Nora B. Calcaterra

A productive NADP+ binding mode of ferredoxin-NADP+ reductase revealed by protein engineering and crystallographic studies.

The flavoenzyme ferredoxin–NADP+ reductase (FNR) catalyzes the production of NADPH during photosynthesis. Whereas the structures of FNRs from spinach leaf and a cyanobacterium as well as many of their homologs have been solved, none of these studies has yielded a productive geometry of the flavin–nicotinamide interaction. Here, we show that this failure occurs because nicotinamide binding to wild type FNR involves the energetically unfavorable displacement of the C-terminal Tyr side chain. We used mutants of this residue (Tyr 308) of pea FNR to obtain the structures of productive NADP+ and NADPH complexes. These structures reveal a unique NADP+ binding mode in which the nicotinamide ring is not parallel to the flavin isoalloxazine ring, but lies against it at an angle of ~30°, with the C4 atom 3 Å from the flavin N5 atom.

Permanent Genetic Resources added to Molecular Ecology Resources Database 1 December 2009-31 January 2010

This article documents the addition of 220 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Allanblackia floribunda, Amblyraja radiata, Bactrocera cucurbitae, Brachycaudus helichrysi, Calopogonium mucunoides, Dissodactylus primitivus, Elodea canadensis, Ephydatia fluviatilis, Galapaganus howdenae howdenae, Hoplostethus atlanticus, Ischnura elegans, Larimichthys polyactis, Opheodrys vernalis, Pelteobagrus fulvidraco, Phragmidium violaceum, Pistacia vera, and Thunnus thynnus. These loci were cross‐tested on the following species: Allanblackia gabonensis, Allanblackia stanerana, Neoceratitis cyanescens, Dacus ciliatus, Dacus demmerezi, Bactrocera zonata, Ceratitis capitata, Ceratitis rosa, Ceratits catoirii, Dacus punctatifrons, Ephydatia mülleri, Spongilla lacustris, Geodia cydonium, Axinella sp., Ischnura graellsii, Ischnura ramburii, Ischnura pumilio, Pistacia integerrima and Pistacia terebinthus.

Cellular nucleic-acid-binding protein, a transcriptional enhancer of c-Myc, promotes the formation of parallel G-quadruplexes

G-rich sequences that contain stretches of tandem guanines can form four-stranded, intramolecular stable DNA structures called G-quadruplexes (termed G4s). Regulation of the equilibrium between single-stranded and G4 DNA in promoter regions is essential for control of gene expression in the cell. G4s are highly stable structures; however, their folding kinetics are slow under physiological conditions. CNBP (cellular nucleic-acid-binding protein) is a nucleic acid chaperone that binds the G4-forming G-rich sequence located within the NHE (nuclease hypersensitivity element) III of the c-Myc proto-oncogene promoter. Several reports have demonstrated that CNBP enhances the transcription of c-Myc in vitro and in vivo; however, none of these reports have assessed the molecular mechanisms responsible for this control. In the present study, by means of Taq polymerase stop assays, electrophoretic mobility-shift assays and CD spectroscopy, we show that CNBP promotes the formation of parallel G4s to the detriment of anti-parallel G4s, and its nucleic acid chaperone activity is required for this effect. These findings are the first to implicate CNBP as a G4-folding modulator and, furthermore, assign CNBP a novel mode-of-action during c-Myc transcriptional regulation.

CNBP: A multifunctional nucleic acid chaperone involved in cell death and proliferation control

Cellular nucleic acid binding protein (CNBP) has been implicated in vertebrate craniofacial development and in myotonic dystrophy type 2 (DM2) and sporadic inclusion body myositis (sIBM) human diseases. In these seemingly unrelated biological processes, CNBP appears to be involved in controlling cell death and proliferation rates. Low levels of CNBP may reduce rate of global protein synthesis, thereby reducing proliferation and increasing apoptosis. Conversely, CNBP might affect transcription of genes required for cell proliferation. Experimental evidences gathered so far make it difficult to ascertain or rule out any of these possibilities. Moreover, both possibilities may not be mutually exclusive. CNBP is a small and strikingly conserved single‐stranded nucleic acid binding protein that is able to bind DNA as well as RNA. CNBP has a broad spectrum of targets, ranging from regulatory sites in gene promoters to translational regulatory elements in mRNA untranslated regions. Biochemical experiments have recently shed light on the possible mechanism of action for CNBP, which may act as a nucleic acid chaperone catalyzing the rearrangement of G‐rich nucleic acid secondary structures likely relevant for transcriptional and/or translational gene regulation. This review focuses on the involvement of CNBP in vertebrate craniofacial development and human DM2 and sIBM diseases, as well as on the biochemical and structural features of CNBP and its cellular and molecular mechanism of action.

