Interspecific variation in mitochondrial serine transfer RNA (UCN) in Euptychiina butterflies (Lepidoptera: Satyrinae): structure and alignment

The nucleotide variation and structural patterns of mitochondrial RNA molecule have been proposed as useful tools in molecular systematics; however, their usefulness is always subject to a proper assessment of homology in the sequence alignment. The present study describes the secondary structure of mitochondrial tRNA for the amino acid serine (UCN) on 13 Euptychiina species and the evaluation of its potential use for evolutionary studies in this group of butterflies. The secondary structure of tRNAs showed variation among the included species except between Hermeuptychia sp1 and sp2. Variation was concentrated in the ribotimidina-pseudouridine-cystosine (TψC), dihydrouridine (DHU) and variable loops and in the DHU and TψC arms. These results suggest this region as a potential marker useful for taxonomic differentiation of species in this group and also confirm the importance of including information from the secondary structure of tRNA to optimize the alignments.     

Determination of the cleavage site of the presequence by mitochondrial processing peptidase on the substrate binding scaffold and the multiple subsites inside a molecular cavity

Mitochondrial processing peptidase (MPP) recognizes a large variety of basic presequences of mitochondrial preproteins and cleaves the single site, often including arginine, at the -2 position (P(2)). To elucidate the recognition and specific processing of the preproteins by MPP, we mutated to alanines at acidic residues conserved in a large internal cavity formed by the MPP subunits, alpha-MPP and beta-MPP, and analyzed the processing efficiencies for various preproteins. We report here that alanine mutations at a subsite in rat beta-MPP interacting with the P(2) arginine cause a shift in the processing site to the C-terminal side of the preprotein. Because of reduced interactions with the P(2) arginine, the mutated enzymes recognize not only the N-terminal authentic cleavage site with P(2) arginine but also the potential C-terminal cleavage site without a P(2) arginine. In fact, it competitively cleaves the two sites of the preprotein. Moreover, the acidified site of alpha-MPP, which binds to the distal basic site in the long presequence, recognized the authentic P(2) arginine as the distal site in compensation for ionic interaction at the proximal site in the mutant MPP. Thus, MPP seems to scan the presequence from beta- to alpha-MPP on the substrate binding scaffold inside the MPP cavity and finds the distal and P(2) arginines on the multiple subsites on both MPP subunits. A possible mechanism for substrate recognition and cleavage is discussed here based on the notable character of a subsite-deficient mutant of MPP in which the substrate specificity is altered.     

Naturally occurring bioactive Cyclobutane-containing (CBC) alkaloids in fungi,fungal endophytes,and plants

This article focuses on the occurrence and biological activities of cyclobutane-containing (CBC) alkaloids obtained from fungi, fungal endophytes, and plants. Naturally occurring CBC alkaloids are of particular interest because many of these compounds display important biological activities and possess antitumour, antibacterial, antimicrobial, antifungal, and immunosuppressive properties. Therefore, these compounds are of great interest in the fields of medicine, pharmacology, medicinal chemistry, and the pharmaceutical industry. Fermentation and production of CBC alkaloids by fungi and/or fungal endophytes is also discussed. This review presents the structures and describes the activities of 98 CBC alkaloids.     

Calcium modulates endopeptidase 24.15 (EC 3.4.24.15) membrane association, secondary structure and substrate specificity

