Mutational analysis of the MEF2B cistrome in human HEK293 cells.

2019 ◽  
Author(s):  
JR Pon
Keyword(s):  
Mutational Analysis ◽  
Hek293 Cells

scholarly journals Neonatal Hypocalcemic Seizures in Offspring of a Mother With Familial Hypocalciuric Hypercalcemia Type 1 (FHH1)

2020 ◽  
Vol 105 (5) ◽  
pp. 1393-1400
Author(s):  
Poonam Dharmaraj ◽  
Caroline M Gorvin ◽  
Astha Soni ◽  
Nick D Nelhans ◽  
Mie K Olesen ◽  
...  
Keyword(s):  
Serum Calcium ◽  
Mutational Analysis ◽  
Mitogen Activated Protein Kinase ◽  
Hek293 Cells ◽  
Familial Hypocalciuric Hypercalcemia ◽  
Loss Of Function ◽  
Hydrogen Bond Interaction ◽  
Benign Condition ◽  
Neonatal Hypocalcemia

Abstract Context Familial hypocalciuric hypercalcemia type 1 (FHH1) is caused by loss-of-function mutations of the calcium-sensing receptor (CaSR) and is considered a benign condition associated with mild-to-moderate hypercalcemia. However, the children of parents with FHH1 can develop a variety of disorders of calcium homeostasis in infancy. Objective The objective of this work is to characterize the range of calcitropic phenotypes in the children of a mother with FHH1. Methods A 3-generation FHH kindred was assessed by clinical, biochemical, and mutational analysis following informed consent. Results The FHH kindred comprised a hypercalcemic man and his daughter who had hypercalcemia and hypocalciuria, and her 4 children, 2 of whom had asymptomatic hypercalcemia, 1 was normocalcemic, and 1 suffered from transient neonatal hypocalcemia and seizures. The hypocalcemic infant had a serum calcium of 1.57 mmol/L (6.28 mg/dL); normal, 2.0 to 2.8 mmol/L (8.0-11.2 mg/dL) and parathyroid hormone of 2.2 pmol/L; normal 1.0 to 9.3 pmol/L, and required treatment with intravenous calcium gluconate infusions. A novel heterozygous p.Ser448Pro CaSR variant was identified in the hypercalcemic individuals, but not the children with hypocalcemia or normocalcemia. Three-dimensional modeling predicted the p.Ser448Pro variant to disrupt a hydrogen bond interaction within the CaSR extracellular domain. The variant Pro448 CaSR, when expressed in HEK293 cells, significantly impaired CaSR-mediated intracellular calcium mobilization and mitogen-activated protein kinase responses following stimulation with extracellular calcium, thereby demonstrating it to represent a loss-of-function mutation. Conclusions Thus, children of a mother with FHH1 can develop hypercalcemia or transient neonatal hypocalcemia, depending on the underlying inherited CaSR mutation, and require investigations for serum calcium and CaSR mutations in early childhood.

scholarly journals Structural Determinants for Heparin Binding in Human Coagulation Factor XI.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2705-2705
Author(s):  
Sergei Shikov ◽  
Wenman Wu ◽  
Peter N. Walsh
Keyword(s):  
Catalytic Domain ◽  
Mutational Analysis ◽  
Solid Phase ◽  
Coagulation Factor ◽  
Hek293 Cells ◽  
Heparin Binding ◽  
Factor Xi ◽  
Dimeric Structure ◽  
Charged Residues ◽  
Positively Charged

