Drosophila melanogaster acetylcholinesterase: Identification and expression of two mutations responsible for cold- and heat-sensitive phenotypes

Ace ^IJ29 and Ac ^IJ40 are cold- and heat-sensitive variants of the gene coding for acetylcholinesterase in Drosophila melanogaster. In the homozygous condition, these mutations are lethal when animals are raised at restrictive temperatures, i.e., below 23° C for Ace ^IJ29 or above 25° C for Ace ^IJ40. The coding regions of the gene in these mutants were sequenced and mutations changing Ser³⁷⁴ to Phe in Ace ^IJ29 and Pro⁷⁵ to Leu in Ace ^IJ40 were found. Acetylcholinesterases bearing these mutations were expressed in Xenopus oocytes and we found that these mutations decrease the secretion rate of the protein most probably by affecting its folding. This phenomenon is exacerbated at restrictive temperatures decreasing the amount of secreted acetylcholinesterase below the lethality threshold. In parallel, the substitution of the conserved Asp²⁴⁸ by an Asn residue completely inhibits the activity of the enzyme and its secretion, preventing the correct folding of the protein in a non-conditional manner.     

Malaria and urbanization in sub-Saharan Africa

There are already 40 cities in Africa with over 1 million inhabitants and the United Nations Environmental Programme estimates that by 2025 over 800 million people will live in urban areas. Recognizing that malaria control can improve the health of the vulnerable and remove a major obstacle to their economic development, the Malaria Knowledge Programme of the Liverpool School of Tropical Medicine and the Systemwide Initiative on Malaria and Agriculture convened a multi-sectoral technical consultation on urban malaria in Pretoria, South Africa from 2nd to 4th December, 2004. The aim of the meeting was to identify strategies for the assessment and control of urban malaria. This commentary reflects the discussions held during the meeting and aims to inform researchers and policy makers of the potential for containing and reversing the emerging problem of urban malaria.     

Sustainable malaria control: transdisciplinary approaches for translational applications

Background

Intravenous (IV) artesunate is the treatment of choice for severe malaria. In Europe, however, no GMP-manufactured product is available and treatment data in European travellers are scarce. Fortunately, artesunate became available in the Netherlands and Belgium through a named patient programme. This is the largest case series of artesunate treated patients with severe malaria in Europe.

Methods

Hospitalized patients treated with IV artesunate between November 2007 and December 2010 in the Netherlands and Belgium were retrospectively evaluated. Patient characteristics, treatment and clinical outcome were recorded on a standardized form and mortality, parasite clearance times and the occurrence of adverse events were evaluated.

Results

Of the 68 treated patients, including 55 with severe malaria, two patients died (2/55 = 3.6%). The mean time to 50% parasite clearance (PCT50), 90% and 99% were 4.4 hours (3.9 - 5.2), 14.8 hours (13.0 - 17.2), and 29.5 hours (25.9 - 34.4) respectively. Artesunate was well tolerated. However, an unusual form of haemolytic anaemia was observed in seven patients. The relationship with artesunate remains uncertain.

Conclusions

Data from the named patient programme demonstrate that IV artesunate is effective and well-tolerated in European travellers lacking immunity. However, increased attention needs to be paid to the possible development of haemolytic anaemia 2-3 weeks after start of treatment. Treatment of IV artesunate should be limited to the period that IV treatment is required and should be followed by a full oral course of an appropriate anti-malarial drug.     

Drosophila acetylcholinesterase. Expression of a functional precursor in Xenopus oocytes.

In insects, acetylcholinesterase is mainly found in the central nervous system. It is expressed in the synapse where it hydrolyzes the neurotransmitter acetylcholine. Maturation of this protein involves several post-translational modifications. The precursor polypeptide is cut at three sites; the N-terminal signal peptide is removed, the C-terminal hydrophobic polypeptide is clipped off and replaced by a glycolipid anchor and the resulting peptide is cut into two polypeptides, corresponding to active subunits. Two of these active subunits are associated to form the final active glycosylated protein. We have expressed the protein via microinjection of an expression vector into Xenopus oocyte nuclei. When the complete cDNA is injected, the acetylcholinesterase formed is biochemically similar to the Drosophila-head acetylcholinesterase. However, the hydrophobic C-terminal peptide is not replaced by a glycolipid anchor. As a consequence, the enzyme is no longer externalized, the proteolytic cutting of the main peptide does not occur and a new polymerization form occurs. Although incompletely processed, this protein is enzymatically active. When a cDNA lacking the coding region of the C-terminal hydrophobic peptide is injected, the resulting acetylcholinesterase is hydrophilic, cleaved into two subunits and secreted into the incubation medium free of contaminants.     

