Nucleotide binding domains of human CFTR: a structural classification of critical residues and disease-causing mutations.

Defective function of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) causes CF, the most frequent lethal inherited disease among the Caucasian population. The structure of this chloride ion channel includes two nucleotide-binding domains (NBDs), whose ATPase activity controls channel gating. Recently, the experimental structures of mouse and human CFTR NBD1 and our model of the human CFTR NBD1/NBD2 heterodimer have provided new insights into specific structural features of the CFTR NBD dimer. In the present work, we provide a structural classification of CF-causing mutations which may complement the existing functional classification. Our analysis also identified amino acid residues which may play a critical role in interdomain interaction and are located at the NBD1-NBD2 interface or on the surface of the dimer. In particular, a cluster of aromatic amino acids, which includes F508 and straddles the two NBDs, might be directly involved in the interaction of the NBD1/NBD2 heterodimer with the channel-forming membrane-spanning domains.Received 24 May 2005; received after revision 13 June 2005; accepted 18 June 2005     

Flavodoxins: sequence, folding, binding, function and beyond

Flavodoxins are electron-transfer proteins involved in a variety of photosynthetic and non-photosynthetic reactions in bacteria, whereas, in eukaryotes, a descendant of the flavodoxin gene helps build multidomain proteins. The redox activity of flavodoxin derives from its bound flavin mononucleotide cofactor (FMN), whose intrinsic properties are profoundly modified by the host apoprotein. This review covers the very exciting last decade of flavodoxin research, in which the folding pathway, the structure and stability of the apoprotein, the mechanism of FMN recognition, the interactions that stabilize the functional complex and tailor the redox potentials, and many details of the binding and electron transfer to partner proteins have been revealed. The next decade should witness an even deeper understanding of the flavodoxin molecule and a greater comprehension of its many physiological roles. The fact that flavodoxin is essential for the survival of some human pathogens could make it a drug target on its own. Received 26 October 2005; received after revision 20 November 2005; accepted 14 December 2005     

Divergence towards a dead end? Cleavage of the divergent domains of ribosomal RNA in apoptosis

In several cases of apoptotic death the large ribosomal subunit 28S rRNA is specifically cleaved. The cleavages appear at specific sites within those domains of the rRNA molecule that have shown exceptional high divergence in evolution (D domains). The cleavages accompany rather than precede apoptosis, and there is a positive, but not complete, correlation between rRNA cleavage and internucleosomal DNa fragmentation. Most cell types studied so far show two alternative cleavage pathways that are mutually exclusive. Cleavage can either start in the D8 domain with secondary cuts within a subdomain of D2 (D2c), or in the D2 domain with subsequent excision of the D2c subdomain. The latter pathway is of particular interest since D2 (unlike D8) is normally inaccessible for RNase attack. That apoptosis specifically affects the ribosomal divergent domains suggests that these domains, which make up roughly 25% of total cellular RNA, might have evolved to serve functions related to apoptosis. Future studies will be directed to test the hypothesis that rRNA fragmentation may be part of an apoptotic program directed against the elimination of illegitimate (viral?) polynucleotides.     

How sphingolipids bind and shape proteins: molecular basis of lipid-protein interactions in lipid shells,rafts and related biomembrane domains

Understanding the molecular mechanisms controlling the association of proteins with lipid rafts is a central issue in cell biology and medicine. A structurally conserved motif (the 'sphingolipid binding domain') has been characterized in unrelated cellular and microbial proteins targeted to lipid rafts. I propose that the structuration of a sphingolipid shell around the sphingolipid binding domain not only extracts the protein from the liquid-disordered phase of the plasma membrane, and ensures its delivery to lipid rafts, but also influences its conformation. The chaperone activity of sphingolipids in shells and rafts may play an important role in infectious and conformational diseases(human immunodeficiency virus-1, prions, Alzheimer).     

Nucleotide-binding domains of human cystic fibrosis transmembrane conductance regulator: detailed sequence analysis and three-dimensional modeling of the heterodimer

The cystic fibrosis transmembrane conductance regulator (CFTR) protein is encoded by the gene that is defective in cystic fibrosis, the most common lethal inherited disease among the Caucasian population. CFTR belongs to the ABC transporter superfamily, whose members form macromolecular architectures composed of two membrane-spanning domains and two nucleotide-binding domains (NBDs). The experimental structures of NBDs from several ABC transporters have recently been solved, opening new avenues for understanding the structure/function relationships and the consequences of some disease-causing mutations of CFTR. Based on a detailed sequence/structure analysis, we propose here a three-dimensional model of the human CFTR NBD heterodimer. This model, which is in agreement with recent experimental data, highlights the specific features of the CFTR asymmetric active sites located at the interface between the two NBDs. Moreover, additional CFTR-specific features can be identified at the subunit interface, which may play critical roles in active site interdependence and are uncommon in other NBD dimers.Received 16 October 2003; received after revision 16 November 2003; accepted 21 November 2003     

Stem cells and their niche: a matter of fate

Embryonic stem cells provide an in vitro model for developmental biologists to study cell fate decisions during ontogenesis, while somatic stem cells allow physiologists to understand tissue homeostasis in the adult. The behavior of stem cells is dependent on an intimate relationship with a supportive niche. This brief review highlights some of the most important recent trends in stem cell biology, focusing in particular on the supportive microenvironments for both embryonic and adult stem cells. Known intrinsic and extrinsic molecular players from the best-characterized stem cell types are summarized, illuminating a number of shared environmental cues among tissues originating from all three embryonic germ layers. Received 6 October 2005; received after revision 27 December 2005; accepted 17 January 2006     