Abstract Previous studies from our laboratory and others have demonstrated that zymogen factor XI (FXI) binds to heparin with moderate (KD ∼110 nM) affinity via residues (K252, K253 and K255) located in the Apple 3 (A3) domain. In contrast, the enzyme, FXIa, was shown to bind to heparin (Biochemistry40: 7569–7580, 2001) with significantly higher affinity (KD ∼9 nM by SPR and ∼1.5 nM by ELISA) via residues (K529, R530 and R532) within the catalytic domain (CD). This interaction potentiates by ∼10-fold the inhibition of FXIa by protease nexin-2. Also, polyanions heparin and dextran sulfate inhibit the catalytic activity of the enzyme factor XIa. The present study was designed to determine the relative contributions of positively charged residues as well as the dimeric structure of FXI to heparin binding. Mutational analysis of full-length FXI expressed in HEK293 cells was based on the following criteria: Conservation of the positively charged residues in FXI among various species; Surface exposure of the residues based on the X-ray crystal structure of FXI (Papagrigoriou E, McEwan P, Walsh PN, Emsley J,Nat. Struct. & Mol. Biol. 2006; 13:557–558); and comparison with human plasma prekallikrein (PK), which does not bind heparin. Two positively charged residues Arg507 (147, chymotrypsin numbering) and Arg532 (173) are conserved in FXI genes of all species for which sequences are available. In human PK, Arg507 is replaced by lysine, while Arg532 is replaced by a neutral glutamine. We have expressed and purified wtFXI, R507A, R532A as well as monomeric C321S/K331A and C321A/I290A. While wtFXI, R507A and R532A demonstrated normal activity in APTT assays; monomeric FXI mutants retained 60-70% activity. The R532A and R507A mutants demonstrated ∼75% decrease in total number of heparin binding sites based on the solid phase ELISA assay using 5F7 monoclonal antibody. Also, the apparent dissociation constants for R507 (11 nM) and R532A (22 nM) were 7 and 11-fold increased respectively compared with 1.6 nM for the wtFXI. We also characterized monomeric FXI C321S/K331A and C321A/I290A proteins for their ability to bind to heparin compared with wtFXI using surface plasmon resonance (SPR). Surprisingly, the monomeric FXI mutants, C321S/K331A and C321A/I290A, which had no mutations in any heparin-binding regions, displayed major defects in binding to heparin by SPR. Although kinetic analysis is challenging due to complex binding kinetics, while Rmax is about 10-fold lower, the off-rate for the binding of the monomeric FXI mutants is drastically increased when compared to that of wtFXI. These results suggest the possibility that the unique dimeric structure of FXI is required for cooperative binding to heparin. Thus, the dimeric structure of FXI and basic residues R507 and R532 in the catalytic domain of factor XI are both necessary for high-affinity heparin binding.

scholarly journals HIV-1 Gag release from yeast reveals ESCRT interaction with the Gag N-terminal protein region

2020 ◽  
Vol 295 (52) ◽  
pp. 17950-17972
Author(s):  
Birgit Meusser ◽  
Bettina Purfuerst ◽  
Friedrich C. Luft
Keyword(s):  
Plasma Membrane ◽  
Mutational Analysis ◽  
Genetic Manipulation ◽  
Membrane Binding ◽  
Hek293 Cells ◽  
Binding Studies ◽  
Site Mutation ◽  
Terminal Protein ◽  
Hiv 1 ◽  
Plasma Membrane Binding

The HIV-1 protein Gag assembles at the plasma membrane and drives virion budding, assisted by the cellular endosomal complex required for transport (ESCRT) proteins. Two ESCRT proteins, TSG101 and ALIX, bind to the Gag C-terminal p6 peptide. TSG101 binding is important for efficient HIV-1 release, but how ESCRTs contribute to the budding process and how their activity is coordinated with Gag assembly is poorly understood. Yeast, allowing genetic manipulation that is not easily available in human cells, has been used to characterize the cellular ESCRT function. Previous work reported Gag budding from yeast spheroplasts, but Gag release was ESCRT-independent. We developed a yeast model for ESCRT-dependent Gag release. We combined yeast genetics and Gag mutational analysis with Gag-ESCRT binding studies and the characterization of Gag-plasma membrane binding and Gag release. With our system, we identified a previously unknown interaction between ESCRT proteins and the Gag N-terminal protein region. Mutations in the Gag-plasma membrane–binding matrix domain that reduced Gag-ESCRT binding increased Gag-plasma membrane binding and Gag release. ESCRT knockout mutants showed that the release enhancement was an ESCRT-dependent effect. Similarly, matrix mutation enhanced Gag release from human HEK293 cells. Release enhancement partly depended on ALIX binding to p6, although binding site mutation did not impair WT Gag release. Accordingly, the relative affinity for matrix compared with p6 in GST-pulldown experiments was higher for ALIX than for TSG101. We suggest that a transient matrix-ESCRT interaction is replaced when Gag binds to the plasma membrane. This step may activate ESCRT proteins and thereby coordinate ESCRT function with virion assembly.

scholarly journals Partial deficiency of 17α-hydroxylase/17,20-lyase caused by a novel missense mutation in the canonical cytochrome heme-interacting motif

