Modèle topologique de la structure d’un antiport vacuolaire de type NHX chez la vigne cultivée (Vitis vinifera)

Botany ◽  
2009 ◽  
Vol 87 (3) ◽  
pp. 339-347 ◽  
Author(s):  
Mohsen Hanana ◽  
Olivier Cagnac ◽  
Ahmed Mliki ◽  
Eduardo Blumwald
Keyword(s):  
Amino Acid ◽  
Vitis Vinifera ◽  
Molecular Mass ◽  
Electrostatic Interactions ◽  
Functional Characterization ◽  
Terminal Region ◽  
Vitis Vinifera L ◽  
Amino Acid Residues ◽  
Transmembrane Segments ◽  
Α Helix

After identifying and isolating a grapevine ( Vitis vinifera L.) NHX vacuolar antiporter and before initializing functional genomic studies, we juged necessary to acquire a minimum of knowledge about the VvNHX1 protein. Thus, we realized a bioinformatic analysis to determine its basic characteristics and to get structural informations that could guide us through the functional characterization. We have determined important physico-chemical parameters (molecular mass, isoelectric point, hydrophobic regions, etc.) and obtained interesting structural data (primary, secondary, and tertiary structures; conserved domains and interaction motives; etc.). The VvNHX1 gene, which encodes this 541 amino-acid protein with a predicted molecular mass of 60 kDa, is made of 14 exons and measures 6.5 kb. The amino-acidic composition of this protein is very important, in particular, for the establishment of the α-helix structure, which represents more than 50% of the protein, but also for charge distribution, which generates critical electrostatic interactions for the ionic flux. The secondary structure of VvNHX1 contains multiple transmembrane α-helix segments that are made of hydrophobic amino-acid residues, thus facilitating its insertion in the membrane. Globally, VvNHX1 has one hydrophobic N-terminal region, made of 10 transmembrane segments with 440 amino-acid residues, and one hydrophilic C-terminal region, made of 100 residues. The region located between the fourth and fifth transmembrane segments represents, with its structure mainly helicoidal and the presence of a favourable electrostatic environment, the pore where cation flux is performed across the membrane. VvNHX1 contains various interaction domains as well as several putative posttranslational modification sites, mainly at the C-terminus but also at the N-terminus, that play an important part in regulating protein activities, influence protein structural stability, or interact with other proteins or signalling molecules.

Peroxisome targeting signal of rat liver acyl-coenzyme A oxidase resides at the carboxy terminus

1989 ◽  
Vol 9 (1) ◽  
pp. 83-91
Author(s):  
S Miyazawa ◽  
T Osumi ◽  
T Hashimoto ◽  
K Ohno ◽  
S Miura ◽  
...  
Keyword(s):  
Amino Acid ◽  
Rat Liver ◽  
Coenzyme A ◽  
Terminal Region ◽  
Proteinase K ◽  
Amino Acid Residues ◽  
C Terminus ◽  
Targeting Signal ◽  
Acyl Coenzyme A

To identify the topogenic signal of peroxisomal acyl-coenzyme A oxidase (AOX) of rat liver, we carried out in vitro import experiments with mutant polypeptides of the enzyme. Full-length AOX and polypeptides that were truncated at the N-terminal region were efficiently imported into peroxisomes, as determined by resistance to externally added proteinase K. Polypeptides carrying internal deletions in the C-terminal region exhibited much lower import activities. Polypeptides that were truncated or mutated at the extreme C terminus were totally import negative. When the five amino acid residues at the extreme C terminus were attached to some of the import-negative polypeptides, the import activities were rescued. Moreover, the C-terminal 199 and 70 amino acid residues of AOX directed fusion proteins with two bacterial enzymes to peroxisomes. These results are interpreted to mean that the peroxisome targeting signal of AOX residues at the C terminus and the five or fewer residues at the extreme terminus have an obligatory function in targeting. The C-terminal internal region also has an important role for efficient import, possibly through a conformational effect.

scholarly journals A Strain of Bacillus thuringiensis Containing a Novel cry7Aa2 Gene that Is Toxic to Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae)

Insects ◽  
2019 ◽  
Vol 10 (9) ◽  
pp. 259 ◽  
Author(s):  
Mikel Domínguez-Arrizabalaga ◽  
Maite Villanueva ◽  
Ana Beatriz Fernandez ◽  
Primitivo Caballero
Keyword(s):  
Amino Acid ◽  
Bacillus Thuringiensis ◽  
Molecular Mass ◽  
Leptinotarsa Decemlineata ◽  
Insect Pests ◽  
Amino Acid Residues ◽  
Cry Protein ◽  
Lc50 Value ◽  
Leptinotarsa Decemlineata Say