Phagocytosis of apoptotic cells: a matter of balance

Efficient clearance of apoptotic cells is required to control homeostasis in normal and pathological circumstances, and inappropriate clearance of cell corpses may lead to autoimmune diseases and inflammation. The multiplicity of phagocytotic mechanisms points to the relevance of removing apoptotic cells. A variety of surface molecules present in either the apoptotic bodies or phagocytes help in attachment and initiation of engulfment. Nonetheless, uncontrolled phagocytosis of apoptotic cells and other particles may lead to tissue injury; therefore, negative signals are important in balancing phagocytotic activity. This review aims at a systematic examination of positive and negative signals that modulate the uptake of apoptotic bodies and the signaling mechanisms involved in the clearance of apoptotic cells.Received 13 November 2004; received after revision 5 March 2005; accepted 8 March 2005     

Porcine factor V: cDNA cloning, gene mapping, three-dimensional protein modeling of membrane binding sites and comparative anatomy of domains

Factor V is a plasma protein essential for blood coagulation. This protein is involved in activated protein C resistance, the most common inherited thrombotic disorder known. We utilized the polymerase chain reaction to clone the porcine factor V gene by generating overlapping clones amplified with primers chosen by comparison with known nucleotide sequences. The porcine factor V cDNA contig encodes a predicted 2258-amino acid protein, making it the largest in comparison to the bovine, human, and murine proteins. Porcine factor V has the highest level of homology with bovine factor V, but also has high levels of conservation of important residues with all the species. Radiation hybrid mapping assigned the porcine factor V gene to chromosome 4. Three-dimensional models of factor V were generated and used to analyze membrane-binding sites in terms of conserved, and therefore likely important residues. Received 3 October 2000; revised 23 November 2000; accepted 6 December 2000     

Automatic recognition of squirrel monkey vocalisations by means of a filterbank

Summary Up to now the problem of classifying squirrel monkey vocalizations has not been solved satisfactorily. The problem is now approached by a method, which consists of 2 phases. At first the monkey vocalizations are compared with the aid of physical criteria and classified by types. According to the classification of types, the membership of the individual vocalizations is tested.     

Transfer of the immunization to a bacterial antigen by RNA

Riassunto L'iniezione ad animali normali di ARN estratto dalla milza di animali immunizzati con antigene H diS. typhi, provoca la comparsa, nel siero degli animali riceventi, di anticorpi diretti contro tale antigene.     

Binding site of activators of the cystic fibrosis transmembrane conductance regulator in the nucleotide binding domains

The use of substances that could activate the defective chloride channels of the mutant cystic fibrosis transmembrane conductance regulator (CFTR) has been suggested as possible therapy for cystic fibrosis. Using epithelia formed by cells stably transfected with wildtype or mutant (G551D, G1349D) CFTR, we estimated the apparent dissociation constant, K_D, of a series of CFTR activators by measuring the increase in the apical membrane current. Modification of apparent K_D of CFTR activators by mutations of the nucleotide-binding domains (NBDs) suggests that the binding site might be in these regions. The human NBD structure was predicted by homology with murine NBD1. An NBD1-NBD2 complex was constructed by overlying monomers to a bacterial ABC transporter NBD dimer in the head-to- tail conformation. Binding sites for CFTR activators were predicted by molecular docking. Comparison of theoretical binding free energy estimated in the model to free energy estimated from the apparent dissociation constants, K_D, resulted in a remarkably good correlation coefficient for one of the putative binding sites, located in the interface between NBD1 and NBD2.Received 21 September 2004; received after revision 6 December 2004; accepted 10 December 2004     

Nucleotide sequence of the gene for the mitochondrial 15S ribosomal RNA of yeast

We have determined the nucleotide sequence of a DNA segment carrying the entire 15S ribosomal RNA gene of yeast mitochondrial genome. Many stretches of sequence are present which are homologous to the E. coli 16S ribosomal RNA gene. The gene sequence can be folded into a secondary structure according to the [1] model on bacterial ribosomal RNAs. The structure reveals a striking similarity between the two RNAs despite the large difference in their base compositions. In the middle of the gene, we found a guanine-cytosine rich sequence that is also present in several other regions of the mitochondrial genome.     

Hairpin RNA: a secondary structure of primary importance

An RNA hairpin is an essential secondary structure of RNA. It can guide RNA folding, determine interactions in a ribozyme, protect messenger RNA (mRNA) from degradation, serve as a recognition motif for RNA binding proteins or act as a substrate for enzymatic reactions. In this review, we have focused on cis-acting RNA hairpins in metazoa, which regulate histone gene expression, mRNA localization and translation. We also review evolution, mechanism of action and experimental use of trans-acting microRNAs, which are coded by short RNA hairpins. Finally, we discuss the existence and effects of long RNA hairpin in animals. We show that several proteins previously recognized to play a role in a specific RNA stem-loop function in cis were also linked to RNA silencing pathways where a different type of hairpin acts in trans. Such overlaps indicate that the relationship between certain mechanisms that recognize different types of RNA hairpins is closer than previously thought. Received 21 November 2005; received after revision 3 January 2006; accepted 11 January 2006     

Automatic recognition of squirrel monkey vocalisations by means of a filterbank.

Up to now the problem of classifying squirrel monkey vocalizations has not been solved satisfactorily. The problems is now approached by a method, which consists of 2 phases. At first the monkey vocalizations are compared with the aid of physical criteria and classified by types. According to the classification of types, the membership of the individual vocalizations is tested.