as lipid uptake following the action of a lipoprotein lipase. Although many structural elements of the lipid transport system of insects are similar to those of mammals, insect lipoprotein-mediated lipid transport was thought to apply only to the latter concept, since the single lipoprotein acts as a reusable lipid shuttle. However, the recent identification of lipoprotein receptors of the LDLR family in insects suggests that lipid transport in these animals may also adopt the first concept. Yet, the endocytic properties of the insect LDLR homologue appear to deviate from those of the mammalian LDLR family members, resulting in the recycling of endocytosed lipoprotein in a transferrin-like manner. This indicates that a hitherto unknown as well as unexpected function can be added to the plethora of functions of LDLR family members. Analysis of the molecular mechanism of the ligand-recycling function of the insect receptor provides also new insight into the possible functioning of the mammalian family members. In the last several years, mammalian and insect lipoprotein-mediated lipid transport systems have been reviewed separately with respect to functioning and lipid delivery. This review, in which new and important developments in the insect field with respect to our understanding of lipid delivery are discussed with a particular focus on the involvement of the LDLR homologue, aims at comparing the two systems, also from an evolutionary biological perspective, and proposes that the two systems are more similar than assumed previously.

Intracellular fate of LDL receptor family members depends on the cooperation between their ligand-binding and EGF domains.

The insect low-density lipoprotein (LDL) receptor (LDLR) homologue LpR mediates endocytosis of an insect lipoprotein (lipophorin) that is structurally related to LDL. Despite these similarities, lipophorin and LDL follow distinct intracellular routes upon endocytosis by their receptors. Whereas LDL is degraded in lysosomes, lipophorin is recycled in a transferrin-like manner. We constructed several hybrid receptors composed of Locusta migratoria LpR and human LDLR regions to identify the domains implicated in LpR-mediated ligand recycling. Additionally, the triadic His562 residue of LDLR, which is putatively involved in ligand uncoupling, was mutated to Asn, corresponding to Asn643 in LpR, to analyse the role of the His triad in receptor functioning. The familial hypercholesterolaemia (FH) class 5 mutants LDLR(H562Y) and LDLR(H190Y) were also analysed in vitro. Fluorescence microscopic investigation and quantification suggest that LpR-mediated ligand recycling involves cooperation between the ligand-binding domain and epidermal growth factor (EGF) domain of LpR, whereas its cytosolic tail does not harbour motifs that affect this process. LDLR residue His562 appears to be essential for LDLR recycling after ligand endocytosis but not for constitutive receptor recycling. Like LDLR(H562N), LDLR(H562Y) did not recycle bound ligand; moreover, the intracellular distribution of both mutant receptors after ligand incubation coincides with that of a lysosomal marker. The LDLR mutant characterization in vitro suggests that LDLR FH class 5 mutations might be divided into two subclasses.

Receptor-mediated endocytosis and intracellular trafficking of lipoproteins and transferrin in insect cells.

While the intracellular pathways of ligands after receptor-mediated endocytosis have been studied extensively in mammalian cells, in insect cells these pathways are largely unknown. We transfected Drosophila Schneider line 2 (S2) cells with the human low-density lipoprotein (LDL) receptor (LDLR) and transferrin (Tf) receptor (TfR), and used endocytosis of LDL and Tf as markers. After endocytosis in mammalian cells, LDL is degraded in lysosomes, whereas Tf is recycled. Fluorescence microscopy analysis revealed that LDL and Tf are internalized by S2 cells transfected with LDLR or TfR, respectively. In transfectants simultaneously expressing LDLR and TfR, both ligands colocalize in endosomes immediately after endocytic uptake, and their location remained unchanged after a chase. Similar results were obtained with Spodoptera frugiperda Sf9 cells that were transfected with TfR, suggesting that Tf is retained intracellularly by both cell lines. The insect lipoprotein, lipophorin, is recycled upon lipophorin receptor (LpR)-mediated endocytosis by mammalian cells, however, not after endocytosis by LpR-expressing S2 transfectants, suggesting that this recycling mechanism is cell-type specific. LpR is endogenously expressed by fat body tissue of Locusta migratoria for a limited period after an ecdysis. A chase following endocytosis of labeled lipophorin by isolated fat body tissue at this developmental stage resulted in a significant decrease of lipophorin-containing vesicles, indicative of recycling of the ligand.

Biosynthesis and secretion of insect lipoprotein: involvement of furin in cleavage of the apoB homolog, apolipophorin-II/I.

