Hydropathy Profile Research Articles

Fusion methodologies for biomedical data

Data fusion methods are powerful tools for integrating the different views of an organism provided by various types of experimental data. We describe various methodologies for integrating and drawing inferences from a collection of biomedical data, primarily focusing on protein and gene expression data. Computational experiments performed using biomedical data, including known protein-protein interactions, hydropathy profiles, gene expression data and amino acid sequences, demonstrate the utility of this approach. Overall, studies agree in that methodologies using carefully selected data of various types to predict particular classes, groups and interactions, perform better than when applied to a single type of data.

Identification and expression analysis of CjLTI, a novel low temperature responsive gene from Caragana jubata

Using rapid amplification of cDNA ends, a full length cDNA (CjLTI) was cloned from apical buds of Caragana jubata, a plant species that grows under extreme cold. The cDNA obtained was 573 bp long consisting of an open reading frame of 351 bp encoding 116 amino acids. Homology analysis did not exhibit significant similarity with any sequence at NCBI database, therefore it was deduced as a novel gene. Secondary structure analysis suggested that the deduced CjLTI contained 25.86% α-helices, 4.31% β-turns, 6.90% extended strands, and 62.93% random coils. The hydropathy profile suggested CjLTI to be a hydrophobic protein having characteristic features of signal peptides at N-terminus. The gene exhibited down-regulation at 5 min of exposure to low temperature (LT, 4 ± 3 °C) followed by a strong up-regulation after 15 min and onwards. Methyl jasmonate (MJ) lead to up-regulation of CjLTI starting at 5 min onwards. The gene exhibited up- and down-regulation of expression pattern in response to abscisic acid (ABA) and salicylic acid (SA). Mild drought stress slightly up-regulated gene expression and at severe drought (up to 115% reduction in leaf water potential) slight down-regulation of gene expression was observed. These results suggested CjLTI to be a LT responsive gene wherein MJ, ABA and SA pathways might be involved in regulating the gene expression.

Novel Hydrophobins from Trichoderma Define a New Hydrophobin Subclass: Protein Properties, Evolution, Regulation and Processing

Hydrophobins are small proteins, characterised by the presence of eight positionally conserved cysteine residues, and are present in all filamentous asco- and basidiomycetes. They are found on the outer surfaces of cell walls of hyphae and conidia, where they mediate interactions between the fungus and the environment. Hydrophobins are conventionally grouped into two classes (class I and II) according to their solubility in solvents, hydropathy profiles and spacing between the conserved cysteines. Here we describe a novel set of hydrophobins from Trichoderma spp. that deviate from this classification in their hydropathy, cysteine spacing and protein surface pattern. Phylogenetic analysis shows that they form separate clades within ascomycete class I hydrophobins. Using T. atroviride as a model, the novel hydrophobins were found to be expressed under conditions of glucose limitation and to be regulated by differential splicing.

Novel Androstenetriol Interacts with the Mitochondrial Translocator Protein and Controls Steroidogenesis

Steroid hormones are metabolically derived from multiple enzymatic transformations of cholesterol. The controlling step in steroid hormone biogenesis is the delivery of cholesterol from intracellular stores to the cytochrome P450 enzyme CYP11A1 in the mitochondrial matrix. The 18-kDa translocator protein (TSPO) plays an integral part in this mitochondrial cholesterol transport. Consistent with its role in intracellular cholesterol movement, TSPO possesses a cholesterol recognition/interaction amino acid consensus (CRAC) motif that has been demonstrated to bind cholesterol. To further investigate the TSPO CRAC motif, we performed molecular modeling studies and identified a novel ligand, 3,17,19-androsten-5-triol (19-Atriol) that inhibits cholesterol binding at the CRAC motif. 19-Atriol could bind a synthetic CRAC peptide and rapidly inhibited hormonally induced steroidogenesis in MA-10 mouse Leydig tumor cells and constitutive steroidogenesis in R2C rat Leydig tumor cells at low micromolar concentrations. Inhibition at these concentrations was not due to toxicity or inhibition of the CYP11A1 enzyme and was reversed upon removal of the compound. In addition, 19-Atriol was an even more potent inhibitor of PK 11195-stimulated steroidogenesis, with activity in the high nanomolar range. This was accomplished without affecting PK 11195 binding or basal steroidogenesis. Finally, 19-Atriol inhibited mitochondrial import and processing of the steroidogenic acute regulatory protein without any effect on TSPO protein levels. In conclusion, we have identified a novel androstenetriol that can interact with the CRAC domain of TSPO, can control hormonal and constitutive steroidogenesis, and may prove to be a useful tool in the therapeutic control of diseases of excessive steroid formation.