The metalloendopeptidase 24.15 (EP24.15) is ubiquitously present in the extracellular environment as a secreted protein. Outside the cell, this enzyme degrades several neuropeptides containing from 5 to 17 amino acids (e.g. gonadotropin releasing hormone, bradykinin, opioids and neurotensin). The constitutive secretion of EP24.15 from glioma C6 cells was demonstrated to be stimulated linearly by reduced concentrations of extracellular calcium. In the present report we demonstrate that extracellular calcium concentration has no effect on the total amount of the extracellular (cell associated + medium) enzyme. Indeed, immuno-cytochemical analyses by confocal and electron microscopy suggested that the absence of calcium favors the enzyme shedding from the plasma membrane into the medium. Two putative calcium-binding sites on EP24.15 (D93 and D159) were altered by site-directed mutagenesis to investigate their possible contribution to binding of the enzyme at the cell surface. These mutated recombinant proteins behave similarly to the wild-type enzyme regarding enzymatic activity, secondary structure, calcium sensitivity and immunoreactivity. However, immunocytochemical analyses by confocal microscopy consistently show a reduced ability of the D93A mutant to associate with the plasma membrane of glioma C6 cells when compared with the wild-type enzyme. These data and the model of the enzyme's structure as determined by X-ray diffraction suggest that D93 is located at the enzyme surface and is consistent with membrane association of EP24.15. Moreover, calcium was also observed to induce a major change in the EP24.15 cleavage site on distinctive fluorogenic substrates. These data suggest that calcium may be an important modulator of ep24.15 cell function.     

Identification of the active site of poly(A)-specific ribonuclease by site-directed mutagenesis and Fe(2+)-mediated cleavage.

Poly(A)-specific ribonuclease (PARN) is the only mammalian exoribonuclease characterized thus far with high specificity for degrading the mRNA poly(A) tail. PARN belongs to the RNase D family of nucleases, a family characterized by the presence of four conserved acidic amino acid residues. Here, we show by site-directed mutagenesis that these residues of human PARN, i.e. Asp(28), Glu(30), Asp(292), and Asp(382), are essential for catalysis but are not required for stabilization of the PARN x RNA substrate complex. We have used iron(II)-induced hydroxyl radical cleavage to map Fe(2+) binding sites in PARN. Two Fe(2+) binding sites were identified, and three of the conserved acidic amino acid residues were important for Fe(2+) binding at these sites. Furthermore, we show that the apparent dissociation constant ((app)K(d)) values for Fe(2+) binding at both sites were affected in PARN polypeptides in which the conserved acidic amino acid residues were substituted to alanine. This suggests that these residues coordinate divalent metal ions. We conclude that the four conserved acidic amino acids are essential residues of the PARN active site and that the active site of PARN functionally and structurally resembles the active site for 3'-exonuclease domain of Escherichia coli DNA polymerase I.     

Collated mutations in mitochondrial DNA (mtDNA) depletion syndrome (excluding the mitochondrial gamma polymerase,POLG1)

These tables list both published and a number of unpublished mutations in genes associated with early onset defects in mitochondrial DNA (mtDNA) maintenance including C10orf2, SUCLG1, SUCLA2, TYMP, RRM2B, MPV17, DGUOK and TK2. The list should not be taken as evidence that any particular mutation is pathogenic. We have included genes known to cause mtDNA depletion, excluding POLG1, because of the existing database (http://tools.niehs.nih.gov/polg/). We have also excluded mutations in C10orf2 associated with dominant adult onset disorders.     

Naturally occurring neuronal plasticity in visual wulst of the Baya weaver, Ploceus philippinus (Linnaeus, 1766)

The visual wulst (VW), the rostro-dorsal surface of the avian telencephalon extending from the midline to the lateral region of the brain, is a laminated “bulge” consisting in four histologically distinct rostro-caudally arranged laminae with a specific sequence: hyperpallium apicale, interstitial nucleus of hyperpallium apicale, hyperpallium intercalatum and hyperpallium densocellulare. The VW has been proposed to be the avian equivalent of the mammalian striate cortex. Various behavioral studies including lesion experiments have indicated the importance of the VW, which receives visual and/or auditory cues. We have investigated qualitatively and quantitatively the fascinating structural changes occurring in VW neurons of the seasonally breeding bird, Ploceus philippinus (Linnaeus, 1766). The Golgi method was used to study the seasonal fluctuations in the neuronal classes of the VW with regard to dendritic thickness, spine morphology and spine density during both the non-breeding and breeding periods of male Baya weaver birds. Significant variations in parameters studied among the various neuronal types located in the different well-demarcated regions of the VW are believed to contribute to the functional differences reported among the wulst regions. Thus, this study extends our view demonstrating naturally occurring neuronal plasticity in a seasonally dynamic avian brain of a bird that hones not only its learning and memorizing system but also its social and sexual system in preparation for the breeding season.     