Cellular nucleic acid binding protein binds G‐rich single‐stranded nucleic acids and may function as a nucleic acid chaperone

Cellular nucleic acid binding protein (CNBP) is a small single‐stranded nucleic acid binding protein made of seven Zn knuckles and an Arg‐Gly rich box. CNBP is strikingly conserved among vertebrates and was reported to play broad‐spectrum functions in eukaryotic cells biology. Neither its biological function nor its mechanisms of action were elucidated yet. The main goal of this work was to gain further insights into the CNBP biochemical and molecular features. We studied Bufo arenarum CNBP (bCNBP) binding to single‐stranded nucleic acid probes representing the main reported CNBP putative targets. We report that, although bCNBP is able to bind RNA and single‐stranded DNA (ssDNA) probes in vitro, it binds RNA as a preformed dimer whereas both monomer and dimer are able to bind to ssDNA. A systematic analysis of variant probes shows that the preferred bCNBP targets contain unpaired guanosine‐rich stretches. These data expand the knowledge about CNBP binding stoichiometry and begins to dissect the main features of CNBP nucleic acid targets. Besides, we show that bCNBP presents a highly disordered predicted structure and promotes the annealing and melting of nucleic acids in vitro. These features are typical of proteins that function as nucleic acid chaperones. Based on these data, we propose that CNBP may function as a nucleic acid chaperone through binding, remodeling, and stabilizing nucleic acids secondary structures. This novel CNBP biochemical activity broadens the field of study about its biological function and may be the basis to understand the diverse ways in which CNBP controls gene expression. J. Cell. Biochem. 103: 1013–1036, 2008.

Inhibition by suramin of mitochondrial ATP synthesis.

Suramin, a drug intensively used in the chemotherapy of African trypanosomiasis and onchocerciasis, is currently being tested in clinical trials for AIDS treatment. Its effects on mitochondrial energy metabolism in mammals were studied. At low concentrations it inhibited ATP synthesis and ATPase activity in submitochondrial particles, as well as ADP-stimulated oxygen consumption and the uncoupler-stimulated ATPase activity in intact rat liver mitochondria. At higher concentrations it also inhibited uncoupled electron transport in both submitochondrial particles and intact mitochondria. From comparison of the kinetic patterns of those inhibitions, evidence suggesting that the adenine nucleotide translocase may be another target for the action of suramin was obtained. The relevance of these findings to the understanding of the biochemical basis of suramin toxicity is discussed.

CNBP mediates neural crest cell expansion by controlling cell proliferation and cell survival during rostral head development

Striking conservation in various organisms suggests that cellular nucleic acid binding protein (CNBP) plays a fundamental biological role across different species. Recently, it was reported that CNBP is required for forebrain formation during chick and mouse embryogenesis. In this study, we have used the zebrafish model system to expand and contextualize the basic understanding of the molecular mechanisms of CNBP activity during vertebrate head development. We show that zebrafish cnbp is expressed in the anterior CNS in a similar fashion as has been observed in early chick and mouse embryos. Using antisense morpholino oligonucleotide knockdown assays, we show that CNBP depletion causes forebrain truncation while trunk development appears normal. A substantial reduction in cell proliferation and an increase in cell death were observed in the anterior regions of cnbp morphant embryos, mainly within the cnbp expression territory. In situ hybridization assays show that CNBP depletion does not affect CNS patterning while it does cause depletion of neural crest derivatives. Our data suggest an essential role for CNBP in mediating neural crest expansion by controlling proliferation and cell survival rather than via a cell fate switch during rostral head development. This possible role of CNBP may not only explain the craniofacial anomalies observed in zebrafish but also those reported for mice and chicken and, moreover, demonstrates that CNBP plays an essential and conserved role during vertebrate head development. J. Cell. Biochem. 102: 1553–1570, 2007.

