scholarly journals Systematic alanine scanning of PAX8 paired domain reveals functional importance of the N-subdomain

2019 ◽  
Vol 62 (3) ◽  
pp. 129-135 ◽  
Author(s):  
Megumi Iwahashi ◽  
Satoshi Narumi
Keyword(s):  
Structure Function ◽  
Congenital Hypothyroidism ◽  
Loss Of Function ◽  
Paired Domain ◽  
Functional Importance ◽  
Alanine Scanning Mutagenesis ◽  
Alanine Scanning ◽  
Pathogenicity Prediction ◽  
Pax Family

Thyroid-specific transcription factor PAX8 has an indispensable role in the thyroid gland development, which is evidenced by the facts thatPAX8/Pax8mutations cause congenital hypothyroidism in humans and mice. More than 90% of knownPAX8mutations were located in the paired domain, suggesting the central role of the domain in exerting the molecular function. Structure-function relationships of PAX8, as well as other PAX family transcription factors, have never been investigated in a systematic manner. Here, we conducted the first alanine scanning mutagenesis study, in which 132 alanine variants located in the paired domain of PAX8 were created and systematically evaluatedin vitro. We found that 76 alanine variants (55%) were loss of function (LOF) variants (defined by <30% activity as compared with wild type PAX8). Importantly, the distribution of LOF variants were skewed, with more frequently observed in the N-subdomain (65% of the alanine variants in the N-subdomain) than in the C-subdomain (45%). Twelve out of 13 alanine variants in residues that have been affected in patients with congenital hypothyroidism were actually LOF, suggesting that the alanine scanning data can be used to evaluate the functional importance of mutated residues. Using ourin vitrodata, we tested the accuracy of seven computational algorithms for pathogenicity prediction, showing that they are sensitive but not specific to evaluate on the paired domain alanine variants. Collectively, our experiment-based data would help better understand the structure-function relationships of the paired domain, and would provide a unique resource for pathogenicity prediction of futurePAX8variants.

scholarly journals Comprehensive Alanine-scanning Mutagenesis of Escherichia coli CsrA Defines Two Subdomains of Critