An ancient prevertebrate Na+-nucleoside cotransporter (hfCNT) from the Pacific hagfish (Eptatretus stouti)

2002 ◽  
Vol 283 (1) ◽  
pp. C155-C168 ◽  
Author(s):  
Sylvia Y. Yao ◽  
Amy M. Ng ◽  
Shaun K. Loewen ◽  
Carol E. Cass ◽  
Stephen A. Baldwin ◽  
...  
Keyword(s):  
Sequence Similarity ◽  
Mammalian Species ◽  
Functional Characterization ◽  
Structural Determinants ◽  
Pathogenic Yeast ◽  
Uridine Uptake ◽  
The Pacific ◽  
Selective System ◽  
Nucleoside Drugs ◽  
Carboxy Terminal

The human concentrative (Na+-linked) plasma membrane transport proteins hCNT1, hCNT2, and hCNT3 are pyrimidine nucleoside-selective (system cit), purine nucleoside-selective (system cif), or broadly selective for both pyrimidine and purine nucleosides (system cib), respectively. All have orthologs in other mammalian species and belong to a gene family (CNT) that has members in insects, nematodes, pathogenic yeast, and bacteria. Here, we report the cDNA cloning and functional characterization of a CNT family member from an ancient marine prevertebrate, the Pacific hagfish ( Eptatretus stouti). This Na+-nucleoside symporter, designated hfCNT, is the first transport protein to be characterized in detail in hagfish and is a 683-amino acid residue protein with 13 predicted transmembrane helical segments (TMs). hfCNT was 52, 50, and 57% identical in sequence to hCNT1, hCNT2, and hCNT3, respectively. Similarity to hCNT3 was particularly marked in the TM 4–13 region. When produced in Xenopus oocytes, hfCNT exhibited the transport properties of system cib, with uridine, thymidine, and inosine apparent K m values of 10–45 μM. The antiviral nucleoside drugs 3′-azido-3′-deoxythymidine, 2′,3′-dideoxycytidine, and 2′,3′-dideoxyinosine were also transported. Simultaneous measurement of uridine-evoked currents and radiolabeled uridine uptake under voltage-clamp conditions gave a Na+-to-uridine coupling ratio of 2:1 (cf. 2:1 for hCNT3 and 1:1 for hCNT1/2). The apparent K 50 value for Na+ activation was >100 mM. A 50:50 chimera between hfCNT and hCNT1 (TMs 7–13 of hfCNT replaced by those of hCNT1) exhibited hCNT1-like cation interactions, establishing that the structural determinants of cation stoichiometry and binding affinity were located within the carboxy-terminal half of the protein. The high degree of sequence similarity between hfCNT and hCNT3 may indicate functional constraints on the primary structure of the transporter and suggests that cib-type CNTs fulfill important physiological functions.

Mutational Analysis of Two Stefin A Epitopes

1999 ◽  
Vol 380 (6) ◽  
Author(s):  
N. Kopitar-Jerala ◽  
R. Jerala ◽  
B. Turk ◽  
F. Gubensek ◽  
V. Turk
Keyword(s):  
Monoclonal Antibodies ◽  
Tertiary Structure ◽  
Mutational Analysis ◽  
Sequence Similarity ◽  
Mammalian Species ◽  
Cross Reactivity ◽  
Binding Affinities ◽  
Amino Acid Residues ◽  
Stefin A ◽  
Carboxy Terminal

AbstractStefin A, an intracellular inhibitor of cysteine proteinases, is expressed most abundantly in epithelial cells and in cells of lymphatic origin. In order to study its role in normal and pathological conditions we have prepared and characterized monoclonal antibodies against recombinant stefin A. Two high affinity monoclonal antibodies (mAbs) (A22 and C52) were tested for binding to free and papain-complexed stefin A and to a chimeric inhibitor, consisting of 61 amino acid residues of stefin A and 37 carboxy-terminal residues of stefin B. mAb A22 recognized not only free stefin A but also stefin A in complex with papain. The mAbs were further tested for their cross-reactivity against stefin A and B isolated from different mammalian species. On the basis of sequence similarity and tertiary structure of human stefin A we have prepared three mutants–Glu33Lys, Asp61Gly and Asn62Tyr–and their reactivity with the mAbs was tested. The binding affinities of mAb A22 for the Asp61Gly and Asn62Tyr mutants were significantly lower, indicating that the two amino acids are part of the stefin A epitope recognized by A22. The binding of both mAbs to the mutants Gly4Arg and Gly4Glu was comparable to wild-type stefin A.