The genome of the Bacillus thuringiensis BM311.1 strain was sequenced and assembled in 359 contigs containing a total of 6,390,221 bp. The plasmidic ORF of a putative cry gene from this strain was identified as a potential novel Cry protein of 1138 amino acid residues with a 98% identity compared to Cry7Aa1 and a predicted molecular mass of 129.4 kDa. The primary structure of Cry7Aa2, which had eight conserved blocks and the classical structure of three domains, differed in 28 amino acid residues from that of Cry7Aa1. The cry7Aa2 gene was amplified by PCR and then expressed in the acrystalliferous strain BMB171. SDS-PAGE analysis confirmed the predicted molecular mass for the Cry7Aa2 protein and revealed that after in vitro trypsin incubation, the protein was degraded to a toxin of 62 kDa. However, when treated with digestive fluids from Leptinotarsa decemlineata larvae, one major proteinase-resistant fragment of slightly smaller size was produced. The spore and crystal mixture produced by the wild-type BM311.1 strain against L. decemlineata neonate larvae resulted in a LC50 value of 18.8 μg/mL, which was statistically similar to the estimated LC50 of 20.8 μg/mL for the recombinant BMB17-Cry7Aa2 strain. In addition, when this novel toxin was activated in vitro with commercial trypsin, the LC50 value was reduced 3.8-fold to LC50 = 4.9 μg/mL. The potential advantages of Cry7Aa2 protoxin compared to Cry7Aa1 protoxin when used in the control of insect pests are discussed.

scholarly journals SLC6A14, a Pivotal Actor on Cancer Stage: When Function Meets Structure

2019 ◽  
Vol 24 (9) ◽  
pp. 928-938 ◽  
Author(s):  
Luca Palazzolo ◽  
Chiara Paravicini ◽  
Tommaso Laurenzi ◽  
Sara Adobati ◽  
Simona Saporiti ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Amino Acids ◽  
Amino Acid ◽  
Molecular Dynamics Simulations ◽  
Electrostatic Interactions ◽  
Amino Acid Residues ◽  
Dibasic Amino Acid ◽  
Novel Target ◽  
Function Relationship ◽  
Dynamics Simulations

SLC6A14 (ATB0,+) is a sodium- and chloride-dependent neutral and dibasic amino acid transporter that regulates the distribution of amino acids across cell membranes. The transporter is overexpressed in many human cancers characterized by an increased demand for amino acids; as such, it was recently acknowledged as a novel target for cancer therapy. The knowledge on the molecular mechanism of SLC6A14 transport is still limited, but some elegant studies on related transporters report the involvement of the 12 transmembrane α-helices in the transport mechanism, and describe structural rearrangements mediated by electrostatic interactions with some pivotal gating residues. In the present work, we constructed a SLC6A14 model in outward-facing conformation via homology modeling and used molecular dynamics simulations to predict amino acid residues critical for substrate recognition and translocation. We docked the proteinogenic amino acids and other known substrates in the SLC6A14 binding site to study both gating regions and the exposed residues involved in transport. Interestingly, some of these residues correspond to those previously identified in other LeuT-fold transporters; however, we could also identify a novel relevant residue with such function. For the first time, by combined approaches of molecular docking and molecular dynamics simulations, we highlight the potential role of these residues in neutral amino acid transport. This novel information unravels new aspects of the human SLC6A14 structure–function relationship and may have important outcomes for cancer treatment through the design of novel inhibitors of SLC6A14-mediated transport.

scholarly journals Genome-Wide Identification of PIFs in Grapes (Vitis vinifera L.) and Their Transcriptional Analysis under Lighting/Shading Conditions

Genes ◽  
2018 ◽  
Vol 9 (9) ◽  
pp. 451 ◽  
Author(s):  
Kekun Zhang ◽  
Ting Zheng ◽  
Xudong Zhu ◽  
Songtao Jiu ◽  
Zhongjie Liu ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Amino Acid ◽  
Vitis Vinifera ◽  
Developmental Stages ◽  
Light Signal ◽  
Transcriptional Analysis ◽  
Vitis Vinifera L ◽  
Syntenic Block ◽  
Transcription Level ◽  
Light Signal Transduction

