Functional analysis of the human concentrative nucleoside transporter-1 variant hCNT1S546P provides insight into the sodium-binding pocket

2012 ◽  
Vol 302 (1) ◽  
pp. C257-C266 ◽  
Author(s):  
Pedro Cano-Soldado ◽  
Edurne Gorraitz ◽  
Ekaitz Errasti-Murugarren ◽  
F. Javier Casado ◽  
M. Pilar Lostao ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Hill Coefficient ◽  
Thymidine Uptake ◽  
Nucleoside Transporter ◽  
Nucleotide Polymorphisms ◽  
Loss Of Function ◽  
Serine Residue ◽  
Concentrative Nucleoside Transporter

SLC28 genes, encoding concentrative nucleoside transporter proteins (CNT), show little genetic variability, although a few single nucleotide polymorphisms (SNPs) have been associated with marked functional disturbances. In particular, human CNT1S546P had been reported to result in negligible thymidine uptake. In this study we have characterized the molecular mechanisms responsible for this apparent loss of function. The hCNT1S546P variant showed an appropriate endoplasmic reticulum export and insertion into the plasma membrane, whereas loss of nucleoside translocation ability affected all tested nucleoside and nucleoside-derived drugs. Site-directed mutagenesis analysis revealed that it is the lack of the serine residue itself responsible for the loss of hCNT1 function. This serine residue is highly conserved, and mutation of the analogous serine in hCNT2 (Ser541) and hCNT3 (Ser568) resulted in total and partial loss of function, respectively. Moreover, hCNT3, the only member that shows a 2Na+/1 nucleoside stoichiometry, showed altered Na+ binding properties associated with a shift in the Hill coefficient, consistent with one Na+ binding site being affected by the mutation. Two-electrode voltage-clamp studies using the hCNT1S546P mutant revealed the occurrence of Na+ leak, which was dependent on the concentration of extracellular Na+ indicating that, although the variant is unable to transport nucleosides, there is an uncoupled sodium transport.

scholarly journals Main Structural Targets for Engineering Lipase Substrate Specificity

Catalysts ◽  
2020 ◽  
Vol 10 (7) ◽  
pp. 747
Author(s):  
Samah Hashim Albayati ◽  
Malihe Masomian ◽  
Siti Nor Hasmah Ishak ◽  
Mohd Shukuri bin Mohamad Ali ◽  
Adam Leow Thean ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Rational Design ◽  
Molecular Mechanisms ◽  
Experimental Studies ◽  
Random Mutagenesis ◽  
Binding Pocket ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Saturation Mutagenesis ◽  
Process Conditions

Microbial lipases represent one of the most important groups of biotechnological biocatalysts. However, the high-level production of lipases requires an understanding of the molecular mechanisms of gene expression, folding, and secretion processes. Stable, selective, and productive lipase is essential for modern chemical industries, as most lipases cannot work in different process conditions. However, the screening and isolation of a new lipase with desired and specific properties would be time consuming, and costly, so researchers typically modify an available lipase with a certain potential for minimizing cost. Improving enzyme properties is associated with altering the enzymatic structure by changing one or several amino acids in the protein sequence. This review detailed the main sources, classification, structural properties, and mutagenic approaches, such as rational design (site direct mutagenesis, iterative saturation mutagenesis) and direct evolution (error prone PCR, DNA shuffling), for achieving modification goals. Here, both techniques were reviewed, with different results for lipase engineering, with a particular focus on improving or changing lipase specificity. Changing the amino acid sequences of the binding pocket or lid region of the lipase led to remarkable enzyme substrate specificity and enantioselectivity improvement. Site-directed mutagenesis is one of the appropriate methods to alter the enzyme sequence, as compared to random mutagenesis, such as error-prone PCR. This contribution has summarized and evaluated several experimental studies on modifying the substrate specificity of lipases.

scholarly journals A Serine Residue in ClC-3 Links Phosphorylation–Dephosphorylation to Chloride Channel Regulation by Cell Volume

1999 ◽  
Vol 113 (1) ◽  
pp. 57-70 ◽  
Author(s):  
Dayue Duan ◽  
Suzanne Cowley ◽  
Burton Horowitz ◽  
Joseph R. Hume
Keyword(s):  
Cell Volume ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Phosphorylation Site ◽  
Cardiac Cells ◽  
Site Directed Mutagenesis ◽  
Cell Swelling ◽  
Transport Systems ◽  
Serine Residue ◽  
Channel Regulation