Transmembrane Topology of Mammalian Planar Cell Polarity Protein Vangl1

Vangl1 and Vangl2 are membrane proteins that play an important role in neurogenesis, and Vangl1/Vangl2 mutations cause neural tube defects in mice and humans. At the cellular level, Vangl proteins regulate the establishment of planar cell polarity (PCP), a process requiring membrane assembly of asymmetrically distributed multiprotein complexes that transmit polarity information to neighboring cells. The membrane topology of Vangl proteins and the protein segments required for structural and functional aspects of multiprotein membrane PCP complexes is unknown. We have used epitope tagging and immunofluorescence to establish the secondary structure of Vangl proteins, including the number, position, and polarity of transmembrane domains. Antigenic hemagglutinin A (HA) peptides (YYDVPDYS) were inserted in predicted intra- or extracellular loops of Vangl1 at positions 18, 64, 139, 178, 213, and 314, and individual mutant variants were stably expressed at the membrane of MDCK polarized cells. The membrane topology of the exofacial HA tag was determined by a combination of immunofluorescence in intact (extracellular epitopes) and permeabilized (intracellular epitopes) cells and by surface labeling. Results indicate that Vangl proteins have a four-transmembrane domain structure with the N-terminal portion (HA18 and HA64) and the large C-terminal portion (HA314) of the protein being intracellular. Topology mapping and hydropathy profiling show that the loop delineated by TMD1-2 (HA139) and TMD3-4 (HA213) is extracellular while the segment separating predicted TMD2-3 (HA178) is intracellular. This secondary structure reveals a compact membrane-associated portion with very short predicted extra- and intracellular loops, while the protein harbors a large intracellular domain.

Discrimination of Golgi Type II Membrane Proteins Based on Their Hydropathy Profiles and the Amino Acid Propensities of Their Transmembrane Regions

Membrane proteins in the Golgi apparatus play important roles in biological functions, predominantly as catalysts related to post-translational modification of protein oligosaccharides. We succeeded in extracting the characteristics of Golgi type II membrane proteins computationally by comparison with those of Golgi no retention proteins, which are mainly localized in the plasma membrane. Golgi type II membrane proteins were detected by combining hydropathy alignment and a position-specific score matrix of the amino acid propensities around the transmembrane region. We achieved 96.2% sensitivity, 93.5% specificity, and a 0.949 success rate in a self-consistency test. In a 5-fold cross-validation test, 88.0% sensitivity, 85.5% specificity, and a 0.867 success rate were achieved.

Identification and Functions of Amino Acid Residues in PotB and PotC Involved in Spermidine Uptake Activity

Amino acid residues on PotB and PotC involved in spermidine uptake were identified by random and site-directed mutagenesis. It was found that Trp(8), Tyr(43), Trp(100), Leu(110), and Tyr(261) in PotB and Trp(46), Asp(108), Glu(169), Ser(196), Asp(198), and Asp(199) in PotC were strongly involved in spermidine uptake and that Tyr(160), Glu(172), and Leu(274) in PotB and Tyr(19), Tyr(88), Tyr(148), Glu(160), Leu(195), and Tyr(211) in PotC were moderately involved in spermidine uptake. Among 11 amino acid residues that were strongly involved in spermidine uptake, Trp(8) in PotB was important for insertion of PotB and PotC into membranes. Tyr(43), Trp(100), and Leu(110) in PotB and Trp(46), Asp(108), Ser(196), and Asp(198) in PotC were found to be involved in the interaction with PotD. Leu(110) and Tyr(261) in PotB and Asp(108), Asp(198), and Asp(199) in PotC were involved in the recognition of spermidine, and Trp(100) and Tyr(261) in PotB and Asp(108), Glu(169), and Asp(198) in PotC were involved in ATPase activity of PotA. Accordingly, Trp(100) in PotB was involved in both PotD recognition and ATPase activity, Leu(110) in PotB was involved in both PotD and spermidine recognition, and Tyr(261) in PotB was involved in both spermidine recognition and ATPase activity. Asp(108) and Asp(198) in PotC were involved in PotD and spermidine recognition as well as ATPase activity. These results suggest that spermidine passage from PotD to the cytoplasm is coupled to the ATPase activity of PotA through a structural change of PotA by its ATPase activity.

