Comparative characterisation of an ecto-5'-nucleotidase (CD73) in non-tumoral MCF10-A breast cells and triple-negative MDA-MB-231 breast cancer cells.

Ecto-5'-nucleotidase (CD73) hydrolyses 5'AMP to adenosine and inorganic phosphate. Breast cancer cells (MDA-MB-231) express high CD73 levels, and this enzyme has been found to play a tumour-promoting role in breast cancer. However, no studies have sought to investigate whether CD73 has differential affinity or substrate preferences between noncancerous and cancerous breast cells. In the present study, we aimed to biochemically characterise ecto-5'-nucleotidase in breast cancer cell lines and assess whether its catalytic function and tumour progression are correlated in breast cancer cells. The results showed that compared to nontumoral breast MCF-10A cells, triple-negative breast cancer MDA-MB-231 cells had a higher ecto-5'-nucleotidase expression level and enzymatic activity. Although ecto-5'-nucleotidase activity in the MDA-MB-231 cell line showed no selectivity among monophosphorylated substrates, 5'AMP was preferred by the MCF-10A cell line. Compared to the MCF-10A cell line, the MDA-MB-231 cell line has better hydrolytic ability, lower substrate affinity, and high inhibitory potential after treatment with a specific CD73 inhibitor α,ß­methylene ADP (APCP). Therefore, we demonstrated that a specific inhibitor of the ecto-5-nucleotidase significantly reduced the migratory and invasive capacity of MDA-MB-231 cells, suggesting that ecto-5-nucleotidase activity might play an important role in metastatic progression.

Lipophorin: The Lipid Shuttle.

Insects need to transport lipids through the aqueous medium of the hemolymph to the organs in demand, after they are absorbed by the intestine or mobilized from the lipid-producing organs. Lipophorin is a lipoprotein present in insect hemolymph, and is responsible for this function. A single gene encodes an apolipoprotein that is cleaved to generate apolipophorin I and II. These are the essential protein constituents of lipophorin. In some physiological conditions, a third apolipoprotein of different origin may be present. In most insects, lipophorin transports mainly diacylglycerol and hydrocarbons, in addition to phospholipids. The fat body synthesizes and secretes lipophorin into the hemolymph, and several signals, such as nutritional, endocrine, or external agents, can regulate this process. However, the main characteristic of lipophorin is the fact that it acts as a reusable shuttle, distributing lipids between organs without being endocytosed or degraded in this process. Lipophorin interacts with tissues through specific receptors of the LDL receptor superfamily, although more recent results have shown that other proteins may also be involved. In this chapter, we describe the lipophorin structure in terms of proteins and lipids, in addition to reviewing what is known about lipoprotein synthesis and regulation. In addition, we reviewed the results investigating lipophorin's function in the movement of lipids between organs and the function of lipophorin receptors in this process.

Genome of Rhodnius prolixus, an insect vector of Chagas disease, reveals unique adaptations to hematophagy and parasite infection.

Rhodnius prolixus not only has served as a model organism for the study of insect physiology, but also is a major vector of Chagas disease, an illness that affects approximately seven million people worldwide. We sequenced the genome of R. prolixus, generated assembled sequences covering 95% of the genome (∼ 702 Mb), including 15,456 putative protein-coding genes, and completed comprehensive genomic analyses of this obligate blood-feeding insect. Although immune-deficiency (IMD)-mediated immune responses were observed, R. prolixus putatively lacks key components of the IMD pathway, suggesting a reorganization of the canonical immune signaling network. Although both Toll and IMD effectors controlled intestinal microbiota, neither affected Trypanosoma cruzi, the causal agent of Chagas disease, implying the existence of evasion or tolerance mechanisms. R. prolixus has experienced an extensive loss of selenoprotein genes, with its repertoire reduced to only two proteins, one of which is a selenocysteine-based glutathione peroxidase, the first found in insects. The genome contained actively transcribed, horizontally transferred genes from Wolbachia sp., which showed evidence of codon use evolution toward the insect use pattern. Comparative protein analyses revealed many lineage-specific expansions and putative gene absences in R. prolixus, including tandem expansions of genes related to chemoreception, feeding, and digestion that possibly contributed to the evolution of a blood-feeding lifestyle. The genome assembly and these associated analyses provide critical information on the physiology and evolution of this important vector species and should be instrumental for the development of innovative disease control methods.