In many mammalian cells, ClC-3 volume-regulated chloride channels maintain a variety of normal cellular functions during osmotic perturbation. The molecular mechanisms of channel regulation by cell volume, however, are unknown. Since a number of recent studies point to the involvement of protein phosphorylation/dephosphorylation in the control of volume-regulated ionic transport systems, we studied the relationship between channel phosphorylation and volume regulation of ClC-3 channels using site-directed mutagenesis and patch-clamp techniques. In native cardiac cells and when overexpressed in NIH/3T3 cells, ClC-3 channels were opened by cell swelling or inhibition of endogenous PKC, but closed by PKC activation, phosphatase inhibition, or elevation of intracellular Ca2+. Site-specific mutational studies indicate that a serine residue (serine51) within a consensus PKC-phosphorylation site in the intracellular amino terminus of the ClC-3 channel protein represents an important volume sensor of the channel. These results provide direct molecular and pharmacological evidence indicating that channel phosphorylation/dephosphorylation plays a crucial role in the regulation of volume sensitivity of recombinant ClC-3 channels and their native counterpart, ICl.vol.

Thyroid-Stimulating Hormone and Thyroid-Stimulating Hormone Receptor Structure-Function Relationships

2002 ◽  
Vol 82 (2) ◽  
pp. 473-502 ◽  
Author(s):  
Mariusz W. Szkudlinski ◽  
Valerie Fremont ◽  
Catherine Ronin ◽  
Bruce D. Weintraub
Keyword(s):  
Structure Function ◽  
Molecular Mechanisms ◽  
Receptor Activation ◽  
Specific Protein ◽  
Thyroid Stimulating Hormone ◽  
Site Directed Mutagenesis ◽  
Loss Of Function ◽  
Glycoprotein Hormone ◽  
Therapeutic Implications ◽  
Stimulating Hormone

This review focuses on recent advances in the structure-function relationships of thyroid-stimulating hormone (TSH) and its receptor. TSH is a member of the glycoprotein hormone family constituting a subset of the cystine-knot growth factor superfamily. TSH is produced by the pituitary thyrotrophs and released to the circulation in a pulsatile manner. It stimulates thyroid functions using specific membrane TSH receptor (TSHR) that belongs to the superfamily of G protein-coupled receptors (GPCRs). New insights into the structure-function relationships of TSH permitted better understanding of the role of specific protein and carbohydrate domains in the synthesis, bioactivity, and clearance of this hormone. Recent progress in studies on TSHR as well as studies on the other GPCRs provided new clues regarding the molecular mechanisms of receptor activation. Such advances are a result of extensive site-directed mutagenesis, peptide and antibody approaches, detailed sequence analyses, and molecular modeling as well as studies on naturally occurring gain- and loss-of-function mutations. This review integrates expanding information on TSH and TSHR structure-function relationships and summarizes current concepts on ligand-dependent and -independent TSHR activation. Special emphasis has been placed on TSH domains involved in receptor recognition, constitutive activity of TSHR, new insights into the evolution of TSH bioactivity, and the development of high-affinity TSH analogs. Such structural, physiological, pathophysiological, evolutionary, and therapeutic implications of TSH-TSHR structure-function studies are frequently discussed in relation to concomitant progress made in studies on gonadotropins and their receptors.

Conserved residues F316 and G476 in the concentrative nucleoside transporter 1 (hCNT1) affect guanosine sensitivity and membrane expression, respectively

2005 ◽  
Vol 288 (1) ◽  
pp. C39-C45 ◽  
Author(s):  
Yurong Lai ◽  
Eun-Woo Lee ◽  
Carl C. Ton ◽  
Shashi Vijay ◽  
Huixia Zhang ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Mdck Cells ◽  
Site Directed Mutagenesis ◽  
Nucleoside Transporter ◽  
Wild Type ◽  
Membrane Expression ◽  
Concentrative Nucleoside Transporter ◽  
Green Fluorescent

The functional significance of two highly conserved amino acid residues, F316 [putative transmembrane domain (TM)7] and G476 (putative TM11), in the concentrative nucleoside transporter hCNT1 (SLC28A1) was examined by performing site-directed mutagenesis. Conservative mutations at these positions (F316A, F316Y, G476A, and G476L) were generated and expressed in Madin-Darby canine kidney (MDCK) cells as fusion polypeptides with green fluorescent protein (GFP). Unlike wild-type hCNT1, G476A-GFP and G476L-GFP were not expressed in the plasma membrane in undifferentiated or differentiated MDCK cells and had no functional activity. Like wild-type hCNT1, F316A-GFP and F316Y-GFP were expressed in the plasma membrane of undifferentiated MDCK cells and in the apical membrane of differentiated MDCK cells. Remarkably, transport of [3H]uridine by F316Y-GFP or F316A-GFP was highly sensitive to inhibition by guanosine. Furthermore, genotyping of exon 11 of hCNT1 (TM7) in a panel of 260 anonymous human DNA samples revealed a novel F316H variant (TT>CA; 1/260). When expressed in MDCK cells, [3H]uridine transport by F316H was also found to be sensitive to inhibition by guanosine (IC50 = 148 μM). The effect of the F316H mutation resembles the N4 type nucleoside transporter phenotype previously reported to be present in human kidneys. We suggest that the N4 transport system is a naturally occurring variant of hCNT1, perhaps at the F316 position. Collectively, our data show that G476 is important for correct membrane targeting, folding, and/or intracellular processing of hCNT1. In addition, we have discovered that hCNT1 displays natural variation at position F316 and that the variant F316H confers on the transporter an unusual sensitivity to inhibition by guanosine.

