scholarly journals A P425R mutation of the proton-coupled folate transporter causing hereditary folate malabsorption produces a highly selective alteration in folate binding

2012 ◽  
Vol 302 (9) ◽  
pp. C1405-C1412 ◽  
Author(s):  
Daniel Sanghoon Shin ◽  
Rongbao Zhao ◽  
Enghui H. Yap ◽  
Andras Fiser ◽  
I. David Goldman
Keyword(s):  
Secondary Structure ◽  
Transmembrane Domain ◽  
Water Soluble ◽  
Wild Type ◽  
Sulfhydryl Reagents ◽  
Functional Changes ◽  
External Loop ◽  
Human Solid Tumors ◽  
Translocation Pathway ◽  
Positively Charged

Proton-coupled folate transporter (PCFT) mediates folate intestinal absorption and transport across the choroid plexus, processes defective in subjects with hereditary folate malabsorption (HFM). PCFT is also widely expressed in human solid tumors where it contributes to the transport of pemetrexed and other antifolates. This study defines the basis for the functional changes due to a P425R mutation detected in a subject with HFM. Among various substitutions, only positively charged mutants (P425R and P425K) lost function but in a highly selective manner. Transport of reduced folates mediated by P425R-PCFT was virtually abolished; the methotrexate influx Kt was increased fivefold (from 2 to 10 μM). In contrast, the pemetrexed influx Kt mediated by P425R-PCFT was decreased 30% compared with wild-type (WT)-PCFT. Methotrexate inhibition of pemetrexed influx was competitive with a Ki for WT-PCFT comparable to its influx Kt. However, the methotrexate influx Ki for P425R-PCFT was ∼15-fold higher than the WT-PCFT influx Kt and threefold higher than the methotrexate influx Kt for the P425R-PCFT mutant. The confirmed secondary structure and homology modeling place the P425 residue at the junction of the 6th external loop and 12th transmembrane domain, remote from the aqueous translocation pathway, a prediction confirmed by the failure to label P425C-PCFT with N-biotinylaminoethyl methanethiosulfonate-biotin and the absence of inhibition of P425C-PCFT function by water-soluble sulfhydryl reagents. Hence, despite its location, the P425R-PCFT mutation produces a conformational change that fully preserves pemetrexed binding but markedly impairs binding of methotrexate and other folates to the carrier.

Abstract 254: Defects in c-kit Receptor Function Affect Myocyte Turnover Resulting in Premature Myocardial Aging in the W/WV Mouse

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Maria-Virginia Caballero ◽  
Sergio Signore ◽  
Ramaswamy Kannappan ◽  
Junghyun Kim ◽  
Barbara Ogórek ◽  
...  
Keyword(s):  
Ejection Fraction ◽  
Ventricular Remodeling ◽  
Transmembrane Domain ◽  
Wall Stress ◽  
Critical Parameters ◽  
Compound Heterozygous ◽  
Wild Type ◽  
Functional Changes ◽  
Kit Receptor ◽  
Myocardial Aging

The objective of this study was to determine whether attenuation of cardiac stem cell (CSC) growth in the adult heart interferes with physiologic cell turnover leading to premature accumulation of senescent myocytes and myocardial aging. For this purpose, W/W V mice, which carry a mutated non-functional c-kit receptor, were studied. The W/W V mouse is a compound heterozygous mutant mouse. The W mutation corresponds to a large amino acid deletion that includes the transmembrane domain of the c-kit receptor while the W V mutation is a point mutation in the kinase domain of this receptor. These genetic modifications reduce c-kit kinase activity by approximately 80-90% affecting hematopoiesis, gametogenesis and melanogenesis. Echocardiographically, at 2-3 months of age, LV diameters and ejection fraction were similar in controls and W/W V mice. However, at 5-6 months and 8-9 months, diastolic and systolic LVDs were larger in W/W V mice than in WT. Ejection fraction decreased with age in W/W V mice. These various aspects of negative ventricular remodeling typically occur with the development of the aging myopathy in mice 24 month-old and older. Moreover, ventricular dilation is commonly associated with wall thinning, both critical parameters of diastolic and systolic wall stress. Importantly, these anatomical and functional changes in W/W V mice were associated with a marked increase in the percentage of CSCs and myocytes expressing the senescence-associated marker p16 INK4a . At two months of age, wild-type mice and W/W V mice had the same number of LV myocytes which were essentially identical in volume. However, in comparison with wild-type mice, W/W V mice at 4 months of age had a decreased number of LV myocytes which were larger in volume. The magnitude of myocyte proliferation at the time of sacrifice was assessed by two markers of the cell cycle, Ki67 and MCM5. On average, cycling myocytes were 3-4-fold higher in wild type mice than in W/W V mice. In contrast, apoptotic myocytes, evaluated by the TdT assay and hairpin 1 in situ ligation, were significantly more frequent in W/W V mice than in WT mice. In conclusion, mutation of the c-kit receptor in CSCs impairs new myocyte formation and promotes accumulation of senescent myocytes.