The biosynthesis of neutral fat-transporting lipoproteins involves the lipidation of their nonexchangeable apolipoprotein. In contrast to its mammalian homolog apolipoprotein B, however, insect apolipophorin-II/I (apoLp-II/I) is cleaved posttranslationally at a consensus substrate sequence for furin, resulting in the appearance of two apolipoproteins in insect lipoprotein. To characterize the cleavage process, a truncated cDNA encoding the N-terminal 38% of Locusta migratoria apoLp-II/I, including the cleavage site, was expressed in insect Sf9 cells. This resulted in the secretion of correctly processed apoLp-II and truncated apoLp-I. The cleavage could be impaired by the furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone (decRVKRcmk) as well as by mutagenesis of the consensus substrate sequence for furin, as indicated by the secretion of uncleaved apoLp-II/I-38. Treatment of L. migratoria fat body, the physiological site of lipoprotein biosynthesis, with decRVKRcmk similarly resulted in the secretion of uncleaved apoLp-II/I, which was integrated in lipoprotein particles of buoyant density identical to wild-type high density lipophorin (HDLp). These results show that apoLp-II/I is posttranslationally cleaved by an insect furin and that biosynthesis and secretion of HDLp can occur independent of this processing step. Structure modeling indicates that the cleavage of apoLp-II/I represents a molecular adaptation in homologous apolipoprotein structures. We propose that cleavage enables specific features of insect lipoproteins, such as low density lipoprotein formation, endocytic recycling, and involvement in coagulation.

Lipophorin receptor-mediated lipoprotein endocytosis in insect fat body cells.

High-density lipophorin (HDLp) in the circulation of insects is able to selectively deliver lipids to target tissues in a nonendocytic manner. In Locusta migratoria, a member of the LDL receptor family has been identified and shown to mediate endocytosis of HDLp in mammalian cells transfected with the cDNA of this receptor. This insect lipophorin receptor (iLR) is temporally expressed in fat body tissue of young adult as well as larval locusts, as shown by Western blot analysis. Fluorescence microscopy revealed that fat body cells internalize fluorescently labeled HDLp and human receptor-associated protein only when iLR is expressed. Expression of iLR is down-regulated on Day 4 after an ecdysis. Consequently, HDLp is no longer internalized. By starving adult locusts immediately after ecdysis, we were able to prolong iLR expression. In addition, expression of the receptor was induced by starving adults after down-regulation of iLR. These results suggest that iLR mediates endocytosis of HDLp in fat body cells, and that expression of iLR is regulated by the demand of fat body tissue for lipids.

Alternative lipid mobilization: the insect shuttle system.

Lipid mobilization in long-distance flying insects has revealed a novel concept for lipid transport in the circulatory system during exercise. Similar to energy generation for sustained locomotion in mammals, the work accomplished by non-stop flight activity is powered by oxidation of free fatty acids (FFA) derived from endogenous reserves of triacylglycerol. The transport form of the lipid, however, is diacylglycerol (DAG), which is delivered to the flight muscles associated with lipoproteins. In the insect system, the multifunctional lipoprotein, high-density lipophorin (HDLp) is loaded with DAG while additionally, multiple copies of the exchangeable apolipoprotein, apoLp-III, associate with the expanding particle. As a result, lipid-enriched low-density lipophorin (LDLp) is formed. At the flight muscles, LDLp-carried DAG is hydrolyzed and FFA are imported into the muscle cells for energy generation. The depletion of DAG from LDLp results in the recovery of both HDLp and apoLp-III, HDLp, identified which are reutilized for another cycle of DAG transport. A receptor for as a novel member of the vertebrate low-density lipoprotein (LDL) receptor family, does not seem to be involved in the lipophorin shuttle mechanism operative during flight activity. In addition, endocytosis of HDLp mediated by the insect receptor does not seem to follow the classical mammalian LDL pathway. Many structural elements of the lipid mobilization system in insects are similar to those in mammals. Domain structures of apoLp-I and apoLp-II, the non-exchangeable apolipoprotein components of HDLp, are related to apoB 100. ApoLp-III is a bundle of five amphipathic alpha-helices that binds to a lipid surface very similar to the four-helix bundle of the N-terminal domain of human apoE. Despite these similarities, the functioning of the insect lipoprotein in energy transport during flight activity is intriguingly different, since the TAG-rich mammalian lipoproteins play no role as a carrier of mobilized lipids during exercise and besides, these lipoproteins are not functioning as a reusable shuttle for lipid transport. On the other hand, the deviant behavior of similar molecules in a different biological system may provide a useful alternative model for studying the molecular basis of processes related to human disorders and disease.