Epithelial Mesenchymal Transition Induces Aberrant Glycosylation through Hexosamine Biosynthetic Pathway Activation.

Deregulated cellular metabolism is a hallmark of tumors. Cancer cells increase glucose and glutamine flux to provide energy needs and macromolecular synthesis demands. Several studies have been focused on the importance of glycolysis and pentose phosphate pathway. However, a neglected but very important branch of glucose metabolism is the hexosamine biosynthesis pathway (HBP). The HBP is a branch of the glucose metabolic pathway that consumes ∼2-5% of the total glucose, generating UDP-GlcNAc as the end product. UDP-GlcNAc is the donor substrate used in multiple glycosylation reactions. Thus, HBP links the altered metabolism with aberrant glycosylation providing a mechanism for cancer cells to sense and respond to microenvironment changes. Here, we investigate the changes of glucose metabolism during epithelial mesenchymal transition (EMT) and the role of O-GlcNAcylation in this process. We show that A549 cells increase glucose uptake during EMT, but instead of increasing the glycolysis and pentose phosphate pathway, the glucose is shunted through the HBP. The activation of HBP induces an aberrant cell surface glycosylation and O-GlcNAcylation. The cell surface glycans display an increase of sialylation α2-6, poly-LacNAc, and fucosylation, all known epitopes found in different tumor models. In addition, modulation of O-GlcNAc levels was demonstrated to be important during the EMT process. Taken together, our results indicate that EMT is an applicable model to study metabolic and glycophenotype changes during carcinogenesis, suggesting that cell glycosylation senses metabolic changes and modulates cell plasticity.

Long-chain acyl-CoA synthetase 2 knockdown leads to decreased fatty acid oxidation in fat body and reduced reproductive capacity in the insect Rhodnius prolixus.

Long-chain acyl-CoA esters are important intermediates in lipid metabolism and are synthesized from fatty acids by long-chain acyl-CoA synthetases (ACSL). The hematophagous insect Rhodnius prolixus, a vector of Chagas' disease, produces glycerolipids in the midgut after a blood meal, which are stored as triacylglycerol in the fat body and eggs. We identified twenty acyl-CoA synthetase genes in R. prolixus, two encoding ACSL isoforms (RhoprAcsl1 and RhoprAcsl2). RhoprAcsl1 transcripts increased in posterior midgut on the second day after feeding, and RhoprAcsl2 was highly transcribed on the tenth day. Both enzymes were expressed in Escherichia coli. Recombinant RhoprACSL1 and RhoprACSL2 had broad pH optima (7.5-9.5 and 6.5-9.5, respectively), were inhibited by triacsin C, and were rosiglitazone-insensitive. Both showed similar apparent Km for palmitic and oleic acid (2-6 µM), but different Km for arachidonic acid (0.5 and 6 µM for RhoprACSL1-Flag and RhoprACSL2-Flag, respectively). The knockdown of RhoprAcsl1 did not result in noticeable phenotypes. However, RhoprACSL2 deficient insects exhibited a 2.5-fold increase in triacylglycerol content in the fat body, and 90% decrease in fatty acid ß-oxidation. RhoprAcsl2 knockdown also resulted in 20% increase in lifespan, delayed digestion, 30% reduced oviposition, and 50% reduction in egg hatching. Laid eggs and hatched nymphs showed remarkable alterations in morphology. In summary, R. prolixus ACSL isoforms have distinct roles on lipid metabolism. Although RhoprACSL1 functions remain unclear, we propose that RhoprACSL2 is the main contributor for the formation of the intracellular acyl-CoA pool channeled for ß-oxidation in the fat body, and is also required for normal reproduction.

Deficiency of glycerol-3-phosphate acyltransferase 1 decreases triacylglycerol storage and induces fatty acid oxidation in insect fat body.