scholarly journals Voltage-Dependent Inactivation of MscS Occurs Independently of the Positively Charged Residues in the Transmembrane Domain

2016 ◽  
Vol 2016 ◽  
pp. 1-6 ◽  
Author(s):  
Takeshi Nomura ◽  
Masahiro Sokabe ◽  
Kenjiro Yoshimura
Keyword(s):  
Voltage Dependence ◽  
Transmembrane Domain ◽  
Wild Type ◽  
Dependent Inactivation ◽  
Mechanical Threshold ◽  
Voltage Dependent ◽  
Arginine Residues ◽  
Tm Domain ◽  
Small Conductance ◽  
Positively Charged

MscS (mechanosensitive channel of small conductance) is ubiquitously found among bacteria and plays a major role in avoiding cell lysis upon rapid osmotic downshock. The gating of MscS is modulated by voltage, but little is known about how MscS senses membrane potential. Three arginine residues (Arg-46, Arg-54, and Arg-74) in the transmembrane (TM) domain are possible to respond to voltage judging from the MscS structure. To examine whether these residues are involved in the voltage dependence of MscS, we neutralized the charge of each residue by substituting with asparagine (R46N, R54N, and R74N). Mechanical threshold for the opening of the expressed wild-type MscS and asparagine mutants did not change with voltage in the range from-40 to +100 mV. By contrast, inactivation process of wild-type MscS was strongly affected by voltage. The wild-type MscS inactivated at +60 to +80 mV but not at-60 to +40 mV. The voltage dependence of the inactivation rate of all mutants tested, that is, R46N, R54N, R74N, and R46N/R74N MscS, was almost indistinguishable from that of the wild-type MscS. These findings indicate that the voltage dependence of the inactivation occurs independently of the positive charges of R46, R54, and R74.

scholarly journals Functional and mechanistic roles of the human proton-coupled folate transporter transmembrane domain 6–7 linker

2016 ◽  
Vol 473 (20) ◽  
pp. 3545-3562 ◽  
Author(s):  
Mike R. Wilson ◽  
Zhanjun Hou ◽  
Lucas J. Wilson ◽  
Jun Ye ◽  
Larry H. Matherly
Keyword(s):  
Secondary Structure ◽  
Targeted Delivery ◽  
Transmembrane Domain ◽  
Surface Expression ◽  
Chimeric Proteins ◽  
Transport Activity ◽  
Wild Type ◽  
Intracellular Loop ◽  
Mutant Proteins ◽  
Sequence Elements

The proton-coupled folate transporter (PCFT; SLC46A1) is a folate–proton symporter expressed in solid tumors and is used for tumor-targeted delivery of cytotoxic antifolates. Topology modeling suggests that the PCFT secondary structure includes 12 transmembrane domains (TMDs) with TMDs 6 and 7 linked by an intracellular loop (positions 236–265) including His247, implicated as functionally important. Single-cysteine (Cys) mutants were inserted from positions 241 to 251 in Cys-less PCFT and mutant proteins were expressed in PCFT-null (R1-11) HeLa cells; none were reactive with 2-aminoethyl methanethiosulfonate biotin, suggesting that the TMD6–7 loop is intracellular. Twenty-nine single alanine mutants spanning the entire TMD6–7 loop were expressed in R1-11 cells; activity was generally preserved, with the exception of the 247, 250, and 251 mutants, partly due to decreased surface expression. Coexpression of PCFT TMD1–6 and TMD7–12 half-molecules in R1-11 cells partially restored transport activity, although removal of residues 252–265 from TMD7–12 abolished transport. Chimeric proteins, including a nonhomologous sequence from a thiamine transporter (ThTr1) inserted into the PCFT TMD6–7 loop (positions 236–250 or 251–265), were active, although replacement of the entire loop with the ThTr1 sequence resulted in substantial loss of activity. Amino acid replacements (Ala, Arg, His, Gln, and Glu) or deletions at position 247 in wild-type and PCFT–ThTr1 chimeras resulted in differential effects on transport. Collectively, our findings suggest that the PCFT TMD6–7 connecting loop confers protein stability and may serve a unique functional role that depends on secondary structure rather than particular sequence elements.

scholarly journals The N-Terminal Transmembrane Domain of λ S Is Required for Holin but Not Antiholin Function

2009 ◽  
Vol 192 (3) ◽  
pp. 725-733 ◽  
Author(s):  
Rebecca White ◽  
Tram Anh T. Tran ◽  
Chelsey A. Dankenbring ◽  
John Deaton ◽  
Ry Young
Keyword(s):  
Cytoplasmic Membrane ◽  
Transmembrane Domain ◽  
Amino Group ◽  
Missense Mutations ◽  
Wild Type Allele ◽  
Wild Type ◽  
Basic Residue ◽  
N Terminus ◽  
Terminal Amino ◽  
Positively Charged

ABSTRACT The λ S gene encodes a holin, S105, and an antiholin, S107, which differs by its Met-Lys N-terminal extension. The model for the lysis-defective character of S107 stipulates that the additional N-terminal basic residue keeps S107 from assuming the topology of S105, which is N-out, C-in, with three transmembrane domains (TMDs). Here we show that the N terminus of S105 retains its fMet residue but that the N terminus of S107 is fully deformylated. This supports the model that in S105, TMD1 inserts into the membrane very rapidly but that in S107, it is retained in the cytoplasm. Further, it reveals that, compared to S105, S107 has two extra positively charged moieties, Lys2 and the free N-terminal amino group, to hinder its penetration into an energized membrane. Moreover, an allele, S105ΔTMD1 , with TMD1 deleted, was found to be defective in lysis, insensitive to membrane depolarization, and dominant to the wild-type allele, indicating that the lysis-defective, antiholin character of S107 is due to the absence of TMD1 from the bilayer rather than to its ectopic localization at the inner face of the cytoplasmic membrane. Finally, the antiholin function of the deletion protein was compromised by the substitution of early-lysis missense mutations in either the deletion protein or parental S105 but restored when both S105ΔTMD1 and holin carried the substitution.

scholarly journals Solubilization of Charged Porphyrins in Interpolyelectrolyte Complexes: A Computer Study

Polymers ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 502
Author(s):  
Karel Šindelka ◽  
Zuzana Limpouchová ◽  
Karel Procházka
Keyword(s):  
Dissipative Particle Dynamics ◽  
Water Soluble ◽  
Coarse Grained ◽  
Core Shell ◽  
Computer Study ◽  
Interpolyelectrolyte Complex ◽  
The Core ◽  
Porphyrin Derivatives ◽  
Oppositely Charged ◽  
Positively Charged

Using coarse-grained dissipative particle dynamics (DPD) with explicit electrostatics, we performed (i) an extensive series of simulations of the electrostatic co-assembly of asymmetric oppositely charged copolymers composed of one (either positively or negatively charged) polyelectrolyte (PE) block A and one water-soluble block B and (ii) studied the solubilization of positively charged porphyrin derivatives (P+) in the interpolyelectrolyte complex (IPEC) cores of co-assembled nanoparticles. We studied the stoichiometric mixtures of 137 A10+B25 and 137 A10−B25 chains with moderately hydrophobic A blocks (DPD interaction parameter aAS=35) and hydrophilic B blocks (aBS=25) with 10 to 120 P+ added (aPS=39). The P+ interactions with other components were set to match literature information on their limited solubility and aggregation behavior. The study shows that the moderately soluble P+ molecules easily solubilize in IPEC cores, where they partly replace PE+ and electrostatically crosslink PE− blocks. As the large P+ rings are apt to aggregate, P+ molecules aggregate in IPEC cores. The aggregation, which starts at very low loadings, is promoted by increasing the number of P+ in the mixture. The positively charged copolymers repelled from the central part of IPEC core partially concentrate at the core-shell interface and partially escape into bulk solvent depending on the amount of P+ in the mixture and on their association number, AS. If AS is lower than the ensemble average ⟨AS⟩n, the copolymer chains released from IPEC preferentially concentrate at the core-shell interface, thus increasing AS, which approaches ⟨AS⟩n. If AS>⟨AS⟩n, they escape into the bulk solvent.

