scholarly journals Functional roles of aspartate residues of the proton-coupled folate transporter (PCFT-SLC46A1); a D156Y mutation causing hereditary folate malabsorption

Blood ◽  
2010 ◽  
Vol 116 (24) ◽  
pp. 5162-5169 ◽  
Author(s):  
Daniel Sanghoon Shin ◽  
Sang Hee Min ◽  
Laura Russell ◽  
Rongbao Zhao ◽  
Andras Fiser ◽  
...  
Keyword(s):  
Small Intestine ◽  
Critical Role ◽  
Surface Expression ◽  
Loss Of Function ◽  
Wild Type ◽  
Intracellular Loop ◽  
Folate Transport ◽  
Functional Roles ◽  
Proximal Small Intestine ◽  
Pakistani Origin

Abstract The proton-coupled folate transporter (PCFT; SLC46A1) mediates folate transport into enterocytes in the proximal small intestine; pcft loss-of-function mutations are the basis for hereditary folate malabsorption. The current study explored the roles of Asp residues in PCFT function. A novel, homozygous, loss-of-function mutation, D156Y, was identified in a child of Pakistani origin with hereditary folate malabsorption. Of the 6 other conserved Asp residues, only one, D109, is shown to be required for function. D156Y, along with a variety of other substitutions at this site (Trp, Phe, Val, Asn, or Lys), lacked function due to instability of the PCFT protein. Substantial function was preserved with Glu, Gly, and, to a lesser extent, with Ser, Thr, and Ala substitutions. This correlated with PCFT bio-tinylated at the cell surface. In contrast, all D109 mutants, including D109E, lacked function irrespective of pH (4.5, 5.5, and 7.4) or substrate concentration (0.5-100μM), despite surface expression comparable to wild-type PCFT. Hence, D156 plays a critical role in PCFT protein stability, and D109, located in the first intracellular loop between the second and third transmembrane domains, is absolutely required for PCFT function.

scholarly journals Role of the glutamate 185 residue in proton translocation mediated by the proton-coupled folate transporter SLC46A1

2009 ◽  
Vol 297 (1) ◽  
pp. C66-C74 ◽  
Author(s):  
Ersin Selcuk Unal ◽  
Rongbao Zhao ◽  
I. David Goldman
Keyword(s):  
Proton Translocation ◽  
Amino Acid Residues ◽  
Loss Of Function ◽  
Wild Type ◽  
Folate Transport ◽  
Rate Limiting Step ◽  
Proximal Small Intestine ◽  
Transport Cycle ◽  
Rate Limiting

The proton-coupled folate transporter (PCFT) SLC46A1 mediates uphill folate transport into enterocytes in proximal small intestine coupled to the inwardly directed proton gradient. Hereditary folate malabsorption is due to loss-of-function mutations in the PCFT gene. This study addresses the functional role of conserved charged amino acid residues within PCFT transmembrane domains with a detailed analysis of the PCFT E185 residue. D156A-, E185A-, E232A-, R148A-, and R376A-PCFT mutants lost function at pH 5.5, as assessed by transient transfection in folate transport-deficient HeLa cells. At pH 7.4, function was preserved only for E185A-PCFT. Loss of function for E185A-PCFT at pH 5.5 was due to an eightfold decrease in the [3H]methotrexate (MTX) influx Vmax; the MTX influx Ktwas identical to that of wild-type (WT)-PCFT (1.5 μM). Consistent with the intrinsic functionality of E185A-PCFT, [3H]MTX influx at pH 5.5 or 7.4 was trans-stimulated in cells preloaded with nonlabeled MTX or 5-formyltetrahydrofolate. Replacement of E185 with Leu, Cys, His, or Gln resulted in a phenotype similar to E185A-PCFT. However, there was greater preservation of activity (∼38% of WT) for the similarly charged E185D-PCFT at pH 5.5. All E185 substitution mutants were biotin accessible at the plasma membrane at a level comparable to WT-PCFT. These observations suggest that the E185 residue plays an important role in the coupled flows of protons and folate mediated by PCFT. Coupling appears to have a profound effect on the maximum rate of transport, consistent with augmentation of a rate-limiting step in the PCFT transport cycle.