A Study of the Evolution of Inverted-Topology Repeats from LeuT-Fold Transporters Using AlignMe

X-ray crystal structures have revealed that numerous secondary transporter proteins originally categorized into different sequence families share similar structures, namely, the LeuT fold. The core of this fold consists of two units of five transmembrane helices, whose conformations have been proposed to exchange to form the two alternate states required for transport. That these two units are related implies that LeuT-like transporters evolved from gene-duplication and fusion events. Thus, the origins of this structural repeat may be relevant to the evolution of transport function. However, the lack of significant sequence similarity requires sensitive sequence search methods for analyzing their evolution. To this end, we developed a software application called AlignMe, which can use various types of input information, such as residue hydrophobicity, to perform pairwise alignments of sequences and/or of hydropathy profiles of (membrane) proteins. We used AlignMe to analyze the evolutionary relationships between repeats of the LeuT fold. In addition, we identified proteins from the so-called DedA family that potentially share a common ancestor with these repeats. DedA domains have been implicated in, e.g., selenite uptake; they are found widely distributed across all kingdoms of life; two or more DedA domains are typically found per genome, and some may adopt dual topologies. These results suggest that DedA proteins existed in ancient organisms and may function as dimers, as required for a would-be ancestor of the LeuT fold. In conclusion, we provide novel insights into the evolution of this important structural motif and thus potentially into the alternating-access mechanism of transport itself.

A Strong Genetic Association between the Tumor Necrosis Factor Locus and Proliferative Vitreoretinopathy: The Retina 4 Project

To assess the genetic contribution to proliferative vitreoretinopathy (PVR) and report the strong association observed in the tumor necrosis factor (TNF) locus. As a component of The Retina 4 Project, a case-controlled, candidate gene association study in the TNF locus was conducted. Blood from 450 patients with (138 cases) and without (312 controls) post-rhegmatogenous retinal detachment (RD) PVR was genotyped to determine polymorphisms located in the TNFα gene. Single nucleotide polymorphisms (SNPs) with correlation coefficients of ≥ 0.8 and a minor allelic frequency of ≥ 10% were studied. Functional SNPs or SNPs previously described in association with other inflammatory diseases were also added for analysis. The SNPlex Genotyping System (Applied Biosystems, Foster City, CA) was used for genotyping. Single nucleotide polymorphism and haplotype analyses were performed. Bioinformatic tools were used to evaluate those SNPs that were significantly associated. Single and haplotypic significant associations with PVR. A total of 11 common tag SNPs in the following genes were analyzed: lymphotoxin alpha (LTA), TNFα, leukocyte-specific transcript 1 (LST1), and the activating natural killer receptor p30 (NCR3). After permutation, there was a significant association in the non-synonymous polymorphism rs2229094(T→C) in the LTA gene (P = 0.0283), which encodes a cysteine to arginine change in the signal peptide. This marker was also present in all significant haplotypic associations and was not observed in any nonsignificant associations. When this SNP was analyzed using bioinformatic tools, the hydropathy profile changed, as well as the transmembrane region and the splicing site predictions. The strong association found in the rs2229094(T→C) of the LTA gene may indicate an important role of this polymorphism in the development of PVR. If supported in extended studies, the rs2229094(T→C) may have significant implications regarding the genetic risk of the retinal repairing process.

