Identification and expression of the first nonmammalian amyloid-beta precursor-like protein APLP2 in the amphibian Xenopus laevis.

The Alzheimer's disease-linked amyloid-beta precursor protein (APP) belongs to a superfamily of proteins, which also comprises the amyloid-beta precursor-like proteins, APLP1 and APLP2. Whereas APP has been identified in both lower and higher vertebrates, thus far, APLP1 and 2 have been characterized only in human and rodents. Here we identify the first nonmammalian APLP2 protein in the South African claw-toed frog Xenopus laevis. The identity between the Xenopus and mammalian APLP2 proteins is approximately 75%, with the highest degree of conservation in a number of amino-terminal regions, the transmembrane domain and the cytoplasmic tail. Furthermore, amino acid residues known to be phosphorylated and glycosylated in mammalian APLP2 are conserved in Xenopus. The availability of the Xenopus APLP2 protein sequence allowed a phylogenetic analysis of APP superfamily members that suggested the occurrence of APP and preAPLP lineages with their separation predating the mammalian-amphibian split. As in mammals, Xenopus APLP2 mRNA was ubiquitously expressed and alternatively spliced forms were detected. However, the expression ratios between the mRNA forms in the various tissues examined were different between Xenopus and mammals, most prominently for the alternatively spliced forms containing the Kunitz protease inhibitor-coding region that were less abundantly expressed than the corresponding mammalian forms. Thus, the identification of APLP2 in Xenopus has revealed evolutionarily conserved regions that may help to delineate functionally important domains, and its overall high degree of conservation suggests an important role for this APP superfamily member.     

The short 5′ untranslated region of the βA3/A1-crystallin mRNA is responsible for leaky ribosomal scanning

Leaky ribosomal scanning allows the expression of multiple proteins from a single mRNA by occasionally skipping the first start codon, and initiating translation at a subsequent one. A3- and A1-crystallin, two members of the -crystallin family of vertebrate eye lens proteins, are produced via this mechanism, of which, until now, only very few examples have been found in eukaryotic genes. Since the two start codons on the A3/A1 messenger lie in the same reading frame, the two translated proteins are identical, except for the 17 residues shorter N-terminal extension of A1-crystallin. It has been suggested that the very short leader (5–7 nucleotides) of the A3/A1 messenger might cause slippage at the first start codon, although the unfavorable context of this start codon might also be responsible. Using transient transfections, we now demonstrate that increasing the length of the leader sequence to 67 nucleotides indeed completely abolishes translation initiation at the second start codon, and thus expression of the A1-crystallin protein. Messengers having a leader of 5, 7 or 14 nucleotides all express both A3- and A1-crystallin at very similar relative levels.     

Structural biology: Paper alert

A selection of interesting papers that were published in the two months before our press date in major journals most likely to report significant results in structural biology.     

Costs of parental care on hunting behaviour of Helobdella papillornata (Euhirudinea: Glossiphoniidae)

Helobdella papillornata, an Australian freshwater leech, feeds primarily on snails and has a high level of parental care involving brooding eggs and young, with direct feeding of young. Parental costs and offspring benefits from these behaviours are poorly understood. A potential cost of parental care may be a change in the time taken to hunt prey. To test this hypothesis, the hunting behaviour of adults without progeny, parents with eggs, and parents with young were compared. We found that parents brooding eggs had a significantly (P = 0.029) longer lag time to begin hunting than parents brooding young, and spent significantly (P = 0.018) less time actively hunting than non-brooding adults. These costs, which may represent lost potential for the parent’s future reproductive success, should be outweighed by the fitness benefits of improved growth and survival of offspring, if parental care is favoured by selection. The hunting costs of care in Helobdella and other benthic, dorsoventrally flattened leeches in the family Glossiphoniidae may be smaller than the costs of brood tending that would be imposed on other freshwater leeches, and this difference may help explain the restriction of care to a single clade of the Euhirudinea.     

Dectin-1 Is Essential for Reverse Transcytosis of Glycosylated SIgA-Antigen Complexes by Intestinal M Cells

Intestinal microfold (M) cells possess a high transcytosis capacity and are able to transport a broad range of materials including particulate antigens, soluble macromolecules, and pathogens from the intestinal lumen to inductive sites of the mucosal immune system. M cells are also the primary pathway for delivery of secretory IgA (SIgA) to the gut-associated lymphoid tissue. However, although the consequences of SIgA uptake by M cells are now well known and described, the mechanisms whereby SIgA is selectively bound and taken up remain poorly understood. Here we first demonstrate that both the Cα1 region and glycosylation, more particularly sialic acid residues, are involved in M cell–mediated reverse transcytosis. Second, we found that SIgA is taken up by M cells via the Dectin-1 receptor, with the possible involvement of Siglec-5 acting as a co-receptor. Third, we establish that transcytosed SIgA is taken up by mucosal CX3CR1⁺ dendritic cells (DCs) via the DC-SIGN receptor. Fourth, we show that mucosal and systemic antibody responses against the HIV p24-SIgA complexes administered orally is strictly dependent on the expression of Dectin-1. Having deciphered the mechanisms leading to specific targeting of SIgA-based Ag complexes paves the way to the use of such a vehicle for mucosal vaccination against various infectious diseases.     

