scholarly journals Transmembrane 163 (TMEM163) protein effluxes zinc

2019 ◽  
Author(s):  
Vanessa B. Sanchez ◽  
Saima Ali ◽  
Adrian Escobar ◽  
Math P. Cuajungco
Keyword(s):  
Protein Function ◽  
Fluorescent Dyes ◽  
Phylogenetic Analyses ◽  
Site Directed Mutagenesis ◽  
Fluorescence Signal ◽  
Multiple Sequence ◽  
Protein Variant ◽  
Cellular Expression ◽  
Zinc Chelator ◽  
Zinc Levels

ABSTRACTRecent investigations of rodent Tmem163 suggest that it binds to and transports zinc as a dimer, and that alanine mutagenesis of its two species-conserved aspartate (D123A/D127A) residues proposed to bind zinc perturbs protein function. Direct corroboration, however, is lacking whether it is an influx or efflux transporter in cells. We hypothesized that human TMEM163 is a zinc effluxer based on its predicted protein characteristics. We used cultured human cell lines that either stably or transiently expressed TMEM163, and pre-loaded the cells with zinc to determine transport activity. We found that TMEM163-expressing cells exhibited significant reduction of intracellular zinc levels as evidenced by two zinc-specific fluorescent dyes and radionuclide zinc-65. The specificity of the fluorescence signal was confirmed upon treatment with TPEN, a high-affinity zinc chelator. Multiple sequence alignment and phylogenetic analyses showed that TMEM163 is related to distinct members of the cation diffusion facilitator (CDF) protein family. To further characterize the efflux function of TMEM163, we substituted alanine in two homologous aspartate residues (D124A/D128A) and performed site-directed mutagenesis of several conserved amino acid residues identified as non-synonymous single nucleotide polymorphism (S61R, S95C, S193P, and E286K). We found a significant reduction of zinc efflux upon cellular expression of D124A/D128A or E286K protein variant when compared with wild-type, suggesting that these particular amino acids are important for normal protein function. Taken together, our findings demonstrate that TMEM163 effluxes zinc and should now be designated ZNT11, as a new member of the mammalian CDF family of zinc efflux transporters.

scholarly journals Mitochondrial copper and phosphate transporter specificity was defined early in the evolution of eukaryotes

2020 ◽  
Author(s):  
Xinyu Zhu ◽  
Aren Boulet ◽  
Katherine M. Buckley ◽  
Casey B. Phillips ◽  
Micah G. Gammon ◽  
...  
Keyword(s):  
Protein Function ◽  
Phylogenetic Signal ◽  
Phylogenetic Analyses ◽  
Phosphate Transport ◽  
Site Directed Mutagenesis ◽  
Specific Gene ◽  
Ancestral State ◽  
Gene Duplications ◽  
Substrate Selection ◽  
Mitochondrial Carrier Family

AbstractMitochondrial carrier family (MCF/SLC25) proteins are selective transporters that maintain the mitochondrial metabolome. Here we combine computational, biochemical and phenotypic approaches to understand substrate selectivity of SLC25A3. In mammals, SLC25A3 transports both copper and phosphate, yet in Saccharomyces cerevisiae the transport of these substrates is partitioned across two paralogs: PIC2, which transports copper, and MIR1, which transports phosphate. To understand whether the ancestral state of this transporter was a single promiscuous transporter that duplicated and gained selectivity, we explored the evolutionary relationships of PIC2 and MIR1 orthologs across the eukaryotic tree of life. Phylogenetic analyses reveal that PIC2-like and MIR1-like orthologs are present in all major eukaryotic supergroups, indicating that the gene duplication that created these paralogs occurred early in eukaryotic evolution. Frequent lineage-specific gene duplications and losses suggest that substrate specificity may be evolutionarily labile. To link this phylogenetic signal to protein function and resolve the residues involved in substrate selection, we used structural modelling and site-directed mutagenesis to identify PIC2 residues involved in copper and phosphate transport activities. Based on these analyses, we generated a Leu175Ala variant of mouse SLC25A3 that retains the ability to transport copper, but not phosphate, and rescues the cytochrome c oxidase defect in SLC25A3 knockout cells. Taken together, this work uses an evolutionary framework to uncover amino acids involved in substrate recognition by MCF proteins responsible for copper and phosphate transport.

scholarly journals Mitochondrial copper and phosphate transporter specificity was defined early in the evolution of eukaryotes

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Xinyu Zhu ◽  
Aren Boulet ◽  
Katherine M Buckley ◽  
Casey B Phillips ◽  
Micah G Gammon ◽  
...  
Keyword(s):  
Protein Function ◽  
Phylogenetic Signal ◽  
Phylogenetic Analyses ◽  
Phosphate Transport ◽  
Site Directed Mutagenesis ◽  
Ancestral State ◽  
Mitochondrial Carrier ◽  
Family Protein ◽  
Mitochondrial Carrier Family ◽  
Transport In Mitochondria

The mitochondrial carrier family protein SLC25A3 transports both copper and phosphate in mammals yet in Saccharomyces cerevisiae the transport of these substrates is partitioned across two paralogs: PIC2 and MIR1. To understand the ancestral state of copper and phosphate transport in mitochondria, we explored the evolutionary relationships of PIC2 and MIR1 orthologs across the eukaryotic tree of life. Phylogenetic analyses revealed that PIC2-like and MIR1-like orthologs are present in all major eukaryotic supergroups, indicating an ancient gene duplication created these paralogs. To link this phylogenetic signal to protein function, we used structural modelling and site-directed mutagenesis to identify residues involved in copper and phosphate transport. Based on these analyses, we generated a L175A variant of mouse SLC25A3 that retains the ability to transport copper but not phosphate. This work highlights the utility of using an evolutionary framework to uncover amino acids involved in substrate recognition by mitochondrial carrier family proteins.

scholarly journals Respiratory syncytial virus B sequence analysis reveals a novel early genotype

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Juan C. Muñoz-Escalante ◽  
Andreu Comas-García ◽  
Sofía Bernal-Silva ◽  
Daniel E. Noyola
Keyword(s):  
Molecular Markers ◽  
Respiratory Syncytial Virus ◽  
Maximum Likelihood ◽  
Respiratory Infections ◽  
Phylogenetic Analyses ◽  
Detection Methods ◽  
Sequence Alignments ◽  
Multiple Sequence ◽  
Individual Gene ◽  
Syncytial Virus

AbstractRespiratory syncytial virus (RSV) is a major cause of respiratory infections and is classified in two main groups, RSV-A and RSV-B, with multiple genotypes within each of them. For RSV-B, more than 30 genotypes have been described, without consensus on their definition. The lack of genotype assignation criteria has a direct impact on viral evolution understanding, development of viral detection methods as well as vaccines design. Here we analyzed the totality of complete RSV-B G gene ectodomain sequences published in GenBank until September 2018 (n = 2190) including 478 complete genome sequences using maximum likelihood and Bayesian phylogenetic analyses, as well as intergenotypic and intragenotypic distance matrices, in order to generate a systematic genotype assignation. Individual RSV-B genes were also assessed using maximum likelihood phylogenetic analyses and multiple sequence alignments were used to identify molecular markers associated to specific genotypes. Analyses of the complete G gene ectodomain region, sequences clustering patterns, and the presence of molecular markers of each individual gene indicate that the 37 previously described genotypes can be classified into fifteen distinct genotypes: BA, BA-C, BA-CC, CB1-THB, GB1-GB4, GB6, JAB1-NZB2, SAB1, SAB2, SAB4, URU2 and a novel early circulating genotype characterized in the present study and designated GB0.

scholarly journals Visualizing monolayers with a water-soluble fluorophore to quantify adsorption, desorption, and the double layer

2015 ◽  
Vol 112 (8) ◽  
pp. E826-E835 ◽  
Author(s):  
Ian C. Shieh ◽  
Joseph A. Zasadzinski
Keyword(s):  
Double Layer ◽  
Fluorescent Dyes ◽  
Selective Adsorption ◽  
Dye Adsorption ◽  
Immiscible Liquid ◽  
Water Soluble ◽  
Fluorescence Signal ◽  
Anionic Phospholipid ◽  
Gas Phases ◽  
Disordered Phase