scholarly journals Ectopic Overexpression of Histone H3K4 Methyltransferase CsSDG36 from Tea Plant Decreases Hyperosmotic Stress Tolerance in Arabidopsis thaliana

2021 ◽  
Vol 22 (10) ◽  
pp. 5064
Author(s):  
Qinghua Chen ◽  
Linghui Guo ◽  
Yanwen Yuan ◽  
Shuangling Hu ◽  
Fei Guo ◽  
...  
Keyword(s):  
Drought Stress ◽  
Transmembrane Domain ◽  
Tea Plant ◽  
Microtubule Assembly ◽  
Pcr Analysis ◽  
Drought Response ◽  
Stomatal Development ◽  
Wild Type ◽  
Over Expression ◽  
Histone H3k4

Histone methylation plays an important regulatory role in the drought response of many plants, but its regulatory mechanism in the drought response of the tea plant remains poorly understood. Here, drought stress was shown to induce lower relative water content and significantly downregulate the methylations of histone H3K4 in the tea plant. Based on our previous analysis of the SET Domain Group (SDG) gene family, the full-length coding sequence (CDS) of CsSDG36 was cloned from the tea cultivar ‘Fuding Dabaicha’. Bioinformatics analysis showed that the open reading frame (ORF) of the CsSDG36 gene was 3138 bp, encoding 1045 amino acids and containing the conserved structural domains of PWWP, PHD, SET and PostSET. The CsSDG36 protein showed a close relationship to AtATX4 of the TRX subfamily, with a molecular weight of 118,249.89 Da, and a theoretical isoelectric point of 8.87, belonging to a hydrophilic protein without a transmembrane domain, probably located on the nucleus. The expression of CsSDG36 was not detected in the wild type, while it was clearly detected in the over-expression lines of Arabidopsis. Compared with the wild type, the over-expression lines exhibited lower hyperosmotic resistance by accelerating plant water loss, increasing reactive oxygen species (ROS) pressure, and increasing leaf stomatal density. RNA-seq analysis suggested that the CsSDG36 overexpression caused the differential expression of genes related to chromatin assembly, microtubule assembly, and leaf stomatal development pathways. qRT-PCR analysis revealed the significant down-regulation of stomatal development-related genes (BASL, SBT1.2(SDD1), EPF2, TCX3, CHAL, TMM, SPCH, ERL1, and EPFL9) in the overexpression lines. This study provides a novel sight on the function of histone methyltransferase CsSDG36 under drought stress.

scholarly journals Large-Scale Synonymous Substitutions in Cucumber Mosaic Virus RNA 3 Facilitate Amino Acid Mutations in the Coat Protein

2018 ◽  
Vol 92 (22) ◽  
Author(s):  
Tomofumi Mochizuki ◽  
Rie Ohara ◽  
Marilyn J. Roossinck
Keyword(s):  
Amino Acid ◽  
Secondary Structure ◽  
Viral Genome ◽  
Large Scale ◽  
Viral Rna ◽  
Wild Type ◽  
Competitive Fitness ◽  
Content Type ◽  
Synonymous Substitutions ◽  
Cp Gene

