JLigand: a graphical tool for the CCP4 template-restraint library

The CCP4 template-restraint library defines restraints for biopolymers, their modifications and ligands that are used in macromolecular structure refinement. JLigand is a graphical editor for generating descriptions of new ligands and covalent linkages

The Structure of Ca2+ Sensor Case16 Reveals the Mechanism of Reaction to Low Ca2+ Concentrations

Here we report the first crystal structure of a high-contrast genetically encoded circularly permuted green fluorescent protein (cpGFP)-based Ca2+ sensor, Case16, in the presence of a low Ca2+ concentration. The structure reveals the positioning of the chromophore within Case16 at the first stage of the Ca2+-dependent response when only two out of four Ca2+-binding pockets of calmodulin (CaM) are occupied with Ca2+ ions. In such a “half Ca2+-bound state”, Case16 is characterized by an incomplete interaction between its CaM-/M13-domains. We also report the crystal structure of the related Ca2+ sensor Case12 at saturating Ca2+ concentration. Based on this structure, we postulate that cpGFP-based Ca2+ sensors can form non-functional homodimers where the CaM-domain of one sensor molecule binds symmetrically to the M13-peptide of the partner sensor molecule. Case12 and Case16 behavior upon addition of high concentrations of free CaM or M13-peptide reveals that the latter effectively blocks the fluorescent response of the sensor. We speculate that the demonstrated intermolecular interaction with endogenous substrates and homodimerization can impede proper functioning of this type of Ca2+ sensors in living cells

Suffix-specific RNAi Leads to Silencing of F Element in Drosophila melanogaster

Separate conserved copies of suffix, a short interspersed Drosophila retroelement (SINE), and also divergent copies in the 3′ untranslated regions of the three genes, have already been described. Suffix has also been identified on the 3′ end of the Drosophila non-LTR F element, where it forms the last conserved domain of the reverse transcriptase (RT). In our current study, we show that the separate copies of suffix are far more actively transcribed than their counterparts on the F element. Transcripts from both strands of suffix are present in RNA preparations during all stages of Drosophila development, providing the potential for the formation of double-stranded RNA and the initiation of RNA interference (RNAi). Using in situ RNA hybridization analysis, we have detected the expression of both sense and antisense suffix transcripts in germinal cells. These sense and antisense transcripts are colocalized in the primary spermatocytes and in the cytoplasm of the nurse cells, suggesting that they form double-stranded RNA. We performed further analyses of suffix-specific small RNAs using northern blotting and SI nuclease protection assays. Among the total RNA preparations isolated from embryos, larvae, pupae and flies, suffix-specific small interfering RNAs (siRNAs) were detected only in pupae. In wild type ovaries, both the siRNAs and longer suffix-specific Piwi-interacting RNAs (piRNAs) were observed, whereas in ovaries of the Dicer-2 mutant, only piRNAs were detected. We further found by 3′ RACE that in pupae and ovaries, F element transcripts lacking the suffix sequence are also present. Our data provide direct evidence that suffix-specific RNAi leads to the silencing of the relative LINE (long interspersed element), F element, and suggests that SINE-specific RNA interference could potentially downregulate a set of genes possessing SINE stretches in their 5′ or 3′ non-coding regions. These data also suggest that double stranded RNAs possessing suffix are processed by both RNAi and an additional silencing mechanism

Both piRNA and siRNA Pathways Are Silencing Transcripts of the Suffix Element in the Drosophila melanogaster Germline and Somatic Cells

In the Drosophila melanogaster germline, the piRNA pathway silences retrotransposons as well as other transcribed repetitive elements. Suffix is an unusual short retroelement that was identified both as an actively transcribed repetitive element and also as an element at the 3′ ends of the Drosophila non-LTR F element. The copies of suffix that are F element-independent are far more actively transcribed than their counterparts on the F element. We studied the patterns of small RNAs targeting both strands of suffix in Drosophila ovaries using an RNase protection assay and the analysis of the corresponding RNA sequences from the libraries of total small RNAs. Our results indicate that suffix sense and antisense transcripts are targeted mainly by 23–29 nucleotides in length piRNAs and also by 21 nucleotides in length siRNAs. Suffix sense transcripts actively form longer RNA species, corresponding either to partial digestion products of the RNAi and Piwi pathways or to another RNA silencing mechanism. Both sense and antisense suffix transcripts accumulated in the ovaries of homozygous spn-E, piwi and aub mutants. These results provide evidence that suffix sense and antisense transcripts in the germ line and soma are targeted by both RNAi and Piwi pathways and that a Dicer-independent pathway of biogenesis of siRNAs could exist in Drosophila cells