Contrast in confocal microscopy of phase-separated monolayers at the air–water interface can be generated by the selective adsorption of water-soluble fluorescent dyes to disordered monolayer phases. Optical sectioning minimizes the fluorescence signal from the subphase, whereas convolution of the measured point spread function with a simple box model of the interface provides quantitative assessment of the excess dye concentration associated with the monolayer. Coexisting liquid-expanded, liquid-condensed, and gas phases could be visualized due to differential dye adsorption in the liquid-expanded and gas phases. Dye preferentially adsorbed to the liquid-disordered phase during immiscible liquid–liquid phase coexistence, and the contrast persisted through the critical point as shown by characteristic circle-to-stripe shape transitions. The measured dye concentration in the disordered phase depended on the phase composition and surface pressure, and the dye was expelled from the film at the end of coexistence. The excess concentration of a cationic dye within the double layer adjacent to an anionic phospholipid monolayer was quantified as a function of subphase ionic strength, and the changes in measured excess agreed with those predicted by the mean-field Gouy–Chapman equations. This provided a rapid and noninvasive optical method of measuring the fractional dissociation of lipid headgroups and the monolayer surface potential.

Morphological redescription and molecular characterization of Trichodina matsu Basson & Van As, 1994 (Ciliophora, Mobilida, Trichodinidae) infecting Tachysurus fulvidraco (Richardson, 1846) from Chongqing, China

Zootaxa ◽  
2021 ◽  
Vol 4995 (2) ◽  
pp. 334-344
Author(s):  
QIAN ZHOU ◽  
FAHUI TANG ◽  
YUANJUN ZHAO
Keyword(s):  
Phylogenetic Analyses ◽  
Molecular Data ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Similar Species ◽  
Sequence Alignments ◽  
Multiple Sequence ◽  
Multiple Sequence Alignments ◽  
Morphological And Molecular Data ◽  
Tachysurus Fulvidraco

During a survey of parasitic ciliates in Chongqing, China, Trichodina matsu Basson & Van As, 1994 was isolated from gills of Tachysurus fulvidraco. Furthermore, the 18S rRNA gene and ITS-5.8S rRNA region of T. matsu were sequenced for the first time and applied for the species identification and comparison with similar species in the present study. Based on the morphological and molecular comparisons, the results indicate that T. matsu is an ectoparasite specific for the Siluriformes catfish. Based on the analyses of genetic distance, multiple sequence alignments, and phylogenetic analyses, no obvious differentiation within populations of T. matsu was found. In addition, the ‘Trichodina hyperparasitis’ (KX904933) in GenBank is a misidentification and appears to be conspecific with T. matsu according to the comparison of morphological and molecular data.  

scholarly journals Embeddings from protein language models predict conservation and variant effects

2021 ◽  
Author(s):  
Céline Marquet ◽  
Michael Heinzinger ◽  
Tobias Olenyi ◽  
Christian Dallago ◽  
Michael Bernhofer ◽  
...  
Keyword(s):  
Protein Function ◽  
Language Models ◽  
Single Amino Acid ◽  
Sequence Alignments ◽  
Multiple Sequence ◽  
Multiple Sequence Alignments ◽  
Human Proteins ◽  
Entire Sequence ◽  
Embedding Methods ◽  
Better Than

Abstract The emergence of SARS-CoV-2 variants stressed the demand for tools allowing to interpret the effect of single amino acid variants (SAVs) on protein function. While Deep Mutational Scanning (DMS) sets continue to expand our understanding of the mutational landscape of single proteins, the results continue to challenge analyses. Protein Language Models (LMs) use the latest deep learning (DL) algorithms to leverage growing databases of protein sequences. These methods learn to predict missing or marked amino acids from the context of entire sequence regions. Here, we explored how to benefit from learned protein LM representations (embeddings) to predict SAV effects. Although we have failed so far to predict SAV effects directly from embeddings, this input alone predicted residue conservation almost as accurately from single sequences as using multiple sequence alignments (MSAs) with a two-state per-residue accuracy (conserved/not) of Q2=80% (embeddings) vs. 81% (ConSeq). Considering all SAVs at all residue positions predicted as conserved to affect function reached 68.6% (Q2: effect/neutral; for PMD) without optimization, compared to an expert solution such as SNAP2 (Q2=69.8). Combining predicted conservation with BLOSUM62 to obtain variant-specific binary predictions, DMS experiments of four human proteins were predicted better than by SNAP2, and better than by applying the same simplistic approach to conservation taken from ConSeq. Thus, embedding methods have become competitive with methods relying on MSAs for SAV effect prediction at a fraction of the costs in computing/energy. This allowed prediction of SAV effects for the entire human proteome (~20k proteins) within 17 minutes on a single GPU.