Structure requirement and identification of a cryptic cleavage site in the mitochondrial processing of a plant F1-ATPase beta-subunit presequence

We sought to determine the structural features involved in the processing of the mitochondrial F1-ATPase beta-subunit (F1beta) presequence (54 residues) from Nicotiana plumbaginifolia. The cleavage efficiency of F1beta presequence mutants linked to the green fluorescent protein (GFP) was evaluated in vivo in tobacco by in situ microscopy and Western blotting. The residue at position -1 (Tyr) was required to be an aromatic residue and the residue at position +2 (Thr) was found to be important for F1beta processing, while, unexpectedly, changing the distal (Arg-15) and proximal (Arg-5) arginine residues did not strongly reduce processing. In addition, results also supported the requirement of a helical structure around the cleavage position. Sequencing of the mature form of a precursor containing the first 30 residues of the F1beta presequence linked to GFP revealed the presence of a cryptic cleavage site between residues 26 and 27, which showed the features of a classical mitochondrial processing site, suggesting dual processing of the F1beta presequence. In vitro processing confirmed these data and showed that processing was sensitive to o-phenanthroline, thus catalyzed by mitochondrial processing peptidase.     

Bovine mitochondrial tRNAPhe, tRNASer (AGY) and tRNASer (UCN): preparation using a new detection method and their properties in aminoacylation

Bovine mitochondrial tRNAPhe, tRNASer (AGY), and tRNASer (UCN) possessing unusual structures were purified using a new hybridization assay system and their properties in aminoacylation were examined. Bovine mitochondrial phenyl-alanyl- and seryl-tRNA synthetases could aminoacylate the same amino acid-specific tRNAs obtained not only from the mitochondria but also from other sources such as E. coli, Thermus thermophilus, bovine and yeast cytosols and archaebacteria, Sulfolobus acidocaldarius. On the contrary, none of both bacterial and cytosolic synthetases could aminoacylate the same amino acid specific tRNAs from the heterologous sources with some exceptions. We consider that the bovine mitochondrial aminoacyl-tRNA synthetases have considerably simple recognition mechanism toward the substrate tRNAs compared with the non-mitochondrial ones. This mechanism may be correlated with the occurrence of structural varieties of the mitochondrial tRNA species with unusual structures.     

Identification and kinetics analysis of a novel heparin-binding site (KEDK) in human tenascin-C

The interaction between tenascin-C (TN-C), a multi-subunit extracellular matrix protein, and heparin was examined using a surface plasmon resonance-based technique on a Biacore system. The aims of the present study were to examine the affinity of fibronectin type III repeats of TN-C fragments (TNIII) for heparin, to investigate the role of the TNIII4 domains in the binding of TN-C to heparin, and to delineate a sequence of amino acids within the TNIII4 domain, which mediates cooperative heparin binding. At a physiological salt concentration, and pH 7.4, TNIII3-5 binds to heparin with high affinity (K(D) = 30 nm). However, a major heparin-binding site in TNIII5 produces a modest affinity binding at a K(D) near 4 microm, and a second site in TNIII4 enhances the binding by several orders of magnitude, although it was far too weak to produce an observable binding of TNIII4 by itself. Moreover, mutagenesis of the KEDK sequence in the TNIII4 domain resulted in the significant reduction of heparin-binding affinity. In addition, residues in the KEDK sequences are conserved in TN-C throughout mammalian evolution. Thus the structure-based sequence alignment, mutagenesis, and sequence conservation data together reveal a KEDK sequence in TNIII4 suggestive of a minor heparin-binding site. Finally, we demonstrate that TNIII4 contains binding sites for heparin sulfate proteoglycan and enhances the heparin sulfate proteoglycan-dependent human gingival fibroblast adhesion to TNIII5, thus providing the biological significance of heparin-binding site of TNIII4. These results suggest that the heparin-binding sites may traverse TNIII4-5 and thus require KEDK in TNIII4 for optimal heparin-binding.     