Characterization of nucleoside transport systems in cultured rat epididymal epithelium

2001 ◽  
Vol 280 (5) ◽  
pp. C1076-C1082 ◽  
Author(s):  
George P. H. Leung ◽  
Jeffrey L. Ward ◽  
Patrick Y. D. Wong ◽  
Chung-Ming Tse
Keyword(s):  
Nucleoside Transport ◽  
Transport Systems ◽  
Thymidine Uptake ◽  
Nucleoside Transporter ◽  
Equilibrative Nucleoside Transporter ◽  
Functional Studies ◽  
Uridine Uptake ◽  
Concentrative Nucleoside Transporter ◽  
Nucleoside Uptake

The nucleoside transport systems in cultured epididymal epithelium were characterized and found to be similar between the proximal (caput and corpus) and distal (cauda) regions of the epididymis. Functional studies revealed that 70% of the total nucleoside uptake was Na+ dependent, while 30% was Na+ independent. The Na+-independent nucleoside transport was mediated by both the equilibrative nitrobenzylthioinosine (NBMPR)-sensitive system (40%) and the NBMPR-insensitive system (60%), which was supported by a biphasic dose response to NBMPR inhibition. The Na+-dependent [3H]uridine uptake was selectively inhibited 80% by purine nucleosides, indicating that the purine nucleoside-selective N1 system is predominant. Since Na+-dependent [3H]guanosine uptake was inhibited by thymidine by 20% and Na+-dependent [3H]thymidine uptake was broadly inhibited by purine and pyrimidine nucleosides, this suggested the presence of the broadly selective N3 system accounting for 20% of Na+-dependent nucleoside uptake. Results of RT-PCR confirmed the presence of mRNA for equilibrative nucleoside transporter (ENT) 1, ENT2, and concentrative nucleoside transporter (CNT) 2 and the absence of CNT1. It is suggested that the nucleoside transporters in epididymis may be important for sperm maturation by regulating the extracellular concentration of adenosine in epididymal plasma.

Sodium-dependent nucleoside transport in the human intestinal brush-border membrane

1997 ◽  
Vol 272 (6) ◽  
pp. G1314-G1320 ◽  
Author(s):  
S. D. Patil ◽  
J. D. Unadkat
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Maximum Velocity ◽  
Membrane Vesicles ◽  
Human Kidney ◽  
Nucleoside Transporters ◽  
Nucleoside Transport ◽  
Hill Coefficient ◽  
Thymidine Uptake ◽  
Nucleoside Transporter

The objective of the study was to determine the identity and kinetic characteristics of nucleoside transporters present in the brush-border membrane of the human jejunum. With use of brush-border membrane vesicles, uptake of [3H]uridine was stimulated two- to threefold by an inwardly directed Na+ gradient and was inhibited by both 100 microM thymidine and 100 microM guanosine nucleosides, which serve as model substrates for purine (N1, cif) and pyrimidine (N2, cit) transporters, respectively. [3H]thymidine and [3H]guanosine transport exhibited an overshoot phenomenon only in the presence of a Na+ gradient. Na(+)-thymidine uptake was inhibited by 100 microM cytidine or thymidine but not by guanosine, inosine, formycin B, or hypoxanthine. [3H]guanosine uptake was inhibited by 100 microM inosine, guanosine, or formycin B but not by thymidine or cytidine. Both adenosine and uridine inhibited uptake of [3H]thymidine and [3H]guanosine to a similar extent, indicating that both N1, cif and N2, cit Na(+)-nucleoside transporters are expressed in human jejunum. Enhanced uptake of Na(+)-thymidine by an inside-negative potential difference generated by K+ and valinomycin provides evidence that nucleoside transport is rheogenic, involving net transfer of a positive charge. The Hill coefficient was unity for all three substrates, indicating a Na(+)-nucleoside coupling stoichiometry of 1:1. At saturating Na+ concentration (150 mM) the kinetic parameters (n = 3-4) Michaelis-Menten constant and maximum velocity for uridine, thymidine, and guanosine uptake were 4.15 +/- 1.79, 2.74 +/- 0.58, 12.02 +/- 1.34 microM and 25.93 +/- 7.38, 16.10 +/- 3.64, 63.92 +/- 10.23 pmol.mg-1.10 s-1, respectively. These results suggest that, in contrast to the human kidney that expresses the N4 nucleoside transporter, the human jejunum expresses both N1 and N2 Na(+)-nucleoside transporters.

