Novel insights into the structure and transport mechanisms of TAPT1

AbstractTransmembrane anterior-posterior transformation protein 1 (TAPT1), encoded by the TAPT1 gene expressed in the basal ciliary body, plays a crucial role in cilia formation as well as axial skeletal patterning. Mutations in this gene have been reported to cause several ciliopathies and osteo-related diseases. Unfortunately, the cellular and molecular pathogenic mechanisms are still unclear also due to the lack of X-ray crystallographic structure and further characterization of TAPT1 protein. In this study, we attempted to characterize this protein by in silico techniques. A 3D structure of TAPT1 was generated by the ab initio method, which was further used for the analysis of the substrate-binding site, to determine pore size and for the prediction of the possible substrate(s). Validation by using different software packages revealed a reliable 3D model of TAPT1. Topology modeling revealed that TAPT1 has eight transmembrane helices with a total number of 27 helices in secondary structure. The amino acid residues H235, R323, K443, N446, S447, L450, K453, S454, Y457, K511, N513, D533, K535, D536, and T538 were found to form the pore surface as well as involved in the binding interaction with the substrate(s). This study predicted flavonoids as the possible substrate for TAPT1, which could further be confirmed by ingenuity pathway analysis. Moreover, our analysis indicated that TAPT1 might localize in the mitochondrial membrane in addition to the ciliary basal body. Our study gives novel insights for TAPT1 structure and its function.

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Cloning and Characterization of Lipase Gene from a Local Isolate of Pseudoxanthomonas sp.

Biosciences Biotechnology Research Asia ◽

10.13005/bbra/2471 ◽

2017 ◽

Vol 14 (2) ◽

pp. 503-507 ◽

Cited By ~ 1

Author(s):

Yogi Yopa Kristia ◽

Syifa F Syihab ◽

Akhmaloka Akhmaloka

Keyword(s):

Crystal Structure ◽

3D Structure ◽

Catalytic Triad ◽

Catalytic Residue ◽

Pseudomonas Sp ◽

Amino Acid Residues ◽

Chromosomal Dna ◽

Lipase Gene

ABSTRACT: Lipase gene from Pseudoxanthomonas sp. was cloned through in vitro amplification from total chromosomal DNA. The gene was sequenced and characterized, coding for 312 amino acid residues. Homological analysis showed that the gene has 98% similarity to lipolytic gene from Uncultured Pseudomonas sp (GenBank No. AKA58891.1). Further analysis appeared that the sequences showed similar unique motifs of lipase sub-family I.1, such as pentapeptide (GHSHG) motif, tetrapeptide (GMLG) motif, and catalytic triad. In additional, 3D structure analysis based on crystal structure of Pseudomonas aeruginose (PDB ID 1ex9) showed that both structure of lipases are similar except on the conformation of catalytic residue of His277 showing to shift more far away compared to that the control.

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Microstructural characterization of CoPtCr/Cr thin film disks by cross-section TEM and elongated probe micro-diffraction

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100088129 ◽

1991 ◽

Vol 49 ◽

pp. 762-763

Author(s):

M.A. Parker ◽

K.E. Johnson ◽

C. Hwang ◽

A. Bermea

Keyword(s):

Magnetic Recording ◽

Electron Probe ◽

Microstructural Characterization ◽

Crystallographic Structure ◽

Recording Media ◽

Layer By Layer ◽

Cross Sectional ◽

New Techniques ◽

Polished Side

We have reported the dependence of the magnetic and recording properties of CoPtCr recording media on the thickness of the Cr underlayer. It was inferred from XRD data that grain-to-grain epitaxy of the Cr with the CoPtCr was responsible for the interaction observed between these layers. However, no cross-sectional TEM (XTEM) work was performed to confirm this inference. In this paper, we report the application of new techniques for preparing XTEM specimens from actual magnetic recording disks, and for layer-by-layer micro-diffraction with an electron probe elongated parallel to the surface of the deposited structure which elucidate the effect of the crystallographic structure of the Cr on that of the CoPtCr.XTEM specimens were prepared from magnetic recording disks by modifying a technique used to prepare semiconductor specimens. After 3mm disks were prepared per the standard XTEM procedure, these disks were then lapped using a tripod polishing device. A grid with a single 1mmx2mm hole was then glued with M-bond 610 to the polished side of the disk.