Effect of substrate binding loop mutations on the structure, kinetics, and inhibition of enoyl acyl carrier protein reductase from Plasmodium falciparum

Enoyl acyl carrier protein reductase (ENR), which catalyzes the final and rate limiting step of fatty acid elongation, has been validated as a potential drug target. Triclosan is known to be an effective inhibitor for this enzyme. We mutated the substrate binding site residue Ala372 of the ENR of Plasmodium falciparum (PfENR) to Methionine and Valine which increased the affinity of the enzyme towards triclosan to almost double, close to that of Escherichia coli ENR (EcENR) which has a Methionine at the structurally similar position of Ala372 of PfENR. Kinetic studies of the mutants of PfENR and the crystal structure analysis of the A372M mutant revealed that a more hydrophobic environment enhances the affinity of the enzyme for the inhibitor. A triclosan derivative showed a threefold increase in the affinity towards the mutants compared to the wild type, due to additional interactions with the A372M mutant as revealed by the crystal structure. The enzyme has a conserved salt bridge which stabilizes the substrate binding loop and appears to be important for the active conformation of the enzyme. We generated a second set of mutants to check this hypothesis. These mutants showed loss of function, except in one case, where the crystal structure showed that the substrate binding loop is stabilized by a water bridge network.     

Studies on the structure of lecithin:cholesterol acyltransferase (LACT)--comparisons of the active site region and secondary structure of the human and the porcine enzymes

1. Chemical modification of essential serine, histidine and cysteine residues of porcine LCAT were accompanied by loss of enzymatic activity. 2. Modification of cysteine with DTNB inactivated the enzyme which could not be reactivated by KCN suggesting direct involvement of the cysteine residue(s) in catalysis. 3. About half of the primary structure of the porcine enzyme was determined. 4. Respective regions of the human and porcine LCAT are highly homologous; especially, the amino-terminus and the region surrounding the DFP-labeled serine residues. 5. The observed primary structure differences represent amino acid substitutions that are projected to induce significant changes in secondary structure.     

Analysis of site-directed mutations in human pro-alpha 2(I) collagen which block cleavage by the C-proteinase

We have used site-directed mutagenesis to obtain human pro alpha 2(I) cDNAs containing novel mutations designed to inhibit cleavage at the C-proteinase site. Deletion of six relatively conserved amino acids which surround the cleavage site did not interfere with assembly of the triple helix in transfected rat cells, but blocked cleavage of the constituent mutated chains by endogenous C-proteinase. Substitution for a conserved Asp, which forms part of the Ala-Asp bond cleaved by C-proteinase, also blocked cleavage by endogenous C-proteinase. The conserved Asp is, therefore, a necessary component of the C-proteinase cleavage site. Incubation in vitro with a purified mouse C-proteinase, confirmed both mutations to be resistant to cleavage by high concentrations of the physiologically relevant enzyme. Mutant pro alpha 2(I) chains, resistant to cleavage by C-proteinase in culture media, were processed in cell layers by a different protease which cleaved telopeptide domains. Naturally occurring mutations at the C-proteinase site have not been described in human patients. The mutations characterized here, further define the C-proteinase cleavage site and provide reagents which may be informative when introduced into transgenic mice.     