Characterization of Recombinant Human Coagulation Factor XFriuli

1996 ◽  
Vol 75 (02) ◽  
pp. 313-317 ◽  
Author(s):  
D J Kim ◽  
A Girolami ◽  
H L James
Keyword(s):  
Catalytic Domain ◽  
Coagulation Factor ◽  
Molecular Size ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Bleeding Tendency ◽  
Factor X ◽  
Amino Terminal ◽  
Normal Plasma ◽  
Plasma Factor

SummaryNaturally occurring plasma factor XFriuli (pFXFr) is marginally activated by both the extrinsic and intrinsic coagulation pathways and has impaired catalytic potential. These studies were initiated to obtain confirmation that this molecule is multi-functionally defective due to the substitution of Ser for Pro at position 343 in the catalytic domain. By the Nelson-Long site-directed mutagenesis procedure a construct of cDNA in pRc/CMV was derived for recombinant factor XFriuli (rFXFr) produced in human embryonic (293) kidney cells. The rFXFr was purified and shown to have a molecular size identical to that of normal plasma factor X (pFX) by gel electrophoretic, and amino-terminal sequencing revealed normal processing cleavages. Using recombinant normal plasma factor X (rFXN) as a reference, the post-translational y-carboxy-glutamic acid (Gla) and (β-hydroxy aspartic acid (β-OH-Asp) content of rFXFr was over 85% and close to 100%, respectively, of expected levels. The specific activities of rFXFr in activation and catalytic assays were the same as those of pFXFr. Molecular modeling suggested the involvement of a new H-bond between the side-chains of Ser-343 and Thr-318 as they occur in anti-parallel (3-pleated sheets near the substrate-binding pocket of pFXFr. These results support the conclusion that the observed mutation in pFXFr is responsible for its dysfunctional activation and catalytic potentials, and that it accounts for the moderate bleeding tendency in the homozygous individuals who possess this variant procoagulant.

scholarly journals Circular RNA mmu_circ_0005019 inhibits fibrosis of cardiac fibroblasts and reverses electrical remodeling of cardiomyocytes

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Na Wu ◽  
Chengying Li ◽  
Bin Xu ◽  
Ying Xiang ◽  
Xiaoyue Jia ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Cardiac Fibroblasts ◽  
Circular Rna ◽  
Protective Role ◽  
Luciferase Reporter ◽  
Loss Of Function ◽  
Candidate Target ◽  
Paired Control ◽  
Reporter Assays ◽  
And Migration

Abstract Background Circular RNA (circRNA) have been reported to play important roles in cardiovascular diseases including myocardial infarction and heart failure. However, the role of circRNA in atrial fibrillation (AF) has rarely been investigated. We recently found a circRNA hsa_circ_0099734 was significantly differentially expressed in the AF patients atrial tissues compared to paired control. We aim to investigate the functional role and molecular mechanisms of mmu_circ_0005019 which is the homologous circRNA in mice of hsa_circ_0099734 in AF. Methods In order to investigate the effect of mmu_circ_0005019 on the proliferation, migration, differentiation into myofibroblasts and expression of collagen of cardiac fibroblasts, and the effect of mmu_circ_0005019 on the apoptosis and expression of Ito, INA and SK3 of cardiomyocytes, gain- and loss-of-function of cell models were established in mice cardiac fibroblasts and HL-1 atrial myocytes. Dual-luciferase reporter assays and RIP were performed to verify the binding effects between mmu_circ_0005019 and its target microRNA (miRNA). Results In cardiac fibroblasts, mmu_circ_0005019 showed inhibitory effects on cell proliferation and migration. In cardiomyocytes, overexpression of mmu_circ_0005019 promoted Kcnd1, Scn5a and Kcnn3 expression. Knockdown of mmu_circ_0005019 inhibited the expression of Kcnd1, Kcnd3, Scn5a and Kcnn3. Mechanistically, mmu_circ_0005019 exerted biological functions by acting as a miR-499-5p sponge to regulate the expression of its target gene Kcnn3. Conclusions Our findings highlight mmu_circ_0005019 played a protective role in AF development and might serve as an attractive candidate target for AF treatment.

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