Cloning and functional characterization of a fatty acid Δ6-desaturase from Oenothera biennis: Production of γ-linolenic acid by heterologous expression in Saccharomyces cerevisiae

Evening primrose Oenothera biennis is grown commercially for γ-linolenic acid (GLA) production by the fatty acid Δ6-desaturase enzymatic reaction. Based on available sequence information, the O. biennis fatty acid Δ6-desaturase gene was cloned, using rapid amplification of cDNA ends methods and reverse transcription-polymerase chain reaction. The deduced amino acid sequence showed high similarity to the plant Δ6-desaturases, which comprised the characteristics of membrane-bound desaturases, including three conserved histidine-rich motifs, hydropathy profile, and a cytochrome b5 domain at the N-terminus. Heterologous expression of the coding sequence in Saccharomyces cerevisiae resulted in the synthesis and accumulation of GLA when provided with linoleic acid substrate.

Did Trypanosomatid Parasites Contain a Eukaryotic Alga–Derived Plastid in Their Evolutionary Past?

The Trypanosomatidae is closely related to euglenids that harbor plastids acquired from a green alga via secondary endosymbiosis. This discovery led to the idea that trypanosomatid parasites contained a green alga-derived plastid in their evolutionary past, an evolutionary scenario that was criticized based on the rarity of plant/plastid/cyanobacterium-like genes in the completely sequenced genomes of Trypanosoma and Leishmania species. Because it is difficult to identify such genes, however, their apparent rarity does not preclude a previous plastid endosymbiosis in the Trypanosomatidae. The genome of the plastid-less apicomplexan Cryptosporidium parvum preserves only a handful of plant/plastid/cyanobacterium-like genes, suggesting massive loss of plastid genes after elimination of its plastid. Additional support for such wholesale gene loss comes from fucoxanthin-containing dinoflagellates. Trypanosomatid nuclear genomes contain cyanobacterium-, green plant-, and haptophyte alga-derived genes, suggesting that they could have possessed a plastid in their evolutionary past; however, these genes also could represent examples of more typical horizontal gene transfer that did not accompany a plastid endosymbiosis. Thus, the presence of host cell genes that were adapted for use in the plastid would be much stronger evidence for a past plastid endosymbiosis in the Trypanosomatidae. Good examples of such genes are those encoding superoxide dismutases (SODs). Trypanosomatid parasites possess 4 iron-containing SODs, with 2 of them, SODA and SODC, targeted to the mitochondrion. In contrast with SODAs with classical single-domain mitochondrial targeting signals, SODCs carry bipartite pre-sequences composed of a signal peptide, followed by a transit peptide. Interestingly, these N-terminal extensions show striking similarities in length, hydropathy profiles, amino acid composition, and targeting properties to pre-sequences of proteins targeted to eukaryotic alga-derived plastids of euglenids and dinoflagellates. In turn, phylogenetic analyses indicate that SODCs originated from a mitochondrion-targeted SOD via gene duplication and were inherited vertically in the trypanosomatid lineage. These data represent a new kind of evidence for a past plastid endosymbiosis in the Trypanosomatidae, but the nature of this plastid remains unclear. It is usually assumed that the trypanosomatid plastid shared a common origin with that of euglenids, but Delta 4 desaturase phylogenies suggest that it could have originated via an independent, tertiary endosymbiosis involving a haptophyte alga. It is also possible that ancestors of the Trypanosomatidae initially possessed a primary plastid that later was replaced by a secondary or tertiary plastid.

Screening a novel Na+/H+antiporter gene from a metagenomic library of halophiles colonizing in the Dagong Ancient Brine Well in China

Metagenomic DNA libraries constructed from the Dagong Ancient Brine Well were screened for genes with Na(+)/H(+) antiporter activity on the antiporter-deficient Escherichia coli KNabc strain. One clone with a stable Na(+)-resistant phenotype was obtained and its Na(+)/H(+) antiporter gene was sequenced and designated as m-nha. The deduced amino acid sequence of M-Nha protein consists of 523 residues with a calculated molecular weight of 58 147 Da and a pI of 5.50, which is homologous with NhaH from Halobacillus dabanensis D-8(T) (92%) and Halobacillus aidingensis AD-6(T) (86%), and with Nhe2 from Bacillus sp. NRRL B-14911 (64%). It had a hydropathy profile with 10 putative transmembrane domains and a long carboxyl terminal hydrophilic tail of 140 amino acid residues, similar to Nhap from Synechocystis sp. and Aphanothece halophytica, as well as NhaG from Bacillus subtilis. The m-nha gene in the antiporter-negative mutant E. coli KNabc conferred resistance to Na(+) and the ability to grow under alkaline conditions. The difference in amino acid sequence and the putative secondary structure suggested that the m-nha isolated from the Dagong Ancient Brine Well in this study was a novel Na(+)/H(+) antiporter gene.