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In Silico Prediction and Validation of Oxygen-Regulated Protein N-myc Downstream Regulated Gene 3 and Virtual Screening of Competitive Inhibitors of L-Lactate as Therapeutics

Letters in Drug Design & Discovery ◽

10.2174/1570180815666180816130621 ◽

2019 ◽

Vol 16 (6) ◽

pp. 637-644

Author(s):

Hongyu Cao ◽

Yanhua Wu ◽

Xingzhi Zhou ◽

Xuefang Zheng ◽

Ge Jiang

Keyword(s):

Active Sites ◽

Competitive Inhibition ◽

Hydrophobic Effect ◽

3D Structure ◽

Amino Acid Residues ◽

In Silico Prediction ◽

Hypoxic Response ◽

Drug Candidates ◽

Competitive Inhibitors ◽

Extracellular Regulated Protein Kinases

Background: N-myc downstream regulated gene 3 (NDRG3) is a newly discovered oxygen-regulated protein which will bind with L-Lactate in hypoxia and further activate Raf (rapidly accelerated fibrosarcoma)-ERK (extracellular regulated protein kinases) pathway, promoting cell growth and angiogenesis. Methods: Competitive inhibition on the binding of NDRG3 and L-Lactate may be potentially a useful strategy for the repression of hypoxic response mediated by NDRG3. The threedimensional (3D) structure of NDRG3 was built by using homology modeling for its crystal structure was not available. Then, L-Lactate was docked into NDRG3, from which we knew it bound with amino acid residues Gln69, His183, Asn189, Ala72 and Pro66 of NDRG3 in the most possible active sites. Approximately 3000 compounds have been virtually screened and the 6 topranked compounds were selected as reference molecules to analyze their interaction relationships, which illustrated that some of them might form electrostatic interaction with Glu70 and Asp187, π-&π stack with Phe75 and Tyr180, hydrogen bonds with Gly71 and Asn189, hydrophobic effect with Ala72 and Ile184. Results: Novel molecules were designed through structural optimization of the 6 top-ranked compounds and subsequently their ADMET properties were predicted. Conclusion: These molecules may be potential drug candidates for the suppression of hypoxic response mediated by NDRG3 and targeted therapy for hypoxia-induced diseases.

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Color-tuning of natural variants of heliorhodopsin

Scientific Reports ◽

10.1038/s41598-020-72125-0 ◽

2021 ◽

Vol 11 (1) ◽

Cited By ~ 1

Author(s):

Se-Hwan Kim ◽

Kimleng Chuon ◽

Shin-Gyu Cho ◽

Ahreum Choi ◽

Seanghun Meas ◽

...

Keyword(s):

3D Structure ◽

Site Directed Mutagenesis ◽

Type I ◽

Transmembrane Helices ◽

Color Tuning ◽

Tuning Mechanism ◽

Spectral Changes ◽

His Tag ◽

Natural Variants ◽

Retinal Chromophore

AbstractMicrobial rhodopsins are distributed through many microorganisms. Heliorhodopsins are newly discovered but have an unclear function. They have seven transmembrane helices similar to type-I and type-II rhodopsins, but they are different in that the N-terminal region of heliorhodopsin is cytoplasmic. We chose 13 representative heliorhodopsins from various microorganisms, expressed and purified with an N-terminal His tag, and measured the absorption spectra. The 13 natural variants had an absorption maximum (λmax) in the range 530–556 nm similar to proteorhodopsin (λmax = 490–525 nm). We selected several candidate residues that influence rhodopsin color-tuning based on sequence alignment and constructed mutants via site-directed mutagenesis to confirm the spectral changes. We found two important residues located near retinal chromophore that influence λmax. We also predict the 3D structure via homology-modeling of Thermoplasmatales heliorhodopsin. The results indicate that the color-tuning mechanism of type-I rhodopsin can be applied to understand the color-tuning of heliorhodopsin.

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Familial hemiplegic migraine type 2 due to a novel missense mutation in ATP1A2

The Journal of Headache and Pain ◽

10.1186/s10194-021-01221-x ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Fabio Antonaci ◽

Sabrina Ravaglia ◽

Gaetano S. Grieco ◽

Stella Gagliardi ◽

Cristina Cereda ◽

...