scholarly journals A Novel Rubi-Like Virus in the Pacific Electric Ray (Tetronarce californica) Reveals the Complex Evolutionary History of the Matonaviridae

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 585
Author(s):  
Rebecca M. Grimwood ◽  
Edward C. Holmes ◽  
Jemma L. Geoghegan
Keyword(s):  
Evolutionary History ◽  
Mammalian Species ◽  
Virus Transmission ◽  
Health Concern ◽  
Virus Species ◽  
Vast Number ◽  
The Pacific ◽  
Positive Sense ◽  
History Of ◽  
Fish Viruses

Rubella virus (RuV) is the causative agent of rubella (“German measles”) and remains a global health concern. Until recently, RuV was the only known member of the genus Rubivirus and the only virus species classified within the Matonaviridae family of positive-sense RNA viruses. Recently, two new rubella-like matonaviruses, Rustrela virus and Ruhugu virus, have been identified in several mammalian species, along with more divergent viruses in fish and reptiles. To screen for the presence of additional novel rubella-like viruses, we mined published transcriptome data using genome sequences from Rubella, Rustrela, and Ruhugu viruses as baits. From this, we identified a novel rubella-like virus in a transcriptome of Tetronarce californica—order Torpediniformes (Pacific electric ray)—that is more closely related to mammalian Rustrela virus than to the divergent fish matonavirus and indicative of a complex pattern of cross-species virus transmission. Analysis of host reads confirmed that the sample analysed was indeed from a Pacific electric ray, and two other viruses identified in this animal, from the Arenaviridae and Reoviridae, grouped with other fish viruses. These findings indicate that the evolutionary history of the Matonaviridae is more complex than previously thought and highlights the vast number of viruses that remain undiscovered.

The Drosophila Polycomb-group gene Enhancer of zeste contains a region with sequence similarity to trithorax

1993 ◽  
Vol 13 (10) ◽  
pp. 6357-6366
Author(s):  
R S Jones ◽  
W M Gelbart
Keyword(s):  
Amino Acids ◽  
Sequence Similarity ◽  
Protein Product ◽  
Polycomb Group ◽  
Acute Leukemias ◽  
Trithorax Group ◽  
Acid Protein ◽  
Enhancer Of Zeste ◽  
Gene Enhancer ◽  
Carboxy Terminal

As is typical of Polycomb-group loci, the Enhancer of zeste [E(z)] gene negatively regulates the segment identity genes of the Antennapedia (ANT-C) and Bithorax (BX-C) gene complexes. A second class of loci, collectively known as the trithorax group, plays an antagonistic role as positive regulators of the ANT-C and BX-C genes. Molecular analysis of the E(z) gene predicts a 760-amino-acid protein product. A region of 116 amino acids near the E(z) carboxy terminus is 41.2% identical (68.4% similar) with a carboxy-terminal region of the trithorax protein. This portion of the trithorax protein is part of a larger region previously shown to share extensive homology with a human protein (ALL-1/Hrx) that is implicated in acute leukemias. Over this same 116 amino acids, E(z) and ALL-1/Hrx are 43.9% identical (68.4% similar). Otherwise, E(z) is not significantly similar to any previously described proteins. As this region of sequence similarity is shared by two proteins with antagonistic functions, we suggest that it may comprise a domain that interacts with a common target, either nucleic acid or protein. Opposite effects on transcription might then be determined by other portions of the two proteins.

scholarly journals Identification of an Oligosaccharide Dehydrogenase from Pycnoporus Cinnabarinus Provides Insights into Fungal Breakdown of Lignocellulose

2021 ◽  
Author(s):  
Gabriele Cerutti ◽  
Elena Gugole ◽  
Linda Celeste Montemiglio ◽  
Annick Turbé-Doan ◽  
Dehbia Chena ◽  
...  
Keyword(s):  
Function Analysis ◽  
Sequence Similarity ◽  
Functional Characterization ◽  
Physiological Role ◽  
Free Form ◽  
Lignocellulose Degradation ◽  
Carbohydrate Active Enzymes ◽  
Recognition Mechanism ◽  
Pycnoporus Cinnabarinus ◽  
Enzymatic Function