Rapid screening of membrane topology of secondary transport proteins

Limited experimental data may be very useful to discriminate between membrane topology models of membrane proteins derived from different methods. A membrane topology screening method is proposed by which the cellular disposition of three positions in a membrane protein are determined, the N- and the C-termini and a position in the middle of the protein. The method involves amplification of the encoding genes or gene fragments by PCR, rapid cloning in dedicated vectors by ligation independent cloning, and determination of the cellular disposition of the three sites using conventional techniques. The N-terminus was determined by labeling with a fluorescent probe, the central position and the C-terminus by the reporter fusion technique using alkaline phosphatase (PhoA) and green fluorescence protein (GFP) as reporters. The method was evaluated using 16 transporter proteins of known function from four different structural classes. For 13 proteins a complete set of three localizations was obtained. The experimental data was used to discriminate between membrane topology models predicted by TMHMM, a widely used predictor using the amino acid sequence as input and by MemGen that uses hydropathy profile alignment and known 3D structures or existing models. It follows that in those cases where the models from the two methods were similar, the models were consistent with the experimental data. In those cases where the models differed, the MemGen model agreed with the experimental data. Three more recent predictors, MEMSAT3, OCTOPUS and TOPCONS showed a significantly higher consistency with the experimental data than observed with TMHMM.

High sequence homology between protein tyrosine acid phosphatase from boar seminal vesicles and human prostatic acid phosphatase.

Boar seminal vesicle protein tyrosine acid phosphatase (PTAP) and human prostatic acid phosphatase (PAP) show high affinity for protein phosphotyrosine residues. The physico-chemical and kinetic properties of the boar and human enzymes are different. The main objective of this study was to establish the nucleotide sequence of cDNA encoding boar PTAP and compare it with that of human PAP cDNA. Also, the amino-acid sequence of boar PTAP was compared with the sequence of human PAP. PTAP was isolated from boar seminal vesicle fluid and sequenced. cDNA to boar seminal vesicle RNA was synthesized, amplified by PCR, cloned in E. coli and sequenced. The obtained N-terminal amino-acid sequence of boar PTAP showed 92% identity with the N-terminal amino-acid sequence of human PAP. The determined sequence of a 354 bp nucleotide fragment (GenBank accession number: GQ184596) showed 90% identity with the corresponding sequence of human PAP. On the basis of this sequence a 118 amino acid fragment of boar PTAP was predicted. This fragment showed 89% identity with the corresponding fragment of human PAP and had a similar hydropathy profile. The compared sequences differ in terms of their isoelectric points and amino-acid composition. This may explain the differences in substrate specificity and inhibitor resistance of boar PTAP and human PAP.

A Conserved Na+ Binding Site of the Sodium-coupled Neutral Amino Acid Transporter 2 (SNAT2)

The SLC38 family of solute transporters mediates the coupled transport of amino acids and Na(+) into or out of cells. The structural basis for this coupled transport process is not known. Here, a profile-based sequence analysis approach was used, predicting a distant relationship with the SLC5/6 transporter families. Homology models using the LeuT(Aa) and Mhp1 transporters of known structure as templates were established, predicting the location of a conserved Na(+) binding site in the center of membrane helices 1 and 8. This homology model was tested experimentally in the SLC38 member SNAT2 by analyzing the effect of a mutation to Thr-384, which is predicted to be part of this Na(+) binding site. The results show that the T384A mutation not only inhibits the anion leak current, which requires Na(+) binding to SNAT2, but also dramatically lowers the Na(+) affinity of the transporter. This result is consistent with a previous analysis of the N82A mutant transporter, which has a similar effect on anion leak current and Na(+) binding and which is also expected to form part of the Na(+) binding site. In contrast, random mutations to other sites in the transporter had little or no effect on Na(+) affinity. Our results are consistent with a cation binding site formed by transmembrane helices 1 and 8 that is conserved among the SLC38 transporters as well as among many other bacterial and plant transporter families of unknown structure, which are homologous to SLC38.