Keyword(s):

Case Reports ◽

3D Structure ◽

Familial Hemiplegic Migraine ◽

Transmembrane Helices ◽

Hemiplegic Migraine ◽

Case Presentation ◽

Italian Family ◽

Protein Prediction ◽

Functional Consequences

Abstract Background The mechanisms of genotype-phenotype interaction in Familiar Hemiplegic migraine type 2 (FHM2) are still far from clear. Different ATP1A2 mutations have been described, with a spectrum of phenotypes ranging from mild to severe. No genotype-phenotype correlations have been attempted. Case presentation We describe an Italian family with FHM and a missense ATP1A2 variant (L425H) not previously described. The clinical picture was mild in all the affected members. Conclusions Co-segregation of the variant with the aura phenotype was complete in this family, suggesting a 100% penetrance. In silico protein prediction softwares indicate that this variant may change the 3D structure of ATPA1A2 at the cytoplasmic loop between the two central transmembrane helices. Milder FHM phenotypes are rarely reported in literature, likely because case reports are biased towards the most severe phenotypes, with milder forms possibly misdiagnosed as sporadic migraine with aura forms (MAs), even with complex auras. Further studies taking into account intra-familiar variability and functional consequences on the channel protein may help clarify genotype-phenotype correlations.

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Identification and characterization of amphibian SLC26A5 using RNA-Seq

BMC Genomics ◽

10.1186/s12864-021-07798-6 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Zhongying Wang ◽

Qixuan Wang ◽

Hao Wu ◽

Zhiwu Huang

Keyword(s):

Amino Acid ◽

Inner Ear ◽

Hair Cells ◽

Rana Catesbeiana ◽

Site Directed Mutagenesis ◽

Amino Acid Residues ◽

Rna Seq ◽

American Bullfrog ◽

Frog Species

Abstract Background Prestin (SLC26A5) is responsible for acute sensitivity and frequency selectivity in the vertebrate auditory system. Limited knowledge of prestin is from experiments using site-directed mutagenesis or domain-swapping techniques after the amino acid residues were identified by comparing the sequence of prestin to those of its paralogs and orthologs. Frog prestin is the only representative in amphibian lineage and the studies of it were quite rare with only one species identified. Results Here we report a new coding sequence of SLC26A5 for a frog species, Rana catesbeiana (the American bullfrog). In our study, the SLC26A5 gene of Rana has been mapped, sequenced and cloned successively using RNA-Seq. We measured the nonlinear capacitance (NLC) of prestin both in the hair cells of Rana’s inner ear and HEK293T cells transfected with this new coding gene. HEK293T cells expressing Rana prestin showed electrophysiological features similar to that of hair cells from its inner ear. Comparative studies of zebrafish, chick, Rana and an ancient frog species showed that chick and zebrafish prestin lacked NLC. Ancient frog’s prestin was functionally different from Rana. Conclusions We mapped and sequenced the SLC26A5 of the Rana catesbeiana from its inner ear cDNA using RNA-Seq. The Rana SLC26A5 cDNA was 2292 bp long, encoding a polypeptide of 763 amino acid residues, with 40% identity to mammals. This new coding gene could encode a functionally active protein conferring NLC to both frog HCs and the mammalian cell line. While comparing to its orthologs, the amphibian prestin has been evolutionarily changing its function and becomes more advanced than avian and teleost prestin.

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Characterization of binding interaction of triclosan and bovine serum albumin

Journal of Environmental Science and Health Part A ◽

10.1080/10934529.2019.1694346 ◽

2019 ◽

Vol 55 (3) ◽

pp. 318-325

Author(s):

Xiaofang Wang ◽

Luyi Zou ◽

Chenyu Mi ◽

Hongyan Yu ◽

Mengxue Dong ◽

...

Keyword(s):

Bovine Serum Albumin ◽

Serum Albumin ◽

Bovine Serum ◽

Binding Interaction

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Molecular Cloning and Characterization of Bifidobacterium bifidum 1,2-α-l-Fucosidase (AfcA), a Novel Inverting Glycosidase (Glycoside Hydrolase Family 95)

Journal of Bacteriology ◽

10.1128/jb.186.15.4885-4893.2004 ◽

2004 ◽

Vol 186 (15) ◽

pp. 4885-4893 ◽

Cited By ~ 154

Author(s):

Takane Katayama ◽

Akiko Sakuma ◽

Takatoshi Kimura ◽

Yutaka Makimura ◽

Jun Hiratake ◽

...

