Regulation of nuclear–cytoplasmic shuttling and function of Family with sequence similarity 13, member A (Fam13a), by B56-containing PP2As and Akt

Recent genome-wide association studies reveal that the FAM13A gene is associated with human lung function and a variety of lung diseases, including chronic obstructive pulmonary disease, asthma, lung cancer, and pulmonary fibrosis. The biological functions of Fam13a, however, have not been studied. In an effort to identify novel substrates of B56-containing PP2As, we found that B56-containing PP2As and Akt act antagonistically to control reversible phosphorylation of Fam13a on Ser-322. We show that Ser-322 phosphorylation acts as a molecular switch to control the subcellular distribution of Fam13a. Fam13a shuttles between the nucleus and cytoplasm. When Ser-322 is phosphorylated by Akt, the binding between Fam13a and 14-3-3 is enhanced, leading to cytoplasmic sequestration of Fam13a. B56-containing PP2As dephosphorylate phospho–Ser-322 and promote nuclear localization of Fam13a. We generated Fam13a-knockout mice. Fam13a-mutant mice are viable and healthy, indicating that Fam13a is dispensable for embryonic development and physiological functions in adult animals. Intriguingly, Fam13a has the ability to activate the Wnt pathway. Although Wnt signaling remains largely normal in Fam13a-knockout lungs, depletion of Fam13a in human lung cancer cells causes an obvious reduction in Wnt signaling activity. Our work provides important clues to elucidating the mechanism by which Fam13a may contribute to human lung diseases.     

The role of circulating serotonin in the development of chronic obstructive pulmonary disease

Background

Cigarette smoking is a major risk factor in the development of age-related chronic obstructive pulmonary disease (COPD). The serotonin transporter (SERT) gene polymorphism has been reported to be associated with COPD, and the degree of cigarette smoking has been shown to be a significant mediator in this relationship. The interrelation between circulating serotonin (5-hydroxytyptamine, 5-HT), cigarette smoking and COPD is however largely unknown. The current study aimed at investigating the mediation effects of plasma 5-HT on cigarette smoking-induced COPD and the relation between plasma 5-HT levels and age.

Methods

The association between plasma 5-HT, age and COPD was analyzed in a total of 62 COPD patients (ever-smokers) and 117 control subjects (healthy non-smokers and ever-smokers). Plasma 5-HT levels were measured by enzyme-linked immuno assay (EIA).

Results

The elevated plasma 5-HT levels were significantly associated with increased odds for COPD (OR = 1.221, 95% CI = 1.123 to 1.319, p<0.0001). The effect remained significant after being adjusted for age and pack-years smoked (OR = 1.271, 95% CI = 1.134 to 1.408, p = 0.0003). Furthermore, plasma 5-HT was found to mediate the relation between pack-years smoked and COPD. A positive correlation (r = 0.303, p = 0.017) was found between plasma 5-HT levels and age in COPD, but not in the control subjects (r = −0.149, p = 0.108).

Conclusion

Our results suggest that cigarette smoke-induced COPD is partially mediated by the plasma levels of 5-HT, and that these become elevated with increased age in COPD. The elevated plasma 5-HT levels in COPD might contribute to the pathogenesis of this disease.     

Nature versus nurture in two highly enantioselective esterases from Bacillus cereus and Thermoanaerobacter tengcongensis

There is an increasing need for the use of biocatalysis to obtain enantiopure compounds as chiral building blocks for drug synthesis such as antibiotics. The principal findings of this study are: (i) the complete sequenced genomes of Bacillus cereus ATCC 14579 and Thermoanaerobacter tengcongensis MB4 contain a hitherto undescribed enantioselective and alkaliphilic esterase (BcEST and TtEST respectively) that is specific for the production of (R)-2-benzyloxy-propionic acid ethyl ester, a key intermediate in the synthesis of levofloxacin, a potent antibiotic; and (ii) directed evolution targeted for increased thermostability of BcEST produced two improved variants, but in either case the 3–5°C increase in the apparent melting temperature (T_m) of the mutants over the native BcEST that has a T_m of 50°C was outperformed by TtEST, a naturally occurring homologue with a T_m of 65°C. Protein modelling of BcEST mapped the S148C and K272R mutations at protein surface and the I88T and Q110L mutations at more buried locations. This work expands the repertoire of characterized members of the α/β-fold hydrolase superfamily. Further, it shows that genome mining is an economical option for new biocatalyst discovery and we provide a rare example of a naturally occurring thermostable biocatalyst that outperforms experimentally evolved homologues that carry out the same hydrolysis.     