Glycerol-3-phosphate acyltransferases (GPAT) catalyze the initial and rate-limiting step for the de novo synthesis of triacylglycerol (TAG). Four mammalian GPAT isoforms have been identified: the mitochondria-associated GPAT1 and 2, and the endoplasmic reticulum (ER)-associated GPAT3 and 4. In the insect Rhodnius prolixus, a vector of Chagas' disease, we previously predicted a mitochondrial-like isoform (RhoprGPAT1) from genomic data. In the current study, we clone the RhoprGPAT1 coding sequence and identify an ER-associated GPAT (RhoprGPAT4) as the second isoform in the insect. RhoprGPAT1 contributes 15% of the total GPAT activity in anterior midgut, 50% in posterior midgut and fat body, and 70% in the ovary. The RhoprGpat1 gene is the predominant transcript in the midgut and fat body. To evaluate the physiological relevance of RhoprGPAT1, we generate RhoprGPAT1-deficient insects. The knockdown of RhoprGpat1 results in 50% and 65% decrease in TAG content in the posterior midgut and fat body, respectively. RhoprGpat1-deficient insects also exhibits impaired lipid droplet expansion and a 2-fold increase in fatty acid ß-oxidation rates in the fat body. We propose that the RhoprGPAT1 mitochondrial-like isoform is required to channel fatty acyl chains towards TAG synthesis and away from ß-oxidation. Such a process is crucial for the insect lipid homeostasis.

In the fed state, autophagy plays a crucial role in assisting the insect vector Rhodnius prolixus mobilize TAG reserves under forced flight activity.

Autophagy is a cellular degradation pathway mediated by highly conserved autophagy-related genes (Atgs). In our previous work, we showed that inhibiting autophagy under starvation conditions leads to significant physiological changes in the insect vector of Chagas disease Rhodnius prolixus; these changes include triacylglycerol (TAG) retention in the fat body, reduced survival and impaired locomotion and flight capabilities. Herein, because it is known that autophagy can be modulated in response to various stimuli, we further investigated the role of autophagy in the fed state, following blood feeding. Interestingly, the primary indicator for the presence of autophagosomes, the lipidated form of Atg8 (Atg8-II), displayed 20%-50% higher autophagic activation in the first 2 weeks after feeding compared to the third week when digestion was complete. Despite the elevated detection of autophagosomes, RNAi-mediated suppression of RpAtg6 and RpAtg8 did not cause substantial changes in TAG or protein levels in the fat body or the flight muscle during blood digestion. We also found that knockdown of RpAtg6 and RpAtg8 led to modest modulations in the gene expression of essential enzymes involved in lipid metabolism and did not significantly stimulate the expression of the chaperones BiP and PDI, which are the main effectors of the unfolded protein response. These findings indicate that impaired autophagy leads to slight disturbances in lipid metabolism and general cell proteostasis. However, the ability of insects to fly during forced flight until exhaustion was reduced by 60% after knockdown of RpAtg6 and RpAtg8. This change was accompanied by TAG and protein increases as well as decreased ATP levels in the fat body and flight muscle, indicating that autophagy during digestion, i.e., under fed conditions, is necessary to sustain high-performance activity.

Deficiency of Brummer lipase disturbs lipid mobilization and locomotion, and impairs reproduction due to defects in the eggshell ultrastructure in the insect vector Rhodnius prolixus.

Rhodnius prolixus is a hematophagous insect, which feeds on large and infrequent blood meals, and is a vector of trypanosomatids that cause Chagas disease. After feeding, lipids derived from blood meal are stored in the fat body as triacylglycerol, which is recruited under conditions of energy demand by lipolysis, where the first step is catalyzed by the Brummer lipase (Bmm), whose orthologue in mammals is the adipose triglyceride lipase (ATGL). Here, we investigated the roles of Bmm in adult Rhodnius prolixus under starvation, and after feeding. Its gene (RhoprBmm) was expressed in all the analyzed insect organs, and its transcript levels in the fat body were not altered by nutritional status. RNAi-mediated knockdown of RhoprBmm caused triacylglycerol retention in the fat body during starvation, resulting in larger lipid droplets and lower ATP levels compared to control females. The silenced females showed decreased flight capacity and locomotor activity. When RhoprBmm knockdown occurred before the blood meal and the insects were fed, the females laid fewer eggs, which collapsed and showed low hatching rates. Their hemolymph had reduced diacylglycerol content and vitellogenin concentration. The chorion (eggshell) of their eggs had no difference in hydrocarbon amounts or in dityrosine crosslinking levels compared to control eggs. However, it showed ultrastructural defects. These results demonstrated that Bmm activity is important not only to guarantee lipid mobilization to maintain energy homeostasis during starvation, but also for the production of viable eggs after a blood meal, by somehow contributing to the right formation of the egg chorion.

