Dissecting muscle and neuronal disorders in a Drosophila model of muscular dystrophy

Perturbation in the Dystroglycan (Dg)-Dystrophin (Dys) complex results in muscular dystrophies and brain abnormalities in human. Here we report that Drosophila is an excellent genetically tractable model to study muscular dystrophies and neuronal abnormalities caused by defects in this complex. Using a fluorescence polarization assay, we show a high conservation in Dg-Dys interaction between human and Drosophila. Genetic and RNAi-induced perturbations of Dg and Dys in Drosophila cause cell polarity and muscular dystrophy phenotypes: decreased mobility, age-dependent muscle degeneration and defective photoreceptor path-finding. Dg and Dys are required in targeting glial cells and neurons for correct neuronal migration. Importantly, we now report that Dg interacts with insulin receptor and Nck/Dock SH2/SH3-adaptor molecule in photoreceptor path-finding. This is the first demonstration of a genetic interaction between Dg and InR.     

G-protein-coupled receptor Gpr1 and G-protein Gpa2 of cAMP-dependent signaling pathway are involved in glucose-induced pexophagy in the yeast Saccharomyces cerevisiae

In yeast cell, glucose induces various changes of cellular metabolism on genetic and metabolic levels. One of such changes is autophagic degradation of dispensable peroxisomes (pexophagy) which occurs in vacuoles. We have found that in Saccharomyces cerevisiae, defect of G-protein-coupled receptor Gpr1 and G-protein Gpa2, both the components of cAMP-signaling pathway, strongly suppressed glucose-induced degradation of matrix peroxisomal protein thiolase. We conclude that proteins Gpr1 and Gpa2 are involved in glucose sensing and signal transduction during pexophagy process in yeast.     

Glucans from fruit bodies of cultivated mushrooms Pleurotus ostreatus and Pleurotus eryngii: Structure and potential prebiotic activity

Cultivated oyster mushrooms (genus Pleurotus) are interesting as a source of biologically active glucans. Partially, β-glucan from Pleurotus sp. (pleuran) has been used as food supplements due to its immunosuppressive activity. Like other dietary fibre components, oyster mushroom polysaccharides can stimulate the growth of colon microorganisms (probiotics), i.e. act as prebiotics. Specific glucans were isolated from stems of Pleurotus ostreatus and Pleurotus eryngii by subsequent boiling water and alkali extraction. Obtained water soluble (L1), alkali soluble (L2) and insoluble (S) fractions were characterised by various analytical methods. Spectroscopic analysis detected glucans in all the fractions: branched 1,3-1,6-β-d-glucan predominated in L1 and S, while linear 1,3-α-d-glucan in L2. Fractions L1 also contained marked amount of proteins partially in complex with glucans; protein content in L2 was insignificant. Effective deproteinisation of L1 and separation of α- and β-glucans in L2 was achieved by the treatment with phenolic reagent. Small amount of chitin was found in S as a component of cell wall chitin–glucan complex. Potential prebiotic activity of extracts L1 and L2 was testing using nine probiotic strains of Lactobacillus, Bifidobacterium and Enterococcus. These probiotics showed different growth characteristics dependently on used extract and strain specificity due to the presence of structurally diverse compounds. The extracts L1 and L2 can be applied to synbiotic construction only for carefully selected probiotic strains. This exploitation of fruit body extracts extends the use of mushrooms P. ostreatus and P. eryngii for human health.     

The ABC transporter ABC40 encodes a phenylacetic acid export system in Penicillium chrysogenum

The filamentous fungus Penicillium chrysogenum is used for the industrial production of β-lactam antibiotics. The pathway for β-lactam biosynthesis has been resolved and involves the enzyme phenylacetic acid CoA ligase that is responsible for the CoA activation of the side chain precursor phenylacetic acid (PAA) that is used for the biosynthesis of penicillin G. To identify ABC transporters related to β-lactam biosynthesis, we analyzed the expression of all 48 ABC transporters present in the genome of P. chryso-genum when grown in the presence and absence of PAA. ABC40 is significantly upregulated when cells are grown or exposed to high levels of PAA. Although deletion of this transporter did not affect β-lactam biosynthesis, it resulted in a significant increase in sensitivity to PAA and other weak acids. It is concluded that ABC40 is involved in weak acid detoxification in P. chrysogenum including resistance to phenylacetic acid.     

The pathway and spatial scale for MscS inactivation

The mechanosensitive channel of small conductance (MscS) is a bacterial tension-driven osmolyte release valve with homologues in many walled eukaryotic organisms. When stimulated by steps of tension in excised patches, Escherichia coli MscS exhibits transient opening followed by reversible adaptation and then complete inactivation. Here, we study properties of the inactivation transition, which renders MscS nonconductive and tension insensitive. Using special pressure protocols we demonstrate that adaptation and inactivation are sequential processes with opposite tension dependencies. In contrast to many eukaryotic channels, which inactivate from the open state, MscS inactivates primarily from the closed state because full openings by preconditioning pulses do not influence the degree of inactivation, and saturating tensions keeping channels open prevent inactivation. The easily opened A98S mutant lacks inactivation completely, whereas the L111S mutant with a right-shifted activation curve inactivates silently before reaching the threshold for opening. This suggests that opening and inactivation are two independent tension-driven pathways, both starting from the closed state. Analysis of tension dependencies for inactivation and recovery rates estimated the in-plane expansion (ΔA) associated with inactivation as 8.5 nm(2), which is about half of the area change for opening. Given that the interhelical contact between the outer TM1-TM2 pairs and the core TM3s is the force-transmitting path from the periphery to the gate, the determined ΔA now can be used as a constraining parameter for the models of the inactivated state in which the lipid-facing TM1-TM2 pairs are displaced and uncoupled from the gate.     