Abstract Background: Fungal glucose dehydrogenases (GDHs) are FAD-dependent enzymes belonging to the glucose-methanol-choline oxidoreductase superfamily. These enzymes are classified in the “Auxiliary Activity” family 3 (AA3) of the Carbohydrate-Active enZymes database, and more specifically in subfamily AA3_2, that also includes the closely related flavoenzymes aryl-alcohol oxidase and glucose 1-oxidase. Based on sequence similarity to known fungal GDHs, an AA3_2 enzyme active on glucose was identified in the genome of Pycnoporus cinnabarinus, a model Basidiomycete able to completely degrade lignin.Results: In our work, substrate screening and functional characterization showed an unexpected preferential activity of this enzyme toward oligosaccharides containing a b(1à3) glycosidic bond, with the highest efficiency observed for the disaccharide laminaribiose. Despite its sequence similarity to GDHs, we defined a novel enzymatic activity, namely oligosaccharide dehydrogenase (ODH), for this enzyme. The crystallographic structures of ODH in the sugar-free form and in complex with glucose and laminaribiose unveiled a peculiar saccharide recognition mechanism which is not shared with previously characterized AA3 oxidoreductases and accounts for ODH preferential activity toward oligosaccharides. The sugar molecules in the active site of ODH are mainly stabilized through CH-p interactions with aromatic residues rather than through hydrogen bonds with highly conserved residues, as observed instead for the fungal glucose dehydrogenases and oxidases characterized to date. Finally, three sugar-binding sites were identified on ODH external surface, which were not previously observed and might be of importance in the physiological scenario.Conclusions: Structure-function analysis of ODH is consistent with its role as an auxiliary enzyme in lignocellulose degradation and unveils yet another enzymatic function within the AA3 family of the Carbohydrate-Active enZymes database. Our findings allow deciphering the molecular determinants of substrate binding and provide insight into the physiological role of ODH, opening new perspectives to exploit biodiversity for lignocellulose transformation into fuels and chemicals.

scholarly journals Solimonas marina sp. nov., isolated from deep seawater of the Pacific Ocean

2021 ◽  
Vol 71 (1) ◽  
Author(s):  
Xiupian Liu ◽  
Qiliang Lai ◽  
Fengqin Sun ◽  
Yaping Du ◽  
Yingbao Gai ◽  
...  
Keyword(s):  
Pacific Ocean ◽  
Type Species ◽  
Sequence Similarity ◽  
Rrna Gene ◽  
Chromosomal Dna ◽  
Deep Seawater ◽  
Content Type ◽  
Link Type ◽  
The Pacific ◽  
The Pacific Ocean

A taxonomic study was carried out on strain C16B3T, which was isolated from deep seawater of the Pacific Ocean. The bacterium was Gram-stain-negative, oxidase- and catalase- positive and rod-shaped. Growth was observed at salinities of 0–8.0 % and at temperatures of 10–45 °C. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain C16B3T belonged to the genus Solimonas , with the highest sequence similarity to Solimonas terrae KIS83-12T (97.2 %), followed by Solimonas variicoloris MN28T (97.0 %) and the other four species of the genus Solimonas (94.5 –96.8 %). The average nucleotide identity and estimated DNA–DNA hybridization values between strain C16B3T and the type strains of the genus Solimonas were 74.05−79.48 % and 19.5–22.5 %, respectively. The principal fatty acids (>5 %) were summed feature 8 (C18 : 1  ω7c/C18 : 1  ω6c; 20.9 %), iso-C16 : 0 (14.6 %), C16 : 1  ω5c (9.4 %), iso-C12 : 0 (8.4 %), summed feature 2 (C14 : 0 3-OH/iso I-C16 : 1 and C12 : 0 aldehyde; 6.8 %) and C16 : 0 (5.5 %). The G+C content of the chromosomal DNA was 65.37 mol%. The respiratory quinone was determined to be Q-8 (100 %). The polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, four unidentified aminolipids, six unidentified phospholipids and one unidentified polar lipid. The combined genotypic and phenotypic data show that strain C16B3T represents a novel species within the genus Solimonas , for which the name Solimonas marina sp. nov. is proposed, with the type strain C16B3T (=MCCC 1A04678T=KCTC 52314T).