Two mutations in mitochondrial ATP6 gene of ATP synthase,related to human cancer,affect ROS,calcium homeostasis and mitochondrial permeability transition in yeast

The relevance of mitochondrial DNA (mtDNA) mutations in cancer process is still unknown. Since the mutagenesis of mitochondrial genome in mammals is not possible yet, we have exploited budding yeast S. cerevisiae as a model to study the effects of tumor-associated mutations in the mitochondrial MTATP6 gene, encoding subunit 6 of ATP synthase, on the energy metabolism. We previously reported that four mutations in this gene have a limited impact on the production of cellular energy. Here we show that two mutations, Atp6-P163S and Atp6-K90E (human MTATP6-P136S and MTATP6-K64E, found in prostate and thyroid cancer samples, respectively), increase sensitivity of yeast cells both to compounds inducing oxidative stress and to high concentrations of calcium ions in the medium, when Om45p, the component of porin complex in outer mitochondrial membrane (OM), was fused to GFP. In OM45-GFP background, these mutations affect the activation of yeast permeability transition pore (yPTP, also called YMUC, yeast mitochondrial unspecific channel) upon calcium induction. Moreover, we show that calcium addition to isolated mitochondria heavily induced the formation of ATP synthase dimers and oligomers, recently proposed to form the core of PTP, which was slower in the mutants. We show the genetic evidence for involvement of mitochondrial ATP synthase in calcium homeostasis and permeability transition in yeast. This paper is a first to show, although in yeast model organism, that mitochondrial ATP synthase mutations, which accumulate during carcinogenesis process, may be significant for cancer cell escape from apoptosis.     

The yeast RNA1 protein, necessary for RNA processing, is homologous to the human ribonuclease/angiogenin inhibitor (RAI)

Summary Mutations in theRNA1 gene ofSaccharomyces cerevisiae, which encodes an essential cytosolic protein, affect the production and processing of all major classes of RNA. The mechanisms underlying these effects are not at all understood. Detailed comparative sequence analyses revealed that the RNA1 protein belongs to a superfamily, the members of which contain repetitive leucine-rich motifs (LRM). Within this superfamily RNA1 is most closely related to the ribonuclease/angiogenin inhibitor (RAI), which is a tightly binding inhibitor of ribonucleolytic activities in mammals. These results not only provide important clues to the structure, function and evolution of the RNAI protein, but also have intriguing implications for possible novel functions of RAI.     

Monitoring of caspase-8/FLICE processing and activation upon Fas stimulation with novel antibodies directed against a cleavage site for caspase-8 and its substrate,FLICE-like inhibitory protein (FLIP)

We generated and characterized novel antibodies specific for a cleavage site of human caspase-8/FLICE and its substrate, FLICE-like inhibitory protein (FLIP). The synthetic peptides used as immunogens were CQGDNYQKGIPVETD (#791) and VSEGQLEDSSLLEVD (#1342), which corresponded to cleaved regions of N-terminal fragments of caspase-8 and FLIP generated by active caspase-8, respectively. Each antibody purified from rabbit antiserum reacted specifically with the immunogen but not with the peptide corresponding to the unproteolyzed form, as assessed by ELISA. In vitro cleavage of GST-FLIP by active caspase-8 generated an N-terminal fragment (GST-p43) and a C-terminal one (p12). Consistent with other in vivo data, the FLIP cleavage site follows the Asp residue, LEVD(376)GPAMKNVEF, identified on N-terminal sequencing of the p12 fragment. #1342-antibody (#1342-Ab) recognized the GST-p43 fragment but not the uncleaved protein, thus confirming its specificity. When the antibodies were used for immunoblotting, flow cytometry, and confocal laser microscopy, the proteolysis of caspase-8 and FLIP, and the subcellular localization of their digests could be monitored in apoptotic U937 cells. Interestingly, a significant increase in the percentage of cells exhibiting caspase-8 and FLIP cleavage was observed upon Fas stimulation in interferon-gamma-treated U937 cells, in which the susceptibility to Fas is extremely enhanced. In contrast, U937 cells treated with vitamin D(3) or all-trans retinoic acid showed Fas-resistance, and caspase-8 processing and FLIP cleavage were strongly inhibited. In conclusion, we established a system based on the cleavage site-directed antibodies to monitor the dynamics of caspase-8 processing and activation during apoptosis. Using this system, we found that Fas-susceptibility changes during U937 differentiation occur upstream of caspase-8 processing/activation.