Modulation of the Purine Pathway for Riboflavin Production in Flavinogenic Recombinant Strain of the Yeast Candida famata

Riboflavin (vitamin B₂) is an indispensable nutrient for humans and animals, since it is the precursor of the essential coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), involved in variety of metabolic reactions. Riboflavin is produced on commercial scale and is used for feed and food fortification purposes, and in medicine. Until recently, the mutant strains of the flavinogenic yeast Candida famata were used in industry for riboflavin production. Guanosine triphosphate is the immediate precursor of riboflavin synthesis. Therefore, the activation of metabolic flux toward purine nucleotide biosynthesis is a promising approach to improve riboflavin production. The phosphoribosyl pyrophosphate synthetase and phosphoribosyl pyrophosphate amidotransferase are the rate limiting enzymes in purine biosynthesis. Corresponding genes PRS3 and ADE4 from yeast Debaryomyces hansenii are modified to avoid feedback inhibition and cooverexpressed on the background of a previously constructed riboflavin overproducing strain of C. famata. Constructed strain accumulates twofold more riboflavin when compared to the parental strain.     

A Naturally Occurring Variant in Human TLR9, P99L, Is Associated with Loss of CpG Oligonucleotide Responsiveness

The innate immune system employs Toll-like receptors (TLRs) for the detection of invading microorganisms based on distinct molecular patterns. For example, TLR9 is activated by microbial DNA and also by short therapeutic CpG-containing oligonucleotides (CpG-ODN). TLR9 activation leads to the production of interferons and the priming of humoral adaptive immune responses. Unfortunately, the principles of ligand recognition by TLR9 are poorly understood, and genetic variants of TLR9, which may affect its function, have not been characterized systematically on the molecular level. We therefore sought to functionally characterize reported single nucleotide polymorphisms of TLR9 in the HEK293 model system. We discovered that two variants, P99L and M400I, are associated with altered receptor function regarding NF-κB activation and cytokine induction. Our investigations show that for the most functionally impaired variant, P99L, the ability to respond to physiological and therapeutic TLR9 ligands is severely compromised. However, CpG-ODN binding is normal. CpG-ODN recognition by TLR9 thus appears to involve two separate events, CpG-ODN binding and sensing. Our studies highlight Pro-99 as a residue important for the latter process. In genotyping studies, we confirmed that both M400I (rs41308230) and P99L (rs5743844) are relatively rare variants of TLR9. Our data add rs41308230 and rs5743844 to the list of functionally important TLR variants and warrant further research into their relevance for infectious disease susceptibility or responsiveness to CpG-ODN-based therapies.     

Protein structure prediction assisted with sparse NMR data in CASP13

CASP13 has investigated the impact of sparse NMR data on the accuracy of protein structure prediction. NOESY and ¹⁵N-¹H residual dipolar coupling data, typical of that obtained for ¹⁵N,¹³C-enriched, perdeuterated proteins up to about 40 kDa, were simulated for 11 CASP13 targets ranging in size from 80 to 326 residues. For several targets, two prediction groups generated models that are more accurate than those produced using baseline methods. Real NMR data collected for a de novo designed protein were also provided to predictors, including one data set in which only backbone resonance assignments were available. Some NMR-assisted prediction groups also did very well with these data. CASP13 also assessed whether incorporation of sparse NMR data improves the accuracy of protein structure prediction relative to nonassisted regular methods. In most cases, incorporation of sparse, noisy NMR data results in models with higher accuracy. The best NMR-assisted models were also compared with the best regular predictions of any CASP13 group for the same target. For six of 13 targets, the most accurate model provided by any NMR-assisted prediction group was more accurate than the most accurate model provided by any regular prediction group; however, for the remaining seven targets, one or more regular prediction method provided a more accurate model than even the best NMR-assisted model. These results suggest a novel approach for protein structure determination, in which advanced prediction methods are first used to generate structural models, and sparse NMR data is then used to validate and/or refine these models.     

Enhanced CPT sensitivity of yeast cells and selective relaxation of Ga14 motif-containing DNA by novel Gal4-topoisomerase I fusion proteins

Human topoisomerase I-B (Top1) efficiently relaxes DNA supercoils during basic cellular processes, and can be transformed into a DNA-damaging agent by antitumour drugs, enzyme mutations and DNA lesions. Here, we describe Gal4-Top1 chimeric proteins (GalTop) with an N-terminal truncation of Top1, and mutations of the Gal4 Zn-cluster and/or Top1 domains that impair their respective DNA-binding activities. Expression levels of chimeras were similar in yeast cells, however, GalTop conferred an increased CPT sensitivity to RAD52- yeast cells as compared to a GalTop with mutations of the Gal4 domain, showing that a functional Gal4 domain can alter in vivo functions of Top1. In vitro enzyme activity was tested with a DNA relaxation assay using negatively supercoiled plasmids with 0 to 5 Gal4 consensus motifs. Only GalTop with a functional Gal4 domain could direct DNA relaxation activity of Top1 specifically to DNA molecules containing Gal4 motifs. By using a substrate competition assay, we could demonstrate that the Gal4-anchored Top1 remains functional and efficiently relax DNA substrates in cis. The enhanced CPT sensitivity of GalTop in yeast cells may then be due to alterations of the chromatin-binding activity of Top1. The GalTop chimeras may indeed mimic a normal mechanism by which Top1 is recruited to chromatin sites in living cells. Such hybrid Top1s may be helpful in further dissecting enzyme functions, and constitute a prototype of a site-specific DNA cutter endowed with high cell lethality.