Triacylglycerol biosynthesis occurs via the glycerol-3-phosphate pathway in the insect Rhodnius prolixus.

Although triacylglycerol (TAG) stores play a critical role in organisms, mechanisms underlying TAG synthesis are poorly understood in invertebrates. In mammals, the synthesis of glycerolipids, including TAG, diacylglycerol (DAG) and phospholipids (PL), occurs predominantly by the glycerol-3-phosphate (G3P) pathway in most cell types, except for in enterocytes. In these cells, the monoacylglycerol (MAG) pathway accounts for the majority of glycerolipid production. The insect Rhodnius prolixus, a vector of Chagas' disease, exhibits a high capacity to produce glycerolipids in the midgut after a blood meal, providing substrates that are transferred to other organs, such as the fat body, which is specialized in TAG production and storage. In this report, the genes required for TAG synthesis were identified in the R. prolixus genome. The genomic data indicated that TAG is synthesized by the G3P pathway, which is the sole pathway for TAG synthesis in this organism. Furthermore, transcription of both the RpGpat and RpDgat genes were upregulated in a diverse number of organs at moments of highest lipid production. In the midgut and fat body, in vitro synthesis of glycerolipids required G3P, but not MAG, as the initial substrate. These results indicate that the G3P pathway is the only route for TAG synthesis in R. prolixus, and its regulation at the transcriptional level can be a determinant of glycerolipid synthesis and TAG formation in insect organs.

Effect of starvation on lipophorin density in fifth instar larval Manduca sexta.

Lipophorin (Lp) is a major insect lipoprotein and is responsible for lipid transport between organs. In this study, the effect of starvation on Lp properties was analyzed in larval Manduca sexta during the fifth instar. Lp hemolymph concentrations in larvae at days 1 and 2 were around 2-3 mg/ml and at day 3 it increased to 8 mg/ml. When larvae were starved for 24 h, they did not grow, but their body mass and hemolymph volume did not decrease significantly. Differences in Lp densities were observed. In fed larvae, from days 1 to 4, two major Lp populations were found with densities of 1.124 ± 0.002 (high density Lp-larval1 , HDLp-L1 ) and 1.141 ± 0.002 g/ml (HDLp-L2 ). When larvae were starved for 24 h, only one Lp population was present, with density 1.114 ± 0.001 g/ml (HDLp-Ls ). When larvae were abdominally ligated at day 1 or 2 of fifth instar, only HDLp-Ls was found after 24 h, indicating that the formation of this HDLp population was not dependent on any factor released by head. On the other hand, larvae that were ligated at day 3 showed the same Lp populations as the fed ones. In 24-h starved larvae, lipid load in Lp was higher as compared to the fed controls. In 24-h ligated larvae Lp lipid content increased when ligation was performed on day 1 or 2, but not on day 3. So, different responses to starvation can be observed depending on the developmental phase of the same larval instar.

Rhodnius prolixus lipophorin: lipid composition and effect of high temperature on physiological role.