Expression, phosphorylation and nuclear localization of the human P1 protein, a homologue of the yeast Mcm 3 replication protein

1995 ◽  
Vol 108 (4) ◽  
pp. 1381-1389 ◽  
Author(s):  
D. Schulte ◽  
R. Burkhart ◽  
C. Musahl ◽  
B. Hu ◽  
C. Schlatterer ◽  
...  
Keyword(s):  
Sequence Similarity ◽  
Protein A ◽  
Fold Increase ◽  
S Phase ◽  
Human Protein ◽  
Replication Protein ◽  
High Sequence Similarity ◽  
Central Regions ◽  
Mcm Proteins ◽  
Carboxy Terminal

The human protein P1 belongs to a newly discovered class of mammalian nuclear proteins with high sequence homology to yeast replication proteins. We present the entire amino acid sequence of the human protein P1 as predicted from the cDNA sequence, and show that P1 shares three central regions of high sequence similarity (about 75%) and a highly hydrophilic carboxy-terminal region with the yeast Mcm3 replication protein. The human genome most probably contains one P1 gene which is activated when HeLa cells progress to S phase, as shown by a several-fold increase in P1-specific mRNA. However, the amounts of P1 protein do not detectably change during this period, but P1 protein becomes phosphorylated at the beginning of S phase. In contrast to the yeast Mcm proteins, which disappear from nuclei after initiation of DNA replication, protein P1 remains in the nucleus during and after S phase. P1 is dispersed in mitotic cells and may be excluded from binding to chromosomes.

scholarly journals An NADH-Dependent Reductase from Eubacterium ramulus Catalyzes the Stereospecific Heteroring Cleavage of Flavanones and Flavanonols

2019 ◽  
Vol 85 (19) ◽  
Author(s):  
Annett Braune ◽  
Michael Gütschow ◽  
Michael Blaut
Keyword(s):  
Sequence Similarity ◽  
Bacterial Species ◽  
Functional Characterization ◽  
Intestinal Bacteria ◽  
Cyclic Ether ◽  
Content Type ◽  
Oxygen Sensitive ◽  
Key Enzyme ◽  
Catalyzed Reactions ◽  
Dietary Flavonoids

ABSTRACT The human intestinal anaerobe Eubacterium ramulus is known for its ability to degrade various dietary flavonoids. In the present study, we demonstrate the cleavage of the heterocyclic C-ring of flavanones and flavanonols by an oxygen-sensitive NADH-dependent reductase, previously described as enoate reductase, from E. ramulus. This flavanone- and flavanonol-cleaving reductase (Fcr) was purified following its heterologous expression in Escherichia coli and further characterized. Fcr cleaved the flavanones naringenin, eriodictyol, liquiritigenin, and homoeriodictyol. Moreover, the flavanonols taxifolin and dihydrokaempferol served as substrates. The catalyzed reactions were stereospecific for the (2R)-enantiomers of the flavanone substrates and for the (2S,3S)-configured flavanonols. The enantioenrichment of the nonconverted stereoisomers allowed for the determination of hitherto unknown flavanone racemization rates. Fcr formed the corresponding dihydrochalcones and hydroxydihydrochalcones in the course of an unusual reductive cleavage of cyclic ether bonds. Fcr did not convert members of other flavonoid subclasses, including flavones, flavonols, and chalcones, the latter indicating that the reaction does not involve a chalcone intermediate. This view is strongly supported by the observed enantiospecificity of Fcr. Cinnamic acids, which are typical substrates of bacterial enoate reductases, were also not reduced by Fcr. Based on the presence of binding motifs for dinucleotide cofactors and a 4Fe-4S cluster in the amino acid sequence of Fcr, a cofactor-mediated hydride transfer from NADH onto C-2 of the respective substrate is proposed. IMPORTANCE Gut bacteria play a crucial role in the metabolism of dietary flavonoids, thereby contributing to their activation or inactivation after ingestion by the human host. Thus, bacterial activities in the intestine may influence the beneficial health effects of these polyphenolic plant compounds. While an increasing number of flavonoid-converting gut bacterial species have been identified, knowledge of the responsible enzymes is still limited. Here, we characterized Fcr as a key enzyme involved in the conversion of flavonoids of several subclasses by Eubacterium ramulus, a prevalent human gut bacterium. Sequence similarity of this enzyme to hypothetical proteins from other flavonoid-degrading intestinal bacteria in databases suggests a more widespread occurrence of this enzyme. Functional characterization of gene products of human intestinal microbiota enables the assignment of metagenomic sequences to specific bacteria and, more importantly, to certain activities, which is a prerequisite for targeted modulation of gut microbial functionality.