Transportable and Non-transportable Inhibitors of L-glutamate Uptake Produce Astrocytic Stellation and Increase EAAT2 Cell Surface Expression

Astrocytic excitatory amino acid transporters (EAATs) regulate excitatory transmission and limit excitotoxicity. Evidence for a functional interface between EAATs and glial fibrillary acidic protein (GFAP) relevant to astrocytic morphology led to investigations of actions of transportable (d-Aspartate (d-Asp) and (2S,3S,4R)-2-(carboxycyclopropyl)glycine (l-CCG-III)) and non-transportable (dl-threo-β-benzyloxyaspartate (dl-TBOA)) inhibitors of Glu uptake in murine astrocytes. d-Asp (1 mM), l-CCG-III (0.5 mM) and dl-TBOA (0.5 mM) produced time-dependent (24–72 h) reductions in ³[H]d-Asp uptake (approximately 30–70%) with little or no gliotoxicity. All drugs induced a profound change in phenotype from cobblestone to stellate morphology and image analysis revealed increases in the intensity of GFAP immunolabelling for l-CCG-III and dl-TBOA. Cytochemistry indicated localized changes in F-actin distribution. Cell surface expression of EAAT2, but not EAAT1, was elevated at 72 h. Blockade of Glu uptake by both types of EAAT inhibitor exerts longer-term effects on astrocytic morphology and a compensatory homeostatic rise in EAAT2 abundance.     

Epigenetic-genetic chromosome dosage approach for fetal trisomy 21 detection using an autosomal genetic reference marker

Background

The putative promoter of the holocarboxylase synthetase (HLCS) gene on chromosome 21 is hypermethylated in placental tissues and could be detected as a fetal-specific DNA marker in maternal plasma. Detection of fetal trisomy 21 (T21) has been demonstrated by an epigenetic-genetic chromosome dosage approach where the amount of hypermethylated HLCS in maternal plasma is normalized using a fetal genetic marker on the Y chromosome as a chromosome dosage reference marker. We explore if this method can be applied on both male and female fetuses with the use of a paternally-inherited fetal single nucleotide polymorphism (SNP) allele on a reference chromosome for chromosome dosage normalization.

Methodology

We quantified hypermethylated HLCS molecules using methylation-sensitive restriction endonuclease digestion followed by real-time or digital PCR analyses. For chromosome dosage analysis, we compared the amount of digestion-resistant HLCS to that of a SNP allele (rs6636, a C/G SNP) that the fetus has inherited from the father but absent in the pregnant mother.

Principal Findings

Using a fetal-specific SNP allele on a reference chromosome, we analyzed 20 euploid and nine T21 placental tissue samples. All samples with the fetal-specific C allele were correctly classified. One sample from each of the euploid and T21 groups were misclassified when the fetal-specific G allele was used as the reference marker. We then analyzed 33 euploid and 14 T21 maternal plasma samples. All but one sample from each of the euploid and T21 groups were correctly classified using the fetal-specific C allele, while correct classification was achieved for all samples using the fetal-specific G allele as the reference marker.

Conclusions

As a proof-of-concept study, we have demonstrated that the epigenetic-genetic chromosome dosage approach can be applied to the prenatal diagnosis of trisomy 21 for both male and female fetuses.     

Structural characterization of the 60-kDa bermuda grass pollen isoallergens, a covalent flavoprotein

Our studies suggest a tripartite structure for the 60-kDa allergen of Bermuda grass pollen (BG60) including a short N-terminal segment, a FAD-binding domain, and a C-terminal domain. The lower molecular weight isoallergens lack the N-terminal segment. The higher protease susceptibility and the lower melting temperature of approximately 20 degrees C of the lower molecular weight isoforms suggest that the N-terminal segment is essential for a compact structure. Database screening reveals that the protease-digested peptide sequences (approximately 180 residues in total) share 40% identity with the plant berberine bridge enzymes. In particular, a 24-residue peptide sequence displays high similarity to a conserved FAD-binding motif. The spectroscopic and SDS-PAGE analyses suggest that the cofactor FAD is covalently linked to the central domain. Therefore, we conclude that BG60 is identified as the first flavinylated allergen.