Mitochondrial Glutamate Carrier GC1 as a Newly Identified Player in the Control of Glucose-stimulated Insulin Secretion

The SLC25 carrier family mediates solute transport across the inner mitochondrial membrane, a process that is still poorly characterized regarding both the mechanisms and proteins implicated. This study investigated mitochondrial glutamate carrier GC1 in insulin-secreting beta-cells. GC1 was cloned from insulin-secreting cells, and sequence analysis revealed hydropathy profile of a six-transmembrane protein, characteristic of mitochondrial solute carriers. GC1 was found to be expressed at the mRNA and protein levels in INS-1E beta-cells and pancreatic rat islets. Immunohistochemistry showed that GC1 was present in mitochondria, and ultrastructural analysis by electron microscopy revealed inner mitochondrial membrane localization of the transporter. Silencing of GC1 in INS-1E beta-cells, mediated by adenoviral delivery of short hairpin RNA, reduced mitochondrial glutamate transport by 48% (p < 0.001). Insulin secretion at basal 2.5 mM glucose and stimulated either by intermediate 7.5 mM glucose or non-nutrient 30 mM KCl was not modified by GC1 silencing. Conversely, insulin secretion stimulated with optimal 15 mM glucose was reduced by 23% (p < 0.005) in GC1 knocked down cells compared with controls. Adjunct of cell-permeant glutamate (5 mM dimethyl glutamate) fully restored the secretory response at 15 mM glucose (p < 0.005). Kinetics of insulin secretion were investigated in perifused isolated rat islets. GC1 silencing in islets inhibited the secretory response induced by 16.7 mM glucose, both during first (-25%, p < 0.05) and second (-33%, p < 0.05) phases. This study demonstrates that insulin-secreting cells depend on GC1 for maximal glucose response, thereby assigning a physiological function to this newly identified mitochondrial glutamate carrier.

Functional Importance of GGXG Sequence Motifs in Putative Reentrant Loops of 2HCT and ESS Transport Proteins

The 2HCT and ESS families are two families of secondary transporters. Members of the two families are unrelated in amino acid sequence but share similar hydropathy profiles, which suggest a similar folding of the proteins in membranes. Structural models show two homologous domains containing five transmembrane segments (TMSs) each, with a reentrant or pore loop between the fourth and fifth TMSs in each domain. Here we show that GGXG sequence motifs present in the putative reentrant loops are important for the activity of the transporters. Mutation of the conserved Gly residues to Cys in the motifs of the Na(+)-citrate transporter CitS in the 2HCT family and the Na(+)-glutamate transporter GltS in the ESS family resulted in strongly reduced transport activity. Similarly, mutation of the variable residue "X" to Cys in the N-terminal half of GltS essentially inactivated the transporter. The corresponding mutations in the N- and C-terminal halves of CitS reduced transport activity to 60 and 25% of that of the wild type, respectively. Residual activity of any of the mutants could be further reduced by treatment with the membrane permeable thiol reagent N-ethylmaleimide (NEM). The X to Cys mutation (S405C) in the cytoplasmic loop in the C-terminal half of CitS rendered the protein sensitive to the bulky, membrane impermeable thiol reagent 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AmdiS) added at the periplasmic side of the membrane, providing further evidence that this part of the loop is positioned between the transmembrane segments. The putative reentrant loop in the C-terminal half of the ESS family does not contain the GGXG motif, but a conserved stretch rich in Gly residues. Cysteine-scanning mutagenesis of a stretch of 18 residues in the GltS protein revealed two residues important for function. Mutant N356C was completely inactivated by treatment with NEM, and mutant P351C appeared to be the counterpart of mutant S405C of CitS; the mutant was inactivated by AmdiS added at the periplasmic side of the membrane. The data support, in general, the structural and mechanistic similarity between the ESS and 2HCT transporters and, more particularly, the two-domain structure of the transporters and the presence and functional importance of the reentrant loops present in each domain. It is proposed that the GGXG motifs are at the vertex of the reentrant loops.