A novel loss-of-function mutation in the proton-coupled folate transporter from a patient with hereditary folate malabsorption reveals that Arg 113 is crucial for function

Blood ◽  
2008 ◽  
Vol 112 (5) ◽  
pp. 2055-2061 ◽  
Author(s):  
Inbal Lasry ◽  
Bluma Berman ◽  
Rachel Straussberg ◽  
Yael Sofer ◽  
Hanna Bessler ◽  
...  
Keyword(s):  
Folic Acid ◽  
Fold Increase ◽  
Loss Of Function ◽  
Transmembrane Helices ◽  
Intracellular Loop ◽  
Acid Growth ◽  
Folate Transport ◽  
Stable Transfectants ◽  
Growth Requirement ◽  
Inactivating Mutations

Abstract Hereditary folate malabsorption (HFM) patients harbor inactivating mutations including R113S in the proton-coupled folate transporter (PCFT), an intestinal folate transporter with optimal activity at acidic pH. Here we identified and characterized a novel R113C mutation residing in the highly conserved first intracellular loop of PCFT. Stable transfectants overexpressing a Myc-tagged wild-type (WT) and mutant R113C PCFT displayed similar transporter targeting to the plasma membrane. However, whereas WT PCFT transfectants showed a 22-fold increase in [3H]folic acid influx at pH 5.5, R113C or mock transfectants showed no increase. Moreover, WT PCFT transfectants displayed a 50% folic acid growth requirement concentration of 7 nM, whereas mock and R113C transfectants revealed 24- to 27-fold higher values. Consistently, upon fluorescein-methotrexate labeling, WT PCFT transfectants displayed a 50% methotrexate displacement concentration of 50 nM, whereas mock and R113C transfectants exhibited 12- to 14-fold higher values. Based on the crystal structure of the homologous Escherichia coli glycerol-3-phosphate transporter, we propose that the cationic R113 residue of PCFT is embedded in a hydrophobic pocket formed by several transmembrane helices that may be part of a folate translocation pore. These findings establish a novel loss of function mutation in HFM residing in an intracellular loop of PCFT crucial for folate transport.

scholarly journals Hypertension resistance polymorphisms in ROMK (Kir1.1) alter channel function by different mechanisms

2010 ◽  
Vol 299 (6) ◽  
pp. F1359-F1364 ◽  
Author(s):  
Liang Fang ◽  
Dimin Li ◽  
Paul A. Welling
Keyword(s):  
Blood Pressure ◽  
Critical Role ◽  
Pressure Control ◽  
Surface Expression ◽  
Protein Product ◽  
Sequence Variants ◽  
Channel Activity ◽  
Loss Of Function ◽  
Channel Function ◽  
Channel Expression

The renal outer medullary K+ (ROMK) channel plays a critical role in renal sodium handling. Recent genome sequencing efforts in the Framingham Heart Study offspring cohort (Ji W, Foo JN, O'Roak BJ, Zhao H, Larson MG, Simon DB, Newton-Cheh C, State MW, Levy D, and Lifton RP. Nat Genet 40: 592–599, 2008) recently revealed an association between suspected loss-of-function polymorphisms in the ROMK channel and resistance to hypertension, suggesting that ROMK activity may also be a determinant of blood pressure control in the general population. Here we examine whether these sequence variants do, in fact, alter ROMK channel function and explore the mechanisms. As assessed by two-microelectrode voltage clamp in Xenopus oocytes, 3/5 of the variants (R193P, H251Y, and T313FS) displayed an almost complete attenuation of whole cell ROMK channel activity. Surface antibody binding measurements of external epitope-tagged channels and analysis of glycosylation-state maturation revealed that these variants prevent channel expression at the plasmalemma, likely as a consequence of retention in the endoplasmic reticulum. The other variants (P166S, R169H) had no obvious effects on the basal channel activity or surface expression but, instead, conferred a gain in regulated-inhibitory gating. As assessed in giant excised patch-clamp studies, apparent phosphotidylinositol 4,5-bisphosphate (PIP2) binding affinity of the variants was reduced, causing channels to be more susceptible to inhibition upon PIP2 depletion. Unlike the protein product of the major ROMK allele, these two variants are sensitive to the inhibitory affects of a G protein-coupled receptor, which stimulates PIP2 hydrolysis. In summary, we have found that hypertension resistance sequence variants inhibit ROMK channel function by different mechanisms, providing new insights into the role of the channel in the maintenance of blood pressure.

scholarly journals Adiponectin deficiency promotes endothelial activation and profoundly exacerbates sepsis-related mortality