ABSTRACTThe effect of large-scale synonymous substitutions in a small icosahedral, single-stranded RNA viral genome on virulence, viral titer, and protein evolution were analyzed. The coat protein (CP) gene of the Fny stain of cucumber mosaic virus (CMV) was modified. We created four CP mutants in which all the codons of nine amino acids in the 5′ or 3′ half of the CP gene were replaced by either the most frequently or the least frequently used synonymous codons in monocot plants. When the dicot host (Nicotiana benthamiana) was inoculated with these four CP mutants, viral RNA titers in uninoculated symptomatic leaves decreased, while all mutants eventually showed mosaic symptoms similar to those for the wild type. The codon adaptation index of these four CP mutants against dicot genes was similar to those of the wild-type CP gene, indicating that the reduction of viral RNA titer was due to deleterious changes of the secondary structure of RNAs 3 and 4. When two 5′ mutants were serially passaged inN. benthamiana, viral RNA titers were rapidly restored but competitive fitness remained decreased. Although no nucleic acid changes were observed in the passaged wild-type CMV, one to three amino acid changes were observed in the synonymously mutated CP of each passaged virus, which were involved in recovery of viral RNA titer of 5′ mutants. Thus, we demonstrated that deleterious effects of the large-scale synonymous substitutions in the RNA viral genome facilitated the rapid amino acid mutation(s) in the CP to restore the viral RNA titer.IMPORTANCERecently, it has been known that synonymous substitutions in RNA virus genes affect viral pathogenicity and competitive fitness by alteration of global or local RNA secondary structure of the viral genome. We confirmed that large-scale synonymous substitutions in the CP gene of CMV resulted in decreased viral RNA titer. Importantly, when viral evolution was stimulated by serial-passage inoculation, viral RNA titer was rapidly restored, concurrent with a few amino acid changes in the CP. This novel finding indicates that the deleterious effects of large-scale nucleic acid mutations on viral RNA secondary structure are readily tolerated by structural changes in the CP, demonstrating a novel part of the adaptive evolution of an RNA viral genome. In addition, our experimental system for serial inoculation of large-scale synonymous mutants could uncover a role for new amino acid residues in the viral protein that have not been observed in the wild-type virus strains.

Functional changes in adenylyl cyclases and associated decreases in relaxation responses in mesenteric arteries from diabetic rats

2005 ◽  
Vol 289 (5) ◽  
pp. H2234-H2243 ◽  
Author(s):  
Takayuki Matsumoto ◽  
Kentaro Wakabayashi ◽  
Tsuneo Kobayashi ◽  
Katsuo Kamata
Keyword(s):  
Mesenteric Artery ◽  
Diabetic Rats ◽  
Functional Change ◽  
Water Soluble ◽  
Mesenteric Arteries ◽  
Diabetic Rat ◽  
Adenylyl Cyclases ◽  
Camp Production ◽  
Functional Changes ◽  
Rat Mesenteric Artery

To assess the functional change in adenylyl cyclases (AC) associated with the diabetic state, we investigated AC-mediated relaxations and cAMP production in mesenteric arteries from rats with streptozotocin (STZ)-induced diabetes. The relaxations induced by the water-soluble forskolin (FSK) analog NKH477, which is a putative AC5 activator, but not by the β-adrenoceptor agonist isoproterenol (Iso) and the AC activator FSK, were reduced in intact diabetic mesenteric artery. In diabetic rats, however, Iso-, FSK-, and NKH477-induced relaxations were attenuated in the presence of inhibitors of nitric oxide synthase and cyclooxygenase. To exclude the influence of phosphodiesterase (PDE), we also examined the relaxations induced by several AC activators in the presence of 3-isobutyl-1-methylxanthine (IBMX; a PDE inhibitor). Under these conditions, the relaxation induced by Iso was greatly impaired in STZ-diabetic rats. This Iso-induced relaxation was significantly attenuated by pretreatment with SQ-22536, an AC inhibitor, in mesenteric rings from age-matched controls but not in those from STZ-diabetic rats. Under the same conditions, the relaxations induced by FSK or NKH477 were impaired in STZ-diabetic rats. Neither FSK- nor A-23187 (a Ca2+ ionophore)-induced cAMP production was significantly different between diabetics and controls. However, cAMP production induced by Iso or NKH477 was significantly impaired in diabetic mesenteric arteries. Expression of mRNAs and proteins for AC5/6 was lower in diabetic mesenteric arteries than in controls. These results suggest that AC-mediated relaxation is impaired in the STZ-diabetic rat mesenteric artery, perhaps reflecting a reduction in AC5/6 activity.