2015 ◽  
Vol 172 (5) ◽  
pp. K19-K25 ◽  
Author(s):  
Petr Rubtsov ◽  
Alexander Nizhnik ◽  
Ivan Dedov ◽  
Natalia Kalinchenko ◽  
Vasily Petrov ◽  
...  
Keyword(s):  
Mutational Analysis ◽  
Gene Mutations ◽  
Ambiguous Genitalia ◽  
Hek293 Cells ◽  
Mild Phenotype ◽  
Sex Development ◽  
Partial Deficiency ◽  
Combined Deficiency

BackgroundDeficiency of 17α-hydroxylase/17,20-lyase is a rare cause of 46,XY disordered sex development.ObjectiveWe characterize in vitro and in vivo effects of two novel CYP17A1 gene mutations identified in a patient with a mild phenotype of CYP17A1 deficiency.Subjects and methodsA 46,XY patient presented with ambiguous genitalia. CYP17A1 deficiency was suspected at 2 months on the basis of steroid analysis performed by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Mutational analysis of the CYP17A1 gene was performed by PCR and Sanger sequencing. To characterize the effect of CYP17A1 mutation on 17α-hydroxylase and 17,20-lyase activities in vitro, HEK293 cells were transiently transfected with CYP17A1 expression plasmids, incubated with progesterone or 17-OH-pregnenolone and concentrations of 17-OH-progesterone or DHEA were then measured in the cell culture medium by LC–MS/MS.ResultsClinical and hormonal findings in the patient were consistent with partial combined deficiency of 17α-hydroxylase/17,20-lyase. The sequencing of the CYP17A1 gene in the patient revealed compound heterozygosity for two novel mutations: c.107delT p.R36fsX107 and p.W121R. After 6-h in vitro culture of transfected HEK293 cells in the presence of 1 μM progesterone, 17α-hydroxylase activity of p.W121R mutant was 60.5±16.3%, while 17,20-lyase activity of mutant measured from the amount of DHEA produced in the presence of 1 μM of 17-OH-pregnenolone was 15.8±2.6% compared with the WT.Conclusionsp.W121R substitution, affecting the first residue in the conserved heme-interacting WXXXR motif of CYP17A1, is associated with partial combined deficiency of 17α-hydroxylase/17,20-lyase.

scholarly journals Mutational Analysis of Compound Heterozygous Mutation P.q6x/p.h232r in Srd5a2 Causing 46,xy Disorder of Sex Development

2022 ◽  
Author(s):  
Hui Yang ◽  
Liwei Li ◽  
Junhong Zhang ◽  
Qing Li ◽  
Li Qiao ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Mutational Analysis ◽  
Catalytic Efficiency ◽  
Hek293 Cells ◽  
Compound Heterozygous Mutation ◽  
Heterozygous Mutation ◽  
Compound Heterozygous ◽  
Disorder Of Sex Development ◽  
Sex Development ◽  
The Family

Abstract Background: Over 100 mutations in the SRD5A2 gene have been identified in subjects with 46,XY disorder of sex development (DSD). Exploration of SRD5A2 mutations and elucidation of the molecular mechanisms behind their effects should reveal the functions of the domains of the 5α-reductase 2 enzyme and identify the cause of 46,XY DSD. Previously, we reported a novel compound heterozygous p.Q6X/p.H232R mutation of the SRD5A2 gene in a case with 46,XY DSD. Whether the compound heterozygous p.Q6X/p.H232R mutation in this gene causes 46,XY DSD requires further exploration. Results: To clarify the cause of 46,XY DSD in the affected family focused on here, SRD5A2 sequencing was performed. Heterozygous p.H232R mutation was identified in the proband’s father, so we concluded that this mutation originated from the paternal side of the family and did not cause 46,XY DSD. Meanwhile, heterozygous p.Q6X mutation was identified in the proband’s mother, maternal uncle, and maternal grandfather, indicating that this mutation originated from maternal side of the family and did not cause 46,XY DSD. To clarify the effect of the p.H232R mutation in SRD5A2 on dihydrotestosterone (DHT) production, p.H232R mutant SRD5A2 plasmids were transfected into HEK293 cells. LC-MS indicated that DHT production decreased compared with that in cells transfected with wild-type SRD5A2.Conclusions: Our findings confirmed that the compound heterozygous p.Q6X/p.H232R mutation in the SRD5A2 gene is the cause of 46,XY DSD. p.H232R mutation reduced DHT production while attenuating the catalytic efficiency of the 5α-reductase 2 enzyme.