2008 ◽  
Vol 295 (3) ◽  
pp. E658-E664 ◽  
Author(s):  
Hwee Teoh ◽  
Adrian Quan ◽  
K. W. Annie Bang ◽  
Guilin Wang ◽  
Fina Lovren ◽  
...  
Keyword(s):  
Adhesion Molecule ◽  
Critical Role ◽  
Cecal Ligation ◽  
Endothelial Activation ◽  
Experimental Sepsis ◽  
Protective Effects ◽  
Loss Of Function ◽  
Wild Type ◽  
Endothelial Adhesion Molecule ◽  
Inflammatory Models

Sepsis is a multifactorial, and often fatal, disorder typically characterized by widespread inflammation and immune activation with resultant endothelial activation. In the present study, we postulated that the adipokine adiponectin serves as a critical modulator of survival and endothelial activation in sepsis. To this aim, we evaluated both loss-of-function (adiponectin gene-deficient mice) and subsequent gain-of-function (recombinant adiponectin reconstitution) strategies in two well-established inflammatory models, cecal ligation perforation (CLP) and thioglyocollate-induced peritonitis. Adipoq−/− mice, subjected to CLP, exhibited a profound (∼8-fold) reduction in survival compared with their wild-type Adipoq+/+ littermates after 48 h. Furthermore, compared with wild-type controls, thioglycollate challenge resulted in a markedly greater influx of peritoneal neutrophils in Adipoq−/− mice accompanied by an excess production of key chemoattractant cytokines (IL-12p70, TNFα, MCP-1, and IL-6) and upregulation of aortic endothelial adhesion molecule VCAM-1 and ICAM-1 expressions. Importantly, all of these effects were blunted by recombinant total adiponectin administration given 3 days prior to thioglycollate challenge. The protective effects of adiponectin were ascribed largely to higher-order adiponectin oligomers, since administration of recombinant C39A trimeric adiponectin did not attenuate endothelial adhesion molecule expression in thioglycollate-challenged Adipoq−/− mice. These data suggest a critical role of adiponectin as a modulator of survival and endothelial inflammation in experimental sepsis and a potential mechanistic link between adiposity and increased sepsis.

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 EPEN-20. EZHIP/CATACOMB COOPERATES WITH PDGF-A AND p53 LOSS TO GENERATE A GENETICALLY ENGINEERED MOUSE MODEL FOR POSTERIOR FOSSA A EPENDYMOMA

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii311-iii311
Author(s):  
Emily Kagan ◽  
Daniel Brat ◽  
Ali Shilatifard ◽  
Andrea Piunti ◽  
Oren Becher
Keyword(s):  
Median Survival ◽  
Critical Role ◽  
Pediatric Brain Tumor ◽  
Genetically Engineered ◽  
Rank Test ◽  
Loss Of Function ◽  
Wild Type ◽  
Term Survival ◽  
Log Rank Test ◽  
Genetically Engineered Mouse Model

Abstract BACKGROUND PFA ependymoma is a pediatric brain tumor with only 30% long-term survival. Recently a gene called CXORF67/EZHIP/CATACOMB (henceforward: CATACOMB) was found to be overexpressed in PFA ependymoma. CATACOMB’s mechanism of action has been found to be analogous to that of the H3K27M mutation as its expression reduces H3K27me3 via inhibition of PRC2 catalytic activity. METHODS We infected NESTIN- or GFAP-expressing neonatal hindbrain progenitors with wild-type CATACOMB or a loss of function (LOF) point mutant (M406K), alone, with PDGFA, and with and without p53 deletion. RESULTS CATACOMB overexpression alone or with p53 loss was insufficient to induce tumorigenesis. CATACOMB overexpression with PDGFA and p53 loss was sufficient to induce tumorigenesis using either the LOF mutant (M406K) or the wild-type CATACOMB in both cells-of-origin. The histology appeared more ependymoma-like when CATACOMB was expressed in GFAP-expressing progenitors. Median survival for the model initiated in NESTIN progenitors was 99.5 days for the CATACOMB mutant (n=26) group and 61 days for the CATACOMB wild-type (n=28; log-rank test p=0.0033). Median survival for the model initiated in GFAP progenitors were 144 days for the CATACOMB mutant (n=19) group and 65 days for the CATACOMB wild-type (n=21; log-rank test is P<0.0013). Immunohistochemistry for H3K27me3 demonstrated that CATACOMB wild-type tumors had reduced H3K27me3 compared to CATACOMB mutant tumors. CONCLUSIONS Disrupting CATACOMB inhibitory activity toward PRC2 significantly increases survival in mice in both models, suggesting this activity plays a critical role in accelerating tumorigenesis. Ependymoma-like histology was more commonly observed in the model initiated in the GFAP-expressing progenitors.