scholarly journals Replacement of both tryptophan residues at 388 and 412 completely abolished cytochalasin B photolabelling of the GLUT1 glucose transporter

1994 ◽  
Vol 302 (2) ◽  
pp. 355-361 ◽  
Author(s):  
K Inukai ◽  
T Asano ◽  
H Katagiri ◽  
M Anai ◽  
M Funaki ◽  
...  
Keyword(s):  
Glucose Transporter ◽  
Cytochalasin B ◽  
Transmembrane Domain ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Site Directed Mutagenesis ◽  
Transport Activity ◽  
Wild Type ◽  
In The Wild ◽  
Glut1 Glucose Transporter

A mutated GLUT1 glucose transporter, a Trp-388, 412 mutant whose tryptophans 388 and 412 were both replaced by leucines, was constructed by site-directed mutagenesis and expressed in Chinese hamster ovary cells. Glucose transport activity was decreased to approx. 30% in the Trp-388, 412 mutant compared with that in the wild type, a similar decrease in transport activity had been observed previously in the Trp-388 mutant and the Trp-412 mutant which had leucine at 388 and 412 respectively. Cytochalasin B labelling of the Trp-388 mutant was only decreased rather than abolished, a result similar to that obtained previously for the Trp-412 mutant. Cytochalasin B labelling was finally abolished completely in the Trp-388, 412 mutant, while cytochalasin B binding to this mutant was decreased to approx. 30% of that of the wild-type GLUT1 at the concentration used for photolabelling. This level of binding is thought to be adequate to detect labelling, assuming that the labelling efficiency of these transporters is similar. These findings suggest that cytochalasin B binds to the transmembrane domain of the glucose transporter in the vicinity of helix 10-11, and is inserted covalently by photoactivation at either the 388 or the 412 site.

Mutation of a single amino acid converts the human water channel aquaporin 5 into an anion channel

2013 ◽  
Vol 305 (6) ◽  
pp. C663-C672 ◽  
Author(s):  
Xue Qin ◽  
Walter F. Boron
Keyword(s):  
Water Channel ◽  
Anion Channel ◽  
Single Amino Acid ◽  
Side Chain ◽  
Wild Type ◽  
Aquaporin 5 ◽  
Anion Conductance ◽  
Electrode Voltage ◽  
Microelectrode Measurements ◽  
Positively Charged

Aquaporin 6 (AQP6) is unique among mammalian AQPs in being an anion channel with negligible water permeability. However, the point mutation Asn60Gly converts AQP6 from an anion channel into a water channel. In the present study of human AQP5, we mutated Leu51 (corresponding to residue 61 in AQP6), the side chain of which faces the central pore. We evaluated function in Xenopus oocytes by two-electrode voltage clamp, video measurements of osmotic H2O permeability ( Pf), microelectrode measurements of surface pH (pHS) to assess CO2 permeability, and surface biotinylation. We found that AQP5-L51R does not exhibit the H2O or CO2 permeability of the wild-type protein but instead has a novel p-chloromercuribenzene sulfonate (pCMBS)-sensitive current. The double mutant AQP5-L51R/C182S renders the conductance insensitive to pCMBS, demonstrating that the current is intrinsic to AQP5. AQP5-L51R has the anion permeability sequence I− > NO3− ≅ NO2− > Br− > Cl− > HCO3− > gluconate. Of the other L51 mutants, L51T (polar uncharged) and L51V (nonpolar) retain H2O and CO2 permeability and do not exhibit anion conductance. L51D and L51E (negatively charged) have no H2O or CO2 permeability. L51K (positively charged) has an intermediate H2O and CO2 permeability and anion conductance. L51H is unusual in having a relatively low CO2 permeability and anion conductance, but a moderate Pf. Thus, positively charged mutations of L51 can convert AQP5 from a H2O/CO2 channel into an anion channel. However, the paradoxical effect of L51H is consistent with the hypothesis that CO2, in part, takes a pathway different from H2O through AQP5.

Sign in / Sign up

Export Citation Format

Share Document