scholarly journals Embeddings from protein language models predict conservation and variant effects

2021 ◽  
Author(s):  
Céline Marquet ◽  
Michael Heinzinger ◽  
Tobias Olenyi ◽  
Christian Dallago ◽  
Kyra Erckert ◽  
...  
Keyword(s):  
Protein Function ◽  
Pearson Correlation ◽  
Performance Measure ◽  
Language Models ◽  
Single Amino Acid ◽  
Data Sets ◽  
Sequence Alignments ◽  
Multiple Sequence ◽  
Multiple Sequence Alignments ◽  
Human Proteins

AbstractThe emergence of SARS-CoV-2 variants stressed the demand for tools allowing to interpret the effect of single amino acid variants (SAVs) on protein function. While Deep Mutational Scanning (DMS) sets continue to expand our understanding of the mutational landscape of single proteins, the results continue to challenge analyses. Protein Language Models (pLMs) use the latest deep learning (DL) algorithms to leverage growing databases of protein sequences. These methods learn to predict missing or masked amino acids from the context of entire sequence regions. Here, we used pLM representations (embeddings) to predict sequence conservation and SAV effects without multiple sequence alignments (MSAs). Embeddings alone predicted residue conservation almost as accurately from single sequences as ConSeq using MSAs (two-state Matthews Correlation Coefficient—MCC—for ProtT5 embeddings of 0.596 ± 0.006 vs. 0.608 ± 0.006 for ConSeq). Inputting the conservation prediction along with BLOSUM62 substitution scores and pLM mask reconstruction probabilities into a simplistic logistic regression (LR) ensemble for Variant Effect Score Prediction without Alignments (VESPA) predicted SAV effect magnitude without any optimization on DMS data. Comparing predictions for a standard set of 39 DMS experiments to other methods (incl. ESM-1v, DeepSequence, and GEMME) revealed our approach as competitive with the state-of-the-art (SOTA) methods using MSA input. No method outperformed all others, neither consistently nor statistically significantly, independently of the performance measure applied (Spearman and Pearson correlation). Finally, we investigated binary effect predictions on DMS experiments for four human proteins. Overall, embedding-based methods have become competitive with methods relying on MSAs for SAV effect prediction at a fraction of the costs in computing/energy. Our method predicted SAV effects for the entire human proteome (~ 20 k proteins) within 40 min on one Nvidia Quadro RTX 8000. All methods and data sets are freely available for local and online execution through bioembeddings.com, https://github.com/Rostlab/VESPA, and PredictProtein.

Fluorescence signaling molecularly imprinted polymers for antibiotics prepared via site-directed post-imprinting introduction of plural fluorescent reporters within the recognition cavity

2016 ◽  
Vol 4 (44) ◽  
pp. 7138-7145 ◽  
Author(s):  
Hirobumi Sunayama ◽  
Takeo Ohta ◽  
Atsushi Kuwahara ◽  
Toshifumi Takeuchi
Keyword(s):  
Molecular Imprinting ◽  
Molecularly Imprinted Polymers ◽  
Fluorescent Dyes ◽  
Specific Binding ◽  
Fluorescence Signal ◽  
Molecularly Imprinted ◽  
Imprinted Polymers ◽  
Fluorescent Reporters ◽  
Binding Event ◽  
Fluorescence Signaling

An antibiotic-imprinted cavity with two different fluorescent dyes was prepared by molecular imprinting and subsequent post-imprinting modifications (PIMs), for the readout of a specific binding event as a fluorescence signal.