scholarly journals Functional roles of the A335 and G338 residues of the proton-coupled folate transporter (PCFT-SLC46A1) mutated in hereditary folate malabsorption

2012 ◽  
Vol 303 (8) ◽  
pp. C834-C842 ◽  
Author(s):  
Daniel Sanghoon Shin ◽  
Rongbao Zhao ◽  
Andras Fiser ◽  
David I. Goldman
Keyword(s):  
Amino Acids ◽  
Homology Modeling ◽  
Choroid Plexus ◽  
Substrate Binding ◽  
Wild Type ◽  
Folate Transport ◽  
Functional Roles ◽  
Proton Coupling ◽  
Functional Defect ◽  
Influx Kinetics

The proton-coupled folate transporter (PCFT-SLC46A1) mediates intestinal folate absorption and folate transport across the choroid plexus, processes defective in hereditary folate malabsorption (HFM). This paper characterizes the functional defect, and the roles of two mutated PCFT residues, associated with HFM (G338R and A335D). The A335D-PCFT and other mutations at this residue result in an unstable protein; when expression of a mutant protein was preserved, function was always retained. The G338R and other charged mutants resulted in an unstable protein; substitutions with small neutral and polar amino acids preserved protein but with impaired function. Pemetrexed and methotrexate (MTX) influx kinetics mediated by the G338C mutant PCFT revealed marked (15- to 20-fold) decreases in Kt and Vmax compared with wild-type PCFT. In contrast, there was only a small (∼2-fold) decrease in the MTX influx Ki and an increase (∼3-fold) in the pemetrexed influx Ki for the G338C-PCFT mutant. Neither a decrease in pH to 4.5, nor an increase to 7.4, restored function of the G338C mutant relative to wild-type PCFT excluding a role for this residue in proton binding or proton coupling. Homology modeling localized the A335 and G338 residues embedded in the 9th transmembrane, consistent with the inaccessibility of the A335C and G338C proteins to MTS reagents. Hence, the loss of intrinsic G338C-PCFT function was due solely to impaired oscillation of the carrier between its conformational states. The data illustrate how alterations in carrier cycling can impact influx Kt without comparable alterations in substrate binding to the carrier.

scholarly journals Neurodegenerative disease mutations in TREM2 reveal a functional surface and distinct loss-of-function mechanisms

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Daniel L Kober ◽  
Jennifer M Alexander-Brett ◽  
Celeste M Karch ◽  
Carlos Cruchaga ◽  
Marco Colonna ◽  
...  
Keyword(s):  
Disease Risk ◽  
Surface Expression ◽  
Patient Specific ◽  
Loss Of Function ◽  
Wild Type ◽  
Functional Surface ◽  
Functional Studies ◽  
Functional Differences ◽  
Risk Variants ◽  
Disease Mutations

Genetic variations in the myeloid immune receptor TREM2 are linked to several neurodegenerative diseases. To determine how TREM2 variants contribute to these diseases, we performed structural and functional studies of wild-type and variant proteins. Our 3.1 Å TREM2 crystal structure revealed that mutations found in Nasu-Hakola disease are buried whereas Alzheimer’s disease risk variants are found on the surface, suggesting that these mutations have distinct effects on TREM2 function. Biophysical and cellular methods indicate that Nasu-Hakola mutations impact protein stability and decrease folded TREM2 surface expression, whereas Alzheimer’s risk variants impact binding to a TREM2 ligand. Additionally, the Alzheimer’s risk variants appear to epitope map a functional surface on TREM2 that is unique within the larger TREM family. These findings provide a guide to structural and functional differences among genetic variants of TREM2, indicating that therapies targeting the TREM2 pathway should be tailored to these genetic and functional differences with patient-specific medicine approaches for neurodegenerative disorders.