Phytochrome-interacting factors (PIFs), as the basic helix–loop–helix (bHLH) transcription factors, are the primary signaling partners for phytochromes (PHY) that play a key role in PHY-mediated light signal transduction. At present, there are few studies on PIFs in fruit trees. In order to clarify the status of PIFs in grapevines, we identified members of the grape PIFs family and conducted phylogenetic and expression analysis. We identified PIF1, PIF3, PIF4, and PIF7 in PIFs families of the grapevine (Vitis vinifera L.), which were distributed on four different chromosomes with similar gene structures. Except for the closer relationship with PIF1 of citrus, PIFs of grape were distant from the other fruit species such as apple, pear, peach, and strawberry. The VvPIFs (except VvPIF4) were located in the syntenic block with those from Arabidopsis thaliana, Solanum lycopersicum, or Citrus sinensis. In addition to PIF1, all PIFs in grapevines have conserved active PHYB binding (APB) sequences. VvPIF1 has a conserved PIF1-specific active PHYA binding (APA) sequence, while amino acid mutations occurred in the specific APA sequence in VvPIF3. Interestingly, two specific motifs were found in the PIF4 amino acid sequence. The photoreceptor-related elements in the VvPIFs promoter region were the most abundant. PIF1, LONG HYPOCOTYL 5 (HY5) and PIF3, PIF4, GIBBERELLIC ACID INSENSITIVE 1 (GAI1) may interact with each other and participate together in light signal transduction. The relative expression levels of the VvPIFs showed diverse patterns in the various organs at different developmental stages, of which PIF4 was most highly expressed. Prior to maturation, the expression of PIF4 and PIF7 in the skin of the different cultivars increased, while the expression of all PIFs in the flesh decreased. The transcription level of PIFs in grape leaves was sensitive to changes in lighting and shading. Shading treatment was beneficial for enhancing the transcription level of VvPIFs, but the effect on VvPIF3 and VvPIF4 was time-controlled. We concluded that PIFs in grapevines are both conservative and species-specific. The identification and analysis of grape PIFs could provide a theoretical foundation for the further construction of grape light regulation networks.

scholarly journals Noncharged amino acid residues at the solvent-exposed positions in the middle and at the C terminus of the α-helix have the same helical propensity

2003 ◽  
Vol 12 (6) ◽  
pp. 1169-1176 ◽  
Author(s):  
Dmitri N. Ermolenko ◽  
John M. Richardson ◽  
George I. Makhatadze
Keyword(s):  
Amino Acid ◽  
Amino Acid Residues ◽  
C Terminus ◽  
Α Helix

scholarly journals Concerted Effects of Amino Acid Substitutions in Conserved Charged Residues and Other Residues in the Cytoplasmic Domain of PomA, a Stator Component of Na+-Driven Flagella

2004 ◽  
Vol 186 (20) ◽  
pp. 6749-6758 ◽  
Author(s):  
Hajime Fukuoka ◽  
Toshiharu Yakushi ◽  
Michio Homma
Keyword(s):  
Amino Acid ◽  
Temperature Shift ◽  
Terminal Region ◽  
Temperature Sensitive ◽  
Amino Acid Substitutions ◽  
Flagellar Motor ◽  
Transmembrane Segments ◽  
Suppressor Mutations ◽  
Essential Components ◽  
Charged Residues

ABSTRACT PomA is a membrane protein that is one of the essential components of the sodium-driven flagellar motor in Vibrio species. The cytoplasmic charged residues of Escherichia coli MotA, which is a PomA homolog, are believed to be required for the interaction of MotA with the C-terminal region of FliG. It was previously shown that a PomA variant with neutral substitutions in the conserved charged residues (R88A, K89A, E96Q, E97Q, and E99Q; AAQQQ) was functional. In the present study, five other conserved charged residues were replaced with neutral amino acids in the AAQQQ PomA protein. These additional substitutions did not affect the function of PomA. However, strains expressing the AAQQQ PomA variant with either an L131F or a T132M substitution, neither of which affected motor function alone, exhibited a temperature-sensitive (TS) motility phenotype. The double substitutions R88A or E96Q together with L131F were sufficient for the TS phenotype. The motility of the PomA TS mutants immediately ceased upon a temperature shift from 20 to 42°C and was restored to the original level approximately 10 min after the temperature was returned to 20°C. It is believed that PomA forms a channel complex with PomB. The complex formation of TS PomA and PomB did not seem to be affected by temperature. Suppressor mutations of the TS phenotype were mapped in the cytoplasmic boundaries of the transmembrane segments of PomA. We suggest that the cytoplasmic surface of PomA is changed by the amino acid substitutions and that the interaction of this surface with the FliG C-terminal region is temperature sensitive.

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