Analysis of N- and O-Glycosylation of Lysosomal Glycoproteins

The vast majority of lysosomal proteins are heavily glycosylated. The present protocol describes the method of analyzing N- and O-linked glycans in lysosomal proteins of interest. The method is based on using deglycosylating enzymes, endoglycosidases, and exoglycosidases. Endoglycosidases catalyze the cleavage of an internal bond in an oligosaccharide, while exoglycosidases remove terminal carbohydrates from glycans. Different types of carbohydrate residues or chains can be removed by specific glycosidases. Removing oligosaccharides with glycosidases increases the electrophoretic mobility of a protein. This increase in mobility depends on the size and number of removed carbohydrate chains. Therefore, the treatment of lysosomal proteins with specific glycosidases followed by a western blot analysis of a protein of interest provides a way to determine which types of glycans are present in the protein by comparing the gel mobility before and after treatment

Role of N-glycosylation in trafficking of apical membrane proteins in epithelia

Polarized distribution of plasma membrane transporters and receptors in epithelia is essential for vectorial functions of epithelia. This polarity is maintained by sorting of membrane proteins into apical or basolateral transport containers in the trans-Golgi network and/or endosomes followed by their delivery to the appropriate plasma membrane domains. Sorting depends on the recognition of sorting signals in proteins by specific sorting machinery. In the present review, we summarize experimental evidence for and against the hypothesis that N-glycans attached to the membrane proteins can act as apical sorting signals. Furthermore, we discuss the roles of N-glycans in the apical sorting event per se and their contribution to folding and quality control of glycoproteins in the endoplasmic reticulum or retention of glycoproteins in the plasma membrane. Finally, we review existing hypotheses on the mechanism of apical sorting and discuss the potential roles of the lectins, VIP36 and galectin-3, as putative apical sorting receptors

P-type ATPases in Health and Disease

P-type ATPases are a large group of evolutionary related ion and lipid pumps that have in common that they catalyze a transient phosphorylated intermediate at a key conserved aspartate residue within the pump in order to function. While all the P-type ATPases perform active transport across cellular membranes, they have different transport specificities and serve diverse physiological functions. The ion pumps of the P-type ATPase family create electrochemical gradients that are essential for transepithelial transport, nutrient uptake and membrane potential. They mediate cellular signaling and provide the ligands for metalloenzymes. Phospholipid flippases, also members of the P-type ATPase superfamily, regulate the asymmetric lipid distribution across the lipid bilayer and are critical for the biogenesis of cell membranes. Since all of these ATPases serve fundamental cellular functions, malfunctioning is associated with various pathophysiological processes and dysfunctions of P-type ATPases are known to contribute to cardiovascular, neurological, renal and metabolic diseases. However, with the ever growing knowledge about the diseases associated with the malfunction of P-type ATPases, they are also promising targets for future drug development. In eukaryotes the most prominent examples of P-type ATPases are the Na+,K+-ATPase (sodium pump), the H+-ATPase (proton pump), the H+,K+-ATPase (proton-potassium pump) and the Ca2+-ATPases (calcium pumps). Mutations in the alpha2 and alpha3 subunit of Na,K-ATPase have been associated with neurological diseases, including rapid-onset dystonia-parkinsonism, familial hemiplegic migraine and alternating hemiplegia of childhood. Dysregulation and loss of expression of Na,K-ATPase and plasma membrane Ca-ATPases may be involved in cancer progression. Malfunctioning of the Ca-ATPases is also thought to contribute to hypertension and neurodegenerative diseases and mutations can cause cardiac dysfunction, deafness, hypertension and cerebellar ataxia. Mutations in the SERCA calcium pumps can cause heart failure, Brody myopathy and Darier disease and mutations in the Cu-ATPase genes cause Menkes and Wilson disease. Deficiencies in phospholipid flippases have been linked to progressive familial intrahepatic cholestasis, obesity, diabetes, hearing loss and neurological diseases

Electrogenic Partial Reactions of the Gastric H,K-ATPase

The fluorescent styryl dye RH421 was used to identify and investigate electrogenic reaction steps of the H,K-ATPase pump cycle. Equilibrium titration experiments were performed with membrane vesicles isolated from hog gastric mucosa, and cytoplasmic and luminal binding of K+ and H+ ions was studied. It was found that the binding and release steps of both ion species in both principal conformations of the ion pump, E1 and P-E2, are electrogenic, whereas the conformation transitions do not contribute significantly to a charge movement within the membrane dielectric. This behavior is in agreement with the transport mechanism found for the Na,K-ATPase and the sarcoplasmic reticulum Ca-ATPase. The data were analyzed on the basis of the Post-Albers reaction cycle. The equilibrium dissociation constants for K1 binding on the cytoplasmic side were 11 and 16 mM. The respective equilibrium dissociation constants on the luminal side were obtained via K+ concentration dependence of the enzyme activity and determined to be 0.11 mM for both luminal binding sites