scholarly journals SAT-404 Neonatal Hypocalcemic Seizures in Offspring of a Mother with Familial Hypocalciuric Hypercalcemia Type 1 (FHH1)

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Fadil M Hannan ◽  
Poonam Dharmaraj ◽  
Caroline M Gorvin ◽  
Astha Soni ◽  
Nick D Nelhans ◽  
...  
Keyword(s):  
Serum Calcium ◽  
Mutational Analysis ◽  
Mitogen Activated Protein Kinase ◽  
Hek293 Cells ◽  
Familial Hypocalciuric Hypercalcemia ◽  
Loss Of Function ◽  
Calcium Sensing Receptor ◽  
Benign Condition ◽  
Calcium Sensing

Abstract Background: Familial hypocalciuric hypercalcemia type 1 (FHH1) is caused by loss-of-function mutations of the calcium-sensing receptor (CaSR), and considered to be a benign condition associated with mild-to-moderate hypercalcemia (1). However, the children of parents with FHH1 can develop a variety of disorders of calcium homeostasis in infancy. Objective: To further characterise the range of calcitropic phenotypes in the children of a mother with FHH1. Methods: We assessed a three generation FHH kindred by clinical, biochemical and mutational analysis following informed consent. Results: The kindred comprised a hypercalcemic male, his daughter who had hypercalcemia and hypocalciuria, and her four children, of whom two had asymptomatic hypercalcemia, one was normocalcemic, and one suffered from transient hypocalcemic seizures during infancy. The hypocalcemic infant had a serum calcium of 1.57 mmol/L (normal, 2.0-2.8) and PTH of 2.2 pmol/L (normal, 1.0-9.3) as a consequence of maternal hypercalcemia, and required treatment with I-V calcium gluconate infusions. Mutational analysis identified a novel heterozygous p.Ser448Pro CaSR variant in the hypercalcemic family members, but not in the children with hypocalcemia or normocalcemia. Three-dimensional modelling using a reported crystal structure of the dimeric CaSR showed the mutated Ser448 residue to be located in the CaSR extracellular domain, and predicted the p.Ser448Pro variant to disrupt a hydrogen bond interaction across the extracellular CaSR dimer interface. The variant Pro448 CaSR, when expressed in HEK293 cells, was shown to significantly impair CaSR-mediated intracellular calcium mobilisation and mitogen-activated protein kinase (MAPK) responses following stimulation with extracellular calcium, thereby demonstrating it to represent a loss-of-function mutation. Conclusion: These studies have identified a novel loss-of-function CaSR mutation which caused asymptomatic hypercalcemia in a mother and her children who had inherited the mutation. However, one child who did not inherit the mutation developed transient neonatal hypocalcemic seizures as a consequence of maternal hypercalcemia. These findings highlight the importance of assessing serum calcium and undertaking CaSR mutational analysis in the newborn offspring of a mother with FHH1. Reference: (1) Hannan FM, Kallay E, Chang W, Brandi ML, Thakker RV. The calcium-sensing receptor in physiology and in calcitropic and noncalcitropic diseases. Nat Rev Endocrinol. 2018; 15(1): 33-51.

scholarly journals Mutational Analysis of a Novel Mutation (p.H232R) in the SRD5A2 Causing 46,XY Disorder of Sex Development

2021 ◽  
Author(s):  
Liwei Li ◽  
Junhong Zhang ◽  
Qing Li ◽  
Li Qiao ◽  
Pengcheng Li ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Mutational Analysis ◽  
Novel Mutation ◽  
Catalytic Efficiency ◽  
Hek293 Cells ◽  
Compound Heterozygous ◽  
Wild Type ◽  
Disorder Of Sex Development ◽  
Maternal Grandfather ◽  
Sex Development