Lipophorin is a major lipoprotein that transports lipids in insects. In Rhodnius prolixus, it transports lipids from midgut and fat body to the oocytes. Analysis by thin-layer chromatography and densitometry identified the major lipid classes present in the lipoprotein as diacylglycerol, hydrocarbons, cholesterol, and phospholipids (PLs), mainly phosphatidylethanolamine and phosphatidylcholine. The effect of preincubation at elevated temperatures on lipophorin capacity to deliver or receive lipids was studied. Transfer of PLs to the ovaries was only inhibited after preincubation of lipophorin at temperatures higher than 55 °C. When it was pretreated at 75 °C, maximal inhibition of phospholipid transfer was observed after 3-min heating and no difference was observed after longer times, up to 60 min. The same activity was also obtained when lipophorin was heated for 20 min at 75 °C at protein concentrations from 0.2 to 10 mg/ml. After preincubation at 55 °C, the same rate of lipophorin loading with PLs at the fat body was still present, and 30% of the activity was observed at 75 °C. The effect of temperature on lipophorin was also analyzed by turbidity and intrinsic fluorescence determinations. Turbidity of a lipophorin solution started to increase after preincubations at temperatures higher than 65 °C. Emission fluorescence spectra were obtained for lipophorin, and the spectral area decreased after preincubations at 85 °C or above. These data indicated no difference in the spectral center of mass at any tested temperature. Altogether, these results demonstrate that lipophorin from R. prolixus is very resistant to high temperatures.

Trypanosoma cruzi: effects of heat shock on ecto-ATPase activity.

In this work, we demonstrate that Trypanosoma cruzi Y strain epimastigotes exhibit Mg2+-dependent ecto-ATPase activity that is stimulated by heat shock. When the epimastigotes were incubated at 37°C for 2h, the ecto-ATPase activity of the cells was 43.95±0.97 nmol Pi/h×10(7) cells, whereas the ecto-ATPase activity of cells that were not exposed to heat shock stress was 16.97±0.30 nmol Pi/h×10(7) cells. The ecto-ATPase activities of cells, that were exposed or not exposed to heat shock stress had approximately the same Km values (2.25±0.26 mM ATP and 1.55±0.23 mM ATP, respectively) and different Vmax values. The heat-shocked cells had higher Vmax values (54.38±3.07 nmol Pi/h×10(7) cells) than the cells that were not exposed to heat shock (19.38±1.76 nmol Pi/h×10(7) cells). We also observed that the ecto-phosphatase and ecto-5'nucleotidase activities of cells that had been incubated at 28°C or 37°C were the same. Interestingly, cycloheximide, an inhibitor of protein synthesis, suppressed the heat shock effect of ecto-ATPase activity on T. cruzi. The Mg2+-dependent ecto-ATPase activity from the Y strain (high virulence) was approximately 2-fold higher than that of Dm28c (a clone with low virulence). In addition, these two strains presented different responses to heat shock with regard to their ecto-ATPase activities; Y strain epimastigotes had a stimulation of 2.52-fold while the Dm28c strain had a 1.71-fold stimulation. In this context, the virulent trypomastigote form of T. cruzi, Dm28c, had an ecto-ATPase activity that was more than 7-fold higher (66.67±5.98 nmol Pi/h×10(7) cells) than that of the insect epimastigote forms (8.91±0.76 nmol Pi/h×10(7) cells). This difference increased to approximately 10-fold when both forms were subjected to heat shock stress (181.14±16.48 nmol Pi/h×10(7) cells for trypomastigotes and 16.71±1.17 nmol Pi/h×10(7) cells for epimastigotes at 37°C). The ecto-ATPase activity of a plasma membrane-enriched fraction obtained from T. cruzi epimastigotes was not increased by heat treatment, which suggested that cytoplasmic components had an influence on enzyme activation by heat shock stress.

Blood meal digestion and changes in lipid reserves are associated with the post-ecdysis development of the flight muscle and ovary in young adults of Rhodnius prolixus.