Association of IREB2 and CHRNA3 polymorphisms with airflow obstruction in severe alpha-1 antitrypsin deficiency

Background

The development of COPD in subjects with alpha-1 antitrypsin (AAT) deficiency is likely to be influenced by modifier genes. Genome-wide association studies and integrative genomics approaches in COPD have demonstrated significant associations with SNPs in the chromosome 15q region that includes CHRNA3 (cholinergic nicotine receptor alpha3) and IREB2 (iron regulatory binding protein 2).We investigated whether SNPs in the chromosome 15q region would be modifiers for lung function and COPD in AAT deficiency.

Methods

The current analysis included 378 PIZZ subjects in the AAT Genetic Modifiers Study and a replication cohort of 458 subjects from the UK AAT Deficiency National Registry. Nine SNPs in LOC123688, CHRNA3 and IREB2 were selected for genotyping. FEV₁ percent of predicted and FEV₁/FVC ratio were analyzed as quantitative phenotypes. Family-based association analysis was performed in the AAT Genetic Modifiers Study. In the replication set, general linear models were used for quantitative phenotypes and logistic regression models were used for the presence/absence of emphysema or COPD.

Results

Three SNPs (rs2568494 in IREB2, rs8034191 in LOC123688, and rs1051730 in CHRNA3) were associated with pre-bronchodilator FEV₁ percent of predicted in the AAT Genetic Modifiers Study. Two SNPs (rs2568494 and rs1051730) were associated with the post-bronchodilator FEV₁ percent of predicted and pre-bronchodilator FEV₁/FVC ratio; SNP-by-gender interactions were observed. In the UK National Registry dataset, rs2568494 was significantly associated with emphysema in the male subgroup; significant SNP-by-smoking interactions were observed.

Conclusions

IREB2 and CHRNA3 are potential genetic modifiers of COPD phenotypes in individuals with severe AAT deficiency and may be sex-specific in their impact.     

Skeletal muscle Nur77 expression enhances oxidative metabolism and substrate utilization

Mitochondrial dysfunction has been implicated in the pathogenesis of type 2 diabetes. Identifying novel regulators of mitochondrial bioenergetics will broaden our understanding of regulatory checkpoints that coordinate complex metabolic pathways. We previously showed that Nur77, an orphan nuclear receptor of the NR4A family, regulates the expression of genes linked to glucose utilization. Here we demonstrate that expression of Nur77 in skeletal muscle also enhances mitochondrial function. We generated MCK-Nur77 transgenic mice that express wild-type Nur77 specifically in skeletal muscle. Nur77-overexpressing muscle had increased abundance of oxidative muscle fibers and mitochondrial DNA content. Transgenic muscle also exhibited enhanced oxidative metabolism, suggestive of increased mitochondrial activity. Metabolomic analysis confirmed that Nur77 transgenic muscle favored fatty acid oxidation over glucose oxidation, mimicking the metabolic profile of fasting. Nur77 expression also improved the intrinsic respiratory capacity of isolated mitochondria, likely due to the increased abundance of complex I of the electron transport chain. These changes in mitochondrial metabolism translated to improved muscle contractile function ex vivo and improved cold tolerance in vivo. Our studies outline a novel role for Nur77 in the regulation of oxidative metabolism and mitochondrial activity in skeletal muscle.     

Shift work and sleep disorder comorbidity tend to go hand in hand

Taking into consideration that shift work has a wide-ranging impact on circadian and sleep functioning, it seems likely that shift work increases the risk of a general sleep disturbance, spread out over a multitude of comorbid sleep disorders. The aim of the present study is to analyze and present the sleep disorder data of 250 shift workers and 971 permanent day workers, taken from a nationally representative sample. Additional data concerning duration, timing, and quality of sleep, daytime functioning and social/family variables were added to the analyses. The results showed that the shift workers experienced significantly more difficulties with the variability of their sleep times, reported more napping and considered themselves more as poor sleepers than the day workers. Most importantly, shift work, in comparison with day work, appeared associated with a significantly higher prevalence of the clinical, International Classification of Sleep Disorders’ defined symptoms of nearly all main sleep disorders (including shift work disorder). For shift workers, the prevalence of a general sleep disturbance was 39.0% (95%CI 33.2 – 45.2), significantly higher than for day workers (24.6%, 95%CI 22.0 – 27.4). Moreover, shift workers were characterized by high levels of sleep disorder comorbidity. In addition, exclusively for shift workers, the prevalence of disordered sleep systematically decreased across decades of life and was considerably higher for single versus partnered shift workers. This study adds to the insight into the interacting factors that determine shift work coping and may play a role in occupational health interventions aimed at reducing sleep problems and thus improving the resilience and tolerance of the shift worker.     

Enzyme-coding genes as molecular clocks: The molecular evolution of animal alpha-amylases

Summary We constructed a cDNA library for the beetle,Tribolium castaneum. This library was screened using a cloned amylase gene fromDrosophila melanogaster as a molecular probe. Beetle amylase cDNA clones were isolated from this bank, and the nucleotide sequence was obtained for a cDNA clone with a coding capacity for 228 amino acids. Both the nucleotide sequence and predicted amino acid sequence were compared to our recent results forD. melanogaster alpha-amylases, along with published sequences for other alpha-amylases. The results show that animal alpha-amylases are highly conserved over their entire length. A borader comparison, which includes plant and microbial alpha-amylase sequences, indicates that parts of the gene are conserved between prokaryotes, plants, and animals. We discuss the potential importance of this and other enzyme-coding genes for the construction of molecular phylogenies and for the study of the general question of molecular clocks in evolution.