Abstract Background: Over 100 mutations in SRD5A2 gene have been identified in subjects with 46,XY DSD. Exploring SRD5A2 mutation and elucidating its molecular mechanism will find the domains function of 5α-reductase 2 enzyme and identify the cause of 46,XY DSD. Previously, we reported a novel compound heterozygous p.H232R/p.Q6X mutation of SRD5A2 gene in a case with 46,XY DSD. Whether the compound heterozygous p.Q6X/p.H232R mutation in SRD5A2 gene causes 46,XY DSD occurrence is needed to be further explored. Results: In order to clarify the cause of 46,XY DSD in the case’s family, SRD5A2 sequencing were performed. The heterozygous p.H232R mutation were identified in the case’s father, so we concluded that the heterozygous p.H232R mutation originated from paternal family and didn’t cause 46,XY DSD occurrence. The heterozygous p.Q6X mutation were identified in the case’s mother, maternal uncle and maternal grandfather, indicating that the heterozygous p.Q6X mutation descended from maternal family and didn’t cause 46,XY DSD occurrence. In order to clarify p.H232R mutation in SRD5A2 on DHT production, p.H232R mutant SRD5A2 plasmids were transfected with HEK293 cells and LC-MS detected that DHT production decreased compared with wild-type SRD5A2 infected ones.Conclusions: Our findings confirmed that the compound heterozygous p.Q6X/p.H232R mutation in SRD5A2 gene is the cause of 46,XY DSD. p.H232R mutation reduced DHT production while attenuated 5α-reductase 2 enzymatic catalytic efficiency.

scholarly journals Mutational analysis of the hepatocyte nuclear factor 4-α (HNF4α/HNF4A)-regulated transcriptome in HEK293 cells

2015 ◽  
Author(s):  
K Grigo ◽  
A Wirsing ◽  
B Lucas ◽  
L Klein-Hitpass ◽  
GU Ryffel
Keyword(s):  
Mutational Analysis ◽  
Hek293 Cells ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Hepatocyte Nuclear Factor 4

scholarly journals Mutational Analysis of the GXXXG/A Motifs in the Human Na+/Taurocholate Co-Transporting Polypeptide NTCP on Its Bile Acid Transport Function and Hepatitis B/D Virus Receptor Function

2021 ◽  
Vol 8 ◽  
Author(s):  
Massimo Palatini ◽  
Simon Franz Müller ◽  
Kira Alessandra Alicia Theresa Lowjaga ◽  
Saskia Noppes ◽  
Jörg Alber ◽  
...  
Keyword(s):  
Bile Acid ◽  
Hepatitis B ◽  
Mutational Analysis ◽  
Hbv Infection ◽  
Hek293 Cells ◽  
Intact Protein ◽  
Receptor Function ◽  
Acid Transport ◽  
Wild Type ◽  
Bile Acid Transport

Homodimerization is essential for plasma membrane sorting of the liver bile acid transporter NTCP and its function as Hepatitis B/D Virus (HBV/HDV) receptor. However, the protein domains involved in NTCP dimerization are unknown. NTCP bears two potential GXXXG/A dimerization motifs in its transmembrane domains (TMDs) 2 and 7. The present study aimed to analyze the role of these GXXXG/A motifs for the sorting, function, and dimerization of NTCP. The NTCP mutants G60LXXXA64L (TMD2), G233LXXXG237L (TMD7) and a double mutant were generated and analyzed for their interaction with wild-type NTCP using a membrane-based yeast-two hybrid system (MYTH) and co-immunoprecipitation (co-IP). In the MYTH system, the TMD2 and TMD7 mutants showed significantly lower interaction with the wild-type NTCP. In transfected HEK293 cells, membrane expression and bile acid transport activity were slightly reduced for the TMD2 mutant but were completely abolished for the TMD7 and the TMD2/7 mutants, while co-IP experiments still showed intact protein-protein interactions. Susceptibility for in vitro HBV infection in transfected HepG2 cells was reduced to 50% for the TMD2 mutant, while the TMD7 mutant was not susceptible for HBV infection at all. We conclude that the GXXXG/A motifs in TMD2 and even more pronounced in TMD7 are important for proper folding and sorting of NTCP, and so indirectly affect glycosylation, homodimerization, and bile acid transport of NTCP, as well as its HBV/HDV receptor function.

scholarly journals Mutational analysis of the hepatocyte nuclear factor 4-α (HNF4α/Hnf4a)-regulated transcriptome in HEK293 cells

2015 ◽  
Author(s):  
B Lucas ◽  
K Grigo ◽  
S Erdmann ◽  
J Lausen ◽  
L Klein-Hitpass ◽  
...  
Keyword(s):  
Mutational Analysis ◽  
Hek293 Cells ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Hepatocyte Nuclear Factor 4
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