A Label-Free Fluorescent AND Logic Gate Aptasensor for Carbohydrate Antigen 15-3 Detection Based on Graphene Oxide

2021 ◽  
Vol 24 ◽  
Author(s):  
Wenxiao Hu ◽  
Yafei Dong ◽  
Luhui Wang ◽  
Yue Wang ◽  
Mengyao Qian ◽  
...  
Keyword(s):  
Fluorescent Dyes ◽  
Logic Gate ◽  
High Sensitivity ◽  
Carbohydrate Antigen ◽  
Fluorescence Signal ◽  
Label Free ◽  
Molecular Logic ◽  
Long Stem ◽  
Middle Ring ◽  
Fluorescent Aptasensor

Background: Molecular logic gate always used fluorescent dyes to realize fluorescence signal. The labeling of the fluorophore is relatively expensive, low yield and singly labeled impuritiesaffects the affinity between the target and the aptamer. Label-free fluorescent aptamer biosensor strategy has attracted widespread interest due to lower cost and simple. Objective: Herein, we have designed a AND logic gate fluorescent aptasensor for detecting carbohydrate antigen 15-3(CA15-3) based on label-free fluorescence signal output. Materials and Methods: A hairpin DNA probe consists of CA15-3 aptamer and partly anti-CA15-3 aptamer sequences as a long stem and G-rich sequences of the middle ring as a quadruplex-forming oligomer. G-rich sequences can fold into a quadruplex by K+, and then G-quadruplex interacts specifically with N-methylmesoporphyrin IX(NMM), leading to a dramatic increase in fluorescence of NMM. With CA15-3 and NMM as the two inputs, the fluorescence intensity of the NMM is the output signal. Lacking of CA15-3 or NMM, there is no significant fluorescence enhancing, and the output of the signal is “0”. The fluorescence signal was dramatically increasing and the output of the signal is “1” only when CA15-3 protein and NMM were added at the same time. Results: This biosensor strategy possessed selectivity, high sensitivity for detecting CA15-3 protein from 10 to 500 U mL-1 and the detection limit was 10 U mL-1, and also showed good reproducibility in spiked human serum. Conclusion: In summary, the proposed AND logic gate fluorescent aptasensor could specifically detect CA15-3.

LARMD: integration of bioinformatic resources to profile ligand-driven protein dynamics with a case on the activation of estrogen receptor

2019 ◽  
Vol 21 (6) ◽  
pp. 2206-2218 ◽  
Author(s):  
Jing-Fang Yang ◽  
Fan Wang ◽  
Yu-Zong Chen ◽  
Ge-Fei Hao ◽  
Guang-Fu Yang
Keyword(s):  
Estrogen Receptor ◽  
Protein Dynamics ◽  
Protein Function ◽  
Inflammatory Diseases ◽  
Great Difficulty ◽  
Vital Role ◽  
Site Directed Mutagenesis ◽  
Bioinformatic Tools ◽  
Wide Range ◽  
User Friendly

Abstract Protein dynamics is central to all biological processes, including signal transduction, cellular regulation and biological catalysis. Among them, in-depth exploration of ligand-driven protein dynamics contributes to an optimal understanding of protein function, which is particularly relevant to drug discovery. Hence, a wide range of computational tools have been designed to investigate the important dynamic information in proteins. However, performing and analyzing protein dynamics is still challenging due to the complicated operation steps, giving rise to great difficulty, especially for nonexperts. Moreover, there is a lack of web protocol to provide online facility to investigate and visualize ligand-driven protein dynamics. To this end, in this study, we integrated several bioinformatic tools to develop a protocol, named Ligand and Receptor Molecular Dynamics (LARMD, http://chemyang.ccnu.edu.cn/ccb/server/LARMD/ and http://agroda.gzu.edu.cn:9999/ccb/server/LARMD/), for profiling ligand-driven protein dynamics. To be specific, estrogen receptor (ER) was used as a case to reveal ERβ-selective mechanism, which plays a vital role in the treatment of inflammatory diseases and many types of cancers in clinical practice. Two different residues (Ile373/Met421 and Met336/Leu384) in the pocket of ERβ/ERα were the significant determinants for selectivity, especially Met336 of ERβ. The helix H8, helix H11 and H7-H8 loop influenced the migration of selective agonist (WAY-244). These computational results were consistent with the experimental results. Therefore, LARMD provides a user-friendly online protocol to study the dynamic property of protein and to design new ligand or site-directed mutagenesis.

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