Redox- and Calmodulin-dependent S-Nitrosylation of the KCNQ1 Channel

Nitric oxide (NO) is a gaseous signal mediator showing numerous important biological effects. NO has been shown in many instances to exhibit its action via the protein S-nitrosylation mechanism, in which binding of NO to Cys residues regulate protein function independently of activation of soluble guanylate cyclase. The direct link between protein S-nitrosylation and functional modulation, however, has been demonstrated only in limited examples. Furthermore, although most proteins have more than one Cys residue, the mechanism by which a certain Cys becomes a specific target residue of S-nitrosylation is poorly understood. We have previously reported that NO regulates currents through the cardiac slowly activating delayed rectifier potassium channel (I(Ks)) irrespective of soluble guanylate cyclase activation. Here we demonstrate using a biotin-switch assay that NO induced S-nitrosylation of the alpha-subunit of the I(Ks) channel, KCNQ1, at Cys(445) in the C terminus. A redox motif flanking Cys(445) and the interaction of KCNQ1 with calmodulin are required for preferential S-nitrosylation of Cys(445). A patch clamp experiment shows that S-nitrosylation of Cys(445) modulates the KCNQ1/KCNE1 channel function. Our data provide a molecular basis of NO-mediated regulation of the I(Ks) channel. This novel regulatory mechanism of the I(Ks) channel may play a role in previously demonstrated NO-mediated phenomenon in cardiac electrophysiology, including shortening in action potential duration in response to intracellular Ca(2+) or sex hormones.

Identification and characterization of a novel ∆6-fatty acid desaturase gene from Rhizopus nigricans

Fatty acid composition of fungi is analysed through the gas chromatography technique. With specific activity a novel enzyme Delta6-fatty acid desaturase was screened and isolated from Rhizopus nigricans. In this study R. nigricans was identified as a fungal species that produced plentiful gamma-linolenic acid. A 1,475 bp full-length cDNA, designated as RnD6D here, with high homology to fungal Delta6-fatty acid desaturase genes was isolated from R. nigricans using reverse transcription polymerase chain reaction and rapid amplification of cDNA ends methods. Sequence analysis indicated that this cDNA sequence had an open reading frame of 1,380 bp encoding a deduced polypeptide of 459 amino acids. Bioinformatics analysis characterized the putative RnD6D protein as a typical membrane-bound desaturase, including three conserved histidine-rich motifs, hydropathy profile and a cytochrome b5-like domain in the N-terminus. The corresponding genomic sequence of RnD6D was 1,689 bp with 4 introns, which was at least one intron more than other fungal Delta6-fatty acid desaturase genes. To elucidate the function of this novel putative desaturase, the coding sequence was expressed in Saccharomyces cerevisiae strain INVScl. A novel peak corresponding to gamma-linolenic acid methyl ester standards was detected with the same retention time, which was absent in the cell transformed with empty vector. The result demonstrated that the coding produced Delta6-fatty acid desaturase activity of RnD6D which led to the accumulation of gamma-linolenic acid. The functionally active RnD6D gene cloned here defined a novel Delta6-fatty acid desaturase from R. nigricans.

Cloning and Characterization of a Novel Aspartic Protease Gene from Marine-Derived Metschnikowia reukaufii and its Expression in E. coli

Metschnikowia reukaufii W6b isolated from marine environment was found to produce a cell-bound acid protease. The full-length cDNA (cDNASAP6 gene) of the acid protease (SAP6) from the marine-derived yeast M. reukaufii W6b was cloned. The insert was 1,755-bp long and contained an open reading frame of 1,527-bp encoding 508 amino acids. The deduced amino acid sequence included a signal peptide of 16 amino acids. The consensus motifs contained a VLLDTGSSDLRM active site and an ALLDSGTTITQF active site. The protein sequence deduced from the cDNASAP6 gene exhibited 12.9% overall identity with Cwp1 of Saccharomyces cerevisiae and a hydropathy profile characteristic of glycosylphosphatidylinositol cell-wall proteins. The cDNASAP6 gene without 48 bp encoding the signal peptide sequence was subcloned into an expression plasmid pET-24a (+) and fused with a 6-His Tag and transformed into Escherichia coli BL21 (DE3) for recombinant expression of the protease. The expressed fusion protein was found to have a unique band with molecular mass of about 54 kDa. The crude acid protease of the culture of the marine yeast strain W6b and the crude recombinant acid protease had milk clotting activity.