Keyword(s):

Amino Acid ◽

Genomic Library ◽

Bifidobacterium Bifidum ◽

Glycoside Hydrolases ◽

Glycoside Hydrolase Family ◽

Amino Acid Residues ◽

Gene Encoding ◽

Sugar Chain ◽

Hydrolysis Of

ABSTRACT A genomic library of Bifidobacterium bifidum constructed in Escherichia coli was screened for the ability to hydrolyze the α-(1→2) linkage of 2′-fucosyllactose, and a gene encoding 1,2-α-l-fucosidase (AfcA) was isolated. The afcA gene was found to comprise 1,959 amino acid residues with a predicted molecular mass of 205 kDa and containing a signal peptide and a membrane anchor at the N and C termini, respectively. A domain responsible for fucosidase activity (the Fuc domain; amino acid residues 577 to 1474) was localized by deletion analysis and then purified as a hexahistidine-tagged protein. The recombinant Fuc domain specifically hydrolyzed the terminal α-(1→2)-fucosidic linkages of various oligosaccharides and a sugar chain of a glycoprotein. The stereochemical course of the hydrolysis of 2′-fucosyllactose was determined to be inversion by using 1H nuclear magnetic resonance. The primary structure of the Fuc domain exhibited no similarity to those of any glycoside hydrolases (GHs) but showed high similarity to those of several hypothetical proteins in a database. Thus, it was revealed that the AfcA protein constitutes a novel inverting GH family (GH family 95).

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The clpB gene of Bifidobacterium breve UCC 2003: transcriptional analysis and first insights into stress induction

Microbiology ◽

10.1099/mic.0.28176-0 ◽

2005 ◽

Vol 151 (9) ◽

pp. 2861-2872 ◽

Cited By ~ 33

Author(s):

Marco Ventura ◽

John G. Kenny ◽

Ziding Zhang ◽

Gerald F. Fitzgerald ◽

Douwe van Sinderen

Keyword(s):

3D Structure ◽

Binding Mode ◽

Dna Interaction ◽

Transcriptional Analysis ◽

Amino Acid Residues ◽

Mobility Shift ◽

Bifidobacterium Breve ◽

Gel Mobility Shift ◽

Slot Blot Analysis

The so-called clp genes, which encode components of the Clp proteolytic complex, are widespread among bacteria. The Bifidobacterium breve UCC 2003 genome contains a clpB gene with significant homology to predicted clpB genes from other members of the Actinobacteridae group. The heat- and osmotic-inducibility of the B. breve UCC 2003 clpB homologue was verified by slot-blot analysis, while Northern blot and primer extension analyses showed that the clpB gene is transcribed as a monocistronic unit with a single promoter. The role of a hspR homologue, known to control the regulation of clpB and dnaK gene expression in other high G+C content bacteria was investigated by gel mobility shift assays. Moreover the predicted 3D structure of HspR provides further insight into the binding mode of this protein to the clpB promoter region, and highlights the key amino acid residues believed to be involved in the protein–DNA interaction.

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Investigation of major amino acid residues of anti-norfloxacin monoclonal antibodies responsible for binding with fluoroquinolones

Scientific Reports ◽

10.1038/s41598-021-96466-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Patamalai Boonserm ◽

Songchan Puthong ◽

Thanaporn Wichai ◽

Sajee Noitang ◽

Pongsak Khunrae ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Amino Acid ◽

3D Structure ◽

Cross Reactivity ◽

Enzyme Linked Immunosorbent Assay ◽

Amino Acid Residues ◽

Variable Regions ◽

Complementarity Determining Regions ◽

Crucial Part ◽

Major Amino Acid

AbstractIt is important to understand the amino acid residues that govern the properties of the binding between antibodies and ligands. We studied the binding of two anti-norfloxacins, anti-nor 132 and anti-nor 155, and the fluoroquinolones norfloxacin, enrofloxacin, ciprofloxacin, and ofloxacin. Binding cross-reactivities tested by an indirect competitive enzyme-linked immunosorbent assay indicated that anti-nor 132 (22–100%) had a broader range of cross-reactivity than anti-nor 155 (62–100%). These cross-reactivities correlated with variations in the numbers of interacting amino acid residues and their positions. Molecular docking was employed to investigate the molecular interactions between the fluoroquinolones and the monoclonal antibodies. Homology models of the heavy chain and light chain variable regions of each mAb 3D structure were docked with the fluoroquinolones targeting the crucial part of the complementarity-determining regions. The fluoroquinolone binding site of anti-nor 155 was a region of the HCDR3 and LCDR3 loops in which hydrogen bonds were formed with TYR (H:35), ASN (H:101), LYS (H:106), ASN (L:92), and ASN (L:93). These regions were further away in anti-nor 132 and could not contact the fluoroquinolones. Another binding region consisting of HIS (L:38) and ASP (H:100) was found for norfloxacin, enrofloxacin, and ciprofloxacin, whereas only ASP (H:100) was found for ofloxacin.

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