Rhodnius prolixus is a hemimetabolous, hematophagous insect, and both nymphs and adults feed exclusively on blood. The blood feeding triggers the molting process and, after five nymphal instar stages, the insect reaches the winged adult form. After the final ecdysis, the young adult still has a lot of blood in the midgut and, thus, we have investigated the changes in protein and lipid contents that are observed in the insect organs as digestion continues after molting. Total midgut protein content decreased during the days after the ecdysis, and digestion was finished fifteen days later. Simultaneously, proteins and triacylglycerols present in the fat body were mobilized, and their contents decreased, whereas they increased in both the ovary and the flight muscle. In order to evaluate the activity of de novo lipogenesis of each organ, the fat body, ovary and flight muscle were incubated in the presence of radiolabeled acetate, and the fat body showed the highest efficiency rate (around 47%) to convert the taken up acetate into lipids. The levels of de novo lipid synthesis in the flight muscle and ovary were very low. When 3H-palmitate was injected into the young females, its incorporation by the flight muscle was higher than by the ovary or the fat body. In the flight muscle, the 3H-palmitate was similarly distributed amongst triacylglycerols, phospholipids, diacylglycerols and free fatty acids, while in the ovary and fat body it was mostly found in triacylglycerols and phospholipids. The flight muscle was not fully developed after the molt, and at day two no lipid droplets were observed. At day five, very small lipid droplets were present, and they increased in size up to day fifteen. The diameter of the muscle fibers also increased from day two to fifteen, as well as the internuclear distance, indicating that muscle hypertrophy occurred along these days. The lipid droplets from the fat body showed a different pattern, and their diameter decreased after day two, but started to increase again at day ten. The data presented herein describes the development of the flight muscle after the final ecdysis, and modifications that occur regarding lipid stores. We show that, after molting, substrates that are present in the midgut and fat body are mobilized and directed to the ovary and flight muscle, for the adults of R. prolixus to be ready to feed and reproduce.

Knockdown of carnitine palmitoyltransferase I (CPT1) reduces fat body lipid mobilization and resistance to starvation in the insect vector Rhodnius prolixus.

The energy stored in fatty acids is essential for several critical activities of insects, such as embryogenesis, oviposition, and flight. Rhodnius prolixus is an obligatory hematophagous hemipteran and vector of Chagas disease, and it feeds infrequently on very large blood meals. As digestion slowly occurs, lipids are synthesized and accumulate in the fat body, mainly as triacylglycerol, in lipid droplets. Between feeding bouts, proper mobilization and oxidation of stored lipids are crucial for survival, and released fatty acids are oxidized by mitochondrial ß-oxidation. Carnitine palmitoyl transferase I (CPT1) is the enzyme that catalyzes the first reaction of the carnitine shuttle, where the activated fatty acid, acyl-CoA, is converted to acyl-carnitine to be transported into the mitochondria. Here, we investigated the role of CPT1 in lipid metabolism and in resistance to starvation in Rhodnius prolixus. The expression of the CPT1 gene (RhoprCpt1) was determined in the organs of adult females on the fourth day after a blood meal, and the flight muscle showed higher expression levels than the ovary, fat body, and anterior and posterior midgut. RhoprCpt1 expression in the fat body dramatically decreased after feeding, and started to increase again 10 days later, but no changes were observed in the flight muscle. ß-oxidation rates were determined in flight muscle and fat body homogenates with the use of 3H-palmitate, and in unfed females, they were higher in the flight muscle. In the fat body, lipid oxidation activity did not show any variation before or at different days after feeding, and was not affected by the presence of etomoxir or malonyl-CoA. We used RNAi and generated RhoprCPT1-deficient insects, which surprisingly did not show a decrease in measured 3H-palmitate oxidation rates. However, the RNAi-knockdown females presented increased amounts of triacylglycerol and larger lipid droplets in the fat body, but not in the flight muscle. When subjected to starvation, these insects had a shorter lifespan. These results indicated that the inhibition of RhoprCpt1 expression compromised lipid mobilization and affected resistance to starvation.

ATP synthase affects lipid metabolism in the kissing bug Rhodnius prolixus beyond its role in energy metabolism.

ATP synthase plays an essential role in mitochondrial metabolism, being responsible for the production of ATP in oxidative phosphorylation. However, recent results have shown that it may also be present in the cell membrane, involved in lipophorin binding to its receptors. Here, we used a functional genetics approach to investigate the roles of ATP synthase in lipid metabolism in the kissing bug Rhodnius prolixus. The genome of R. prolixus encodes five nucleotide-binding domain genes of the ATP synthase α and ß family, including the α and ß subunits of ATP synthase (RpATPSynα and RpATPSynß), and the catalytic and non-catalytic subunits of the vacuolar ATPase (RpVha68 and RpVha55). These genes were expressed in all analyzed organsn highest in the ovaries, fat body and flight muscle. Feeding did not regulate the expression of ATP synthases in the posterior midgut or fat body. Furthermore, ATP synthase is present in the fat body's mitochondrial and membrane fractions. RpATPSynß knockdown by RNAi impaired ovarian development and reduced egg-laying by approximately 85%. Furthermore, the lack of RpATPSynß increased the amount of triacylglycerol in the fat body due to increased de novo fatty acid synthesis and reduced transfer of lipids to lipophorin. RpATPSynα knockdown had similar effects, with altered ovarian development, reduced oviposition, and triacylglycerol accumulation in the fat body. However, ATP synthases knockdown had only a slight effect on the amount of ATP in the fat body. These results support the hypothesis that ATP synthase has a direct role in lipid metabolism and lipophorin physiology, which are not directly due to changes in energy metabolism.

Silencing of the 20S proteasomal subunit-α6 triggers full oogenesis arrest and increased mRNA levels of the selective autophagy adaptor protein p62/SQSTM1 in the ovary of the vector Rhodnius prolixus.

The high reproductive rates of insects contribute significantly to their ability to act as vectors of a variety of vector-borne diseases. Therefore, it is strategically critical to find molecular targets with biotechnological potential through the functional study of genes essential for insect reproduction. The ubiquitin-proteasome system is a vital degradative pathway that contributes to the maintenance of regular eukaryotic cell proteostasis. This mechanism involves the action of enzymes to covalently link ubiquitin to proteins that are meant to be delivered to the 26S proteasome and broken down. The 26S proteasome is a large protease complex (including the 20S and 19S subcomplexes) that binds, deubiquitylates, unfolds, and degrades its substrates. Here, we used bioinformatics to identify the genes that encode the seven α and ß subunits of the 20S proteasome in the genome of R. prolixus and learned that those transcripts are accumulated into mature oocytes. To access proteasome function during oogenesis, we conducted RNAi functional tests employing one of the 20S proteasome subunits (Prosα6) as a tool to suppress 20S proteasomal activity. We found that Prosα6 silencing resulted in no changes in TAG buildup in the fat body and unaffected availability of yolk proteins in the hemolymph of vitellogenic females. Despite this, the silencing of Prosα6 culminated in the impairment of oocyte maturation at the early stages of oogenesis. Overall, we discovered that proteasome activity is especially important for the signals that initiate oogenesis in R. prolixus and discuss in what manner further investigations on the regulation of proteasome assembly and activity might contribute to the unraveling of oogenesis molecular mechanisms and oocyte maturation in this vector.

Gene identification and enzymatic properties of a membrane-bound trehalase from the ovary of Rhodnius prolixus.

Trehalose represents the main hemolymph sugar in most insects and its metabolic availability is regulated by trehalase. In this study, trehalase activity associated with the reproductive system was investigated in the insect Rhodnius prolixus, a hematophagous hemipteran vector of Chagas' disease. A single-copy gene that encodes a membrane-bound trehalase (RpTre-2) was identified in the genome of R. prolixus. RpTre-2 deduced amino acid sequence is closely related to other insect membrane-bound trehalases. The expression of this gene was detected in all analyzed organs, including ovary, where total trehalase enzymatic activity was determined, and was highest at day 7 after blood meal. Ovary membranes showed a major trehalase specific activity, which confirmed the presence of a membrane-bound trehalase in this insect. This trehalase activity seemed not to be regulated at transcriptional level, as the expression of RpTre-2 gene in the ovary did not change over the days after feeding. Similarly, ovarian follicles at different developmental stages did not show any variation in the transcription level of this gene. The RpTre-2 kinetic parameters were also investigated. Activity was highest at pH 5.5 and followed Michaelis-Menten kinetics, with an apparent K(m) = 1.42 ± 0.36 mM and Vmax = 167.90 ± 12.91 nmol/mg protein/h. These data reveal the presence of a membrane-bound trehalase in R. prolixus that is active in ovary and probably takes part in the insect carbohydrate metabolism associated with the reproductive process.

LDL uptake by Leishmania amazonensis: involvement of membrane lipid microdomains.