Fishing the Molecular Bases of Treacher Collins Syndrome

Treacher Collins syndrome (TCS) is an autosomal dominant disorder of craniofacial development, and mutations in the TCOF1 gene are responsible for over 90% of TCS cases. The knowledge about the molecular mechanisms responsible for this syndrome is relatively scant, probably due to the difficulty of reproducing the pathology in experimental animals. Zebrafish is an emerging model for human disease studies, and we therefore assessed it as a model for studying TCS. We identified in silico the putative zebrafish TCOF1 ortholog and cloned the corresponding cDNA. The derived polypeptide shares the main structural domains found in mammals and amphibians. Tcof1 expression is restricted to the anterior-most regions of zebrafish developing embryos, similar to what happens in mouse embryos. Tcof1 loss-of-function resulted in fish showing phenotypes similar to those observed in TCS patients, and enabled a further characterization of the mechanisms underlying craniofacial malformation. Besides, we initiated the identification of potential molecular targets of treacle in zebrafish. We found that Tcof1 loss-of-function led to a decrease in the expression of cellular proliferation and craniofacial development. Together, results presented here strongly suggest that it is possible to achieve fish with TCS-like phenotype by knocking down the expression of the TCOF1 ortholog in zebrafish. This experimental condition may facilitate the study of the disease etiology during embryonic development.

Dissecting CNBP, a Zinc-Finger Protein Required for Neural Crest Development, in Its Structural and Functional Domains

Cellular nucleic-acid-binding protein (CNBP) plays an essential role in forebrain and craniofacial development by controlling cell proliferation and survival to mediate neural crest expansion. CNBP binds to single-stranded nucleic acids and displays nucleic acid chaperone activity in vitro. The CNBP family shows a conserved modular organization of seven Zn knuckles and an arginine-glycine-glycine (RGG) box between the first and second Zn knuckles. The participation of these structural motifs in CNBP biochemical activities has still not been addressed. Here, we describe the generation of CNBP mutants that dissect the protein into regions with structurally and functionally distinct properties. Mutagenesis approaches were followed to generate: (i) an amino acid replacement that disrupted the fifth Zn knuckle; (ii) N-terminal deletions that removed the first Zn knuckle and the RGG box, or the RGG box alone; and (iii) a C-terminal deletion that eliminated the three last Zn knuckles. Mutant proteins were overexpressed in Escherichia coli, purified, and used to analyze their biochemical features in vitro, or overexpressed in Xenopus laevis embryos to study their function in vivo during neural crest cell development. We found that the Zn knuckles are required, but not individually essential, for CNBP biochemical activities, whereas the RGG box is essential for RNA-protein binding and nucleic acid chaperone activity. Removal of the RGG box allowed CNBP to preserve a weak single-stranded-DNA-binding capability. A mutant mimicking the natural N-terminal proteolytic CNBP form behaved as the RGG-deleted mutant. By gain-of-function and loss-of-function experiments in Xenopus embryos, we confirmed the participation of CNBP in neural crest development, and we demonstrated that the CNBP mutants lacking the N-terminal region or the RGG box alone may act as dominant negatives in vivo. Based on these data, we speculate about the existence of a specific proteolytic mechanism for the regulation of CNBP biochemical activities during neural crest development.

Primary structure and developmental expression of Bufo arenarum cellular nucleic acid‐binding protein: Changes in subcellular localization during early embryogenesis

A Bufo arenarum cellular nucleic acid‐binding protein (bCNBP) full‐length cDNA was cloned. bCNBP is a 19.4 kDa protein containing seven CCHC zinc finger motifs, an RGG box and a Ser‐rich region. Amino acid comparisons showed high values of homology in vertebrates and smaller values in insects or inferior eukaryotes. Northern blot analysis during oogenesis and early development revealed two transcripts with different expressions of pattern behavior. One of them is present in all stages analyzed, whereas the other is only detected from the beginning of zygotic transcription. Immunocytochemistry assays carried out on sections of ovary and early embryos showed that there was no specific staining of previtellogenic oocytes. In early vitellogenic oocytes, in oocytes at stages V/VI and in embryos at early blastula stage, reaction was observed inside the cytoplasm. At mid‐blastula stage, CNBP was mainly detected in the epiblast. At the late gastrula stage, two layers of cells were stained in the archenteron roof, in which the internal one presented as strong staining. Nuclei in this layer were stained even stronger than the cytoplasm. Changes in mRNA expression patterns, accompanied by changes in subcellular localization, suggest that CNBP might interact with both nuclear and cytoplasmic nucleic acids.