MicroRNA-21-5p as a novel therapeutic target for osteoarthritis

Rheumatology ◽  
2019 ◽  
Vol 58 (8) ◽  
pp. 1485-1497 ◽  
Author(s):  
Xiao-bo Wang ◽  
Feng-chao Zhao ◽  
Lin-hong Yi ◽  
Jin-long Tang ◽  
Zheng-ya Zhu ◽  
...  
Keyword(s):  
Mirna Expression ◽  
Medial Meniscus ◽  
Critical Role ◽  
Cartilage Degradation ◽  
Mirna Expression Profile ◽  
Conditional Knockout ◽  
Luciferase Reporter ◽  
Loss Of Function ◽  
Wild Type ◽  
Functional Pathway

Abstract Objective Growing evidence indicates that microRNAs (miRNA) play a critical role in the pathogenesis of OA, and overexpressing or silencing miRNA expression in OA models can contribute to the development of miRNA-based therapeutics. The objective of this study was to determine whether intra-articular injection of miRNA can inhibit OA progression. Methods The miRNA expression profile was determined in OA cartilage tissues and controls. Functional analysis of the miRNAs on extracellular matrix degradation was performed after miRNA mimic or inhibitor transfection. Luciferase reporter assays and western blotting were employed to determine miRNA targets. To investigate the functional mechanism of miR-21-5p in OA development, miR-21-5pfl/flCol2a1-CreER and wild-type mice were subject to surgical destabilization of the medial meniscus. Therapeutically, wild-type mice undergoing surgical destabilization of the medial meniscus were treated with intra-articular injection of agomir- and antagomir-21-5p. Results We found that expression of miR-21-5p was significantly up-regulated in OA cartilage tissues. The articular cartilage degradation of miR-21-5p conditional knockout mice was significantly alleviated compared with that of wild-type mice in spontaneous and destabilization of the medial meniscus models. Through gain-of-function and loss-of-function studies, miR-21-5p was shown to significantly affect matrix synthesis genes expression, and chondrocyte proliferation and apoptosis. Further, fibroblast growth factor 18 (FGF18) was identified as a target of miR-21-5p. Intra-articular injection of antagomir-21-5p significantly attenuated the severity of experimental OA. Clinically, FGF18 expression level was correlated with miR-21-5p expression and a modified Mankin scale. Conclusion Our findings reveal a miRNA functional pathway important for OA development, highlighting miRNA-21-5p silencing as an attractive therapeutic regimen in future clinical trials involving patients with OA.

scholarly journals In Vitro Analyses of Novel HCN4 Gene Mutations

2018 ◽  
Vol 49 (3) ◽  
pp. 1238-1248 ◽  
Author(s):  
Melina Möller ◽  
Nicole Silbernagel ◽  
Eva Wrobel ◽  
Birgit Stallmayer ◽  
Elsie Amedonu ◽  
...  
Keyword(s):  
Sinus Node ◽  
Membrane Surface ◽  
Functional Characterization ◽  
Surface Expression ◽  
Confocal Imaging ◽  
Xenopus Laevis Oocytes ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Wild Type ◽  
Adrenergic Stimulation

Background/Aims: The hyperpolarization-activated cyclic nucleotide-gated cation channel HCN4 contributes significantly to the generation of basic cardiac electrical activity in the sinus node and is a mediator of modulation by β–adrenergic stimulation. Heterologous expression of sick sinus syndrome (SSS) and bradycardia associated mutations within the human HCN4 gene results in altered channel function. The main aim was to describe the functional characterization of three (two novel and one known) missense mutations of HCN4 identified in families with SSS. Methods: Here, the two-electrode voltage clamp technique on Xenopus laevis oocytes and confocal imaging on transfected COS7 cells respectively, were used to analyze the functional effects of three HCN4 mutations; R378C, R550H, and E1193Q. Membrane surface expressions of wild type and the mutant channels were assessed by confocal microscopy, chemiluminescence assay, and Western blot in COS7 and HeLa cells. Results: The homomeric mutant channels R550H and E1193Q showed loss of function through increased rates of deactivation and distinctly reduced surface expression in all three homomeric mutant channels. HCN4 channels containing R550H and E1193Q mutant subunits only showed minor effects on the voltage dependence and rates of activation/deactivation. In contrast, homomeric R378C exerted a left-shifted activation curve and slowed activation kinetics. These effects were reduced in heteromeric co-expression of R378C with wild-type (WT) channels. Conclusion: Dysfunction of homomeric/heteromeric mutant HCN4-R378C, R550H, and E1193Q channels in the present study was primarily caused by loss of function due to decreased channel surface expression.

Sign in / Sign up

Export Citation Format

Share Document