Molecular cloning, chromosomal organization, and functional characterization of a sodium-dicarboxylate cotransporter from mouse kidney

2000 ◽  
Vol 279 (3) ◽  
pp. F482-F490 ◽  
Author(s):  
Ana M. Pajor ◽  
Nina N. Sun
Keyword(s):  
Sequence Similarity ◽  
Genomic Structure ◽  
Functional Characterization ◽  
Krebs Cycle ◽  
Initial Step ◽  
Xenopus Laevis Oocytes ◽  
Chromosome 11 ◽  
Inward Currents ◽  
Urinary Citrate

The sodium-dicarboxylate cotransporter of the renal proximal tubule, NaDC-1, reabsorbs filtered Krebs cycle intermediates and plays an important role in the regulation of urinary citrate concentrations.1 Low urinary citrate is a risk factor for the development of kidney stones. As an initial step in the characterization of NaDC-1 regulation, the genomic structure and functional properties of the mouse Na+-dicarboxylate cotransporter (mNaDC-1) were determined. The gene coding for mNaDC-1, Slc13a2, is found on chromosome 11. The gene is ∼24.9 kb in length and contains 12 exons. The mRNA coding for mNaDC-1 is found in kidney and small intestine. Expression of mNaDC-1 in Xenopus laevis oocytes results in increased transport of di- and tricarboxylates. The Michaelis-Menten constant ( K m) for succinate was 0.35 mM, and the K m for citrate was 0.6 mM. The transport of citrate was stimulated by acidic pH, whereas the transport of succinate was insensitive to pH changes. Transport by mNaDC-1 is electrogenic, and substrates produced inward currents in the presence of sodium. The sodium affinity was relatively high in mNaDC-1, with half-saturation constants for sodium of 10 mM (radiotracer experiments) and 28 mM at −50 mV (2-electrode voltage clamp experiments). Lithium acts as a potent inhibitor of transport, but it can also partially substitute for sodium. In conclusion, the mNaDC-1 is related in sequence and function to the other NaDC-1 orthologs. However, its function more closely resembles the rabbit and human orthologs rather than the rat NaDC-1, with which it shares higher sequence similarity.

scholarly journals An aphid effector promotes barley susceptibility through suppression of defence gene expression

2020 ◽  
Vol 71 (9) ◽  
pp. 2796-2807 ◽  
Author(s):  
Carmen Escudero-Martinez ◽  
Patricia A Rodriguez ◽  
Shan Liu ◽  
Pablo A Santos ◽  
Jennifer Stephens ◽  
...  
Keyword(s):  
Gene Expression ◽  
Sequence Similarity ◽  
Rhopalosiphum Padi ◽  
Functional Characterization ◽  
Aphid Species ◽  
Transgenic Barley ◽  
Defence Gene Expression ◽  
Hormone Signalling ◽  
Plant Susceptibility

Abstract Aphids secrete diverse repertoires of effectors into their hosts to promote the infestation process. While ‘omics’ approaches facilitated the identification and comparison of effector repertoires from a number of aphid species, the functional characterization of these proteins has been limited to dicot (model) plants. The bird cherry-oat aphid Rhopalosiphum padi is a pest of cereal crops, including barley. Here, we extend efforts to characterize aphid effectors with regard to their role in promoting susceptibility to the R. padi–barley interaction. We selected three R. padi effectors based on sequence similarity to previously characterized Myzus persicae effectors and assessed their subcellular localization, expression, and role in promoting plant susceptibility. Expression of R. padi effectors RpC002 and Rp1 in transgenic barley lines enhanced plant susceptibility to R. padi but not M. persicae, for which barley is a poor host. Characterization of Rp1 transgenic barley lines revealed reduced gene expression of plant hormone signalling genes relevant to plant–aphid interactions, indicating that this effector enhances susceptibility by suppressing plant defences in barley. Our data suggest that some aphid effectors specifically function when expressed in host species, and feature activities that benefit their corresponding aphid species.

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