Leishmania amazonensis lacks a de novo mechanism for cholesterol synthesis and therefore must scavenge this lipid from the host environment. In this study we show that the L. amazonensis takes up and metabolizes human LDL(1) particles in both a time and dose-dependent manner. This mechanism implies the presence of a true LDL receptor because the uptake is blocked by both low temperature and by the excess of non-labelled LDL. This receptor is probably associated with specific microdomains in the membrane of the parasite, such as rafts, because this process is blocked by methyl-ß-cyclodextrin (MCBD). Cholesteryl ester fluorescently-labeled LDL (BODIPY-cholesteryl-LDL) was used to follow the intracellular distribution of this lipid. After uptake it was localized in large compartments along the parasite body. The accumulation of LDL was analyzed by flow cytometry using FITC-labeled LDL particles. Together these data show for the first time that L. amazonensis is able to compensate for its lack of lipid synthesis through the use of a lipid importing machinery largely based on the uptake of LDL particles from the host. Understanding the details of the molecular events involved in this mechanism may lead to the identification of novel targets to block Leishmania infection in human hosts.

Silencing of Mitochondrial Trifunctional Protein A Subunit (HADHA) Increases Lipid Stores, and Reduces Oviposition and Flight Capacity in the Vector Insect Rhodnius prolixus.

Rhodnius prolixus is an obligatory hematophagous insect, vector of Chagas disease. After blood meal, lipids are absorbed, metabolized, synthesized, and accumulated in the fat body. When necessary, stored lipids are mobilized, transported to other organs, or are oxidized to provide energy. Mitochondrial ß-oxidation is a cyclic conserved pathway, where degradation of long-chain fatty acids occurs to contribute to cellular energetic demands. Three of its reactions are catalyzed by the mitochondrial trifunctional protein (MTP), which is composed by hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunits alpha and beta (HADHA and HADHB, respectively). Here, we investigated the role of HADHA in lipid metabolism and reproduction of Rhodnius prolixus females. The expression of HADHA gene (RhoprHadha) was determined in the organs of starving adult insects. The flight muscle and ovary had higher expression levels when compared to the anterior and posterior midguts or the fat body. RhoprHadha gene expression was upregulated by blood meal in the flight muscle and fat body. We generated insects with RNAi-mediated knockdown of RhoprHadha to address the physiological role of this gene. RhoprHadha deficiency resulted in higher triacylglycerol content and larger lipid droplets in the fat body during starvation. After feeding, lifespan of the knockdown females was not affected, but they exhibited a decrease in oviposition, although hatching was the same in both groups. Silenced females showed lower forced flight capacity than the control ones, and their fat bodies had lower gene expression levels of Brummer lipase (RhoprBmm) and long-chain acyl-CoA synthetase 2 (RhoprAcsl2). Taken together, these findings indicate that HADHA is important to guarantee successful reproduction and efficient mobilization of lipid stores during starvation and flight.

Deficiency of Acetyl-CoA Carboxylase Impairs Digestion, Lipid Synthesis, and Reproduction in the Kissing Bug Rhodnius prolixus.

Rhodnius prolixus is a hematophagous insect, vector of Chagas disease. After feeding, as blood is slowly digested, amino acids are used as substrates to fuel lipid synthesis, and adult females accumulate lipids in the fat body and produce eggs. In order to evaluate the importance of de novo fatty acid synthesis for this insect metabolism, we generated acetyl-CoA carboxylase (ACC) deficient insects. The knockdown (AccKD) females had delayed blood digestion and a shorter lifespan. Their fat bodies showed reduced de novo lipogenesis activity, did not accumulate triacylglycerol during the days after blood meal, and had smaller lipid droplets. At 10 days after feeding, there was a general decrease in the amounts of neutral lipids and phospholipids in the fat body. In the hemolymph, no difference was observed in lipid composition at 5 days after blood meal, but at day ten, there was an increase in hydrocarbon content and a decrease in phospholipids. Total protein concentration and amino acid composition were not affected. The AccKD females laid 60% fewer eggs than the control ones, and only 7% hatched (89% for control), although their total protein and triacylglycerol contents were not different. Scanning electron microscopy of the egg surface showed that chorion (eggshell) from the eggs laid by the AccKD insects had an altered ultrastructural pattern when compared to control ones. These results show that ACC has a central role in R. prolixus nutrient homeostasis, and its appropriate activity is important to digestion, lipid synthesis and storage, and reproductive success.