Initiator elements function to determine the activity state of BX-C enhancers

A >300 kb cis-regulatory region is required for the proper expression of the three bithorax complex (BX-C) homeotic genes. Based on genetic and transgenic analysis, a model has been proposed in which the numerous BX-C cis-regulatory elements are spatially restricted through the activation or repression of parasegment-specific chromatin domains. Particular early embryonic enhancers, called initiators, have been proposed to control this complex process. Here, in order to better understand the process of domain activation, we have undertaken a systematic in situ dissection of the iab-6 cis-regulatory domain using a new method, called InSIRT. Using this method, we create and genetically characterize mutations affecting iab-6 function, including mutations specifically modifying the iab-6 initiator. Through our mutagenesis of the iab-6 initiator, we provide strong evidence that initiators function not to directly control homeotic gene expression but rather as domain control centers to determine the activity state of the enhancers and silencers within a cis-regulatory domain.     

Separation of Anopheles merus from freshwater Anopheles gambiae by salinity tolerance test and morphological characters

The separation methods for Anopheles merus from freshwater A. gambiae s.l. involving the use of salinity tolerance test, sensilla coeloconica, palpal ratio and palpal bands were evaluated for a period of one year on a total of about 340 mosquitoes. The salinity tolerance test method was found to be quite simple and reliable but unsuitable in disease transmission studies due to an interval of 2-3 days between the collection and dissection periods and also due to the fact that only a fraction of the mosquito sample is generally identified by this method. Although significantly higher proportions of sensilla coeloconica and palpal ratio were observed in A. merus as compared to freshwater A. gambiae s.l. these characters were found quite unreliable due to their overlapping between two mosquito groups. Sensilla coeloconica and palpal ratio used separately could separate respective percentages of 11.4 and 11.8 A. merus from freshwater A. gambiae s.l., while in combination they separated up to 40.9%. Percentages 4-banded palp mosquitoes accounted for about 32% in A. merus and 19% in freshwater A. gambiae s.l. All these characters also displayed some seasonal variations in the two mosquito groups.     

Post-translational modifications of Drosophila acetylcholinesterase. In vitro mutagenesis and expression in Xenopus oocytes.

Drosophila acetylcholinesterase (EC 3.1.1.7) is a 150-kDa glycoprotein anchored in plasmic membranes via a glycolipid. It is composed of two active subunits which are themselves made of two noncovalently linked polypeptides of 18 and 55 kDa resulting from the proteolysis of a single precursor of 75 kDa. Active Drosophila acetylcholinesterase can be expressed in Xenopus oocytes as an excreted protein. We have identified some of the amino acids essential in post-translational modifications of the protein by site-directed mutagenesis and expression of mutants in this system. The intersubunit disulfide bond involves cysteine at position 615. Cleavage of the 75-kDa precursor, as observed in Drosophila, originates from a hydrophilic peptide (in position 148 to 180) which does not exist in cholinesterase sequences from vertebrates. This cleavage is associated with excretion out of the cell. Drosophila acetylcholinesterase exhibits four effective sites of asparagine-linked glycosylation in positions 126, 174, 331, and 531. We show that glycosylations and dimerization protect the protein against proteolytic digestion. In contrast, none of these post-translational modifications significantly affects the activity of acetylcholinesterase or affinity for its substrate.     

PLEKHA5, PLEKHA6, and PLEKHA7 bind to PDZD11 to target the Menkes ATPase ATP7A to the cell periphery and regulate copper homeostasis

Copper homeostasis is crucial for cellular physiology and development, and its dysregulation leads to disease. The Menkes ATPase ATP7A plays a key role in copper efflux, by trafficking from the Golgi to the plasma membrane upon cell exposure to elevated copper, but the mechanisms that target ATP7A to the cell periphery are poorly understood. PDZD11 interacts with the C-terminus of ATP7A, which contains sequences involved in ATP7A trafficking, but the role of PDZD11 in ATP7A localization is unknown. Here we identify PLEKHA5 and PLEKHA6 as new interactors of PDZD11 that bind to the PDZD11 N-terminus through their WW domains similarly to the junctional protein PLEKHA7. Using CRISPR-KO kidney epithelial cells, we show by immunofluorescence microscopy that WW-PLEKHAs (PLEKHA5, PLEKHA6, PLEKHA7) recruit PDZD11 to distinct plasma membrane localizations and that they are required for the efficient anterograde targeting of ATP7A to the cell periphery in elevated copper conditions. Pull-down experiments show that WW-PLEKHAs promote PDZD11 interaction with the C-terminus of ATP7A. However, WW-PLEKHAs and PDZD11 are not necessary for ATP7A Golgi localization in basal copper, ATP7A copper-induced exit from the Golgi, and ATP7A retrograde trafficking to the Golgi. Finally, measuring bioavailable and total cellular copper, metallothionein-1 expression, and cell viability shows that WW-PLEKHAs and PDZD11 are required for maintaining low intracellular copper levels when cells are exposed to elevated copper. These data indicate that WW-PLEKHAs-PDZD11 complexes regulate the localization and function of ATP7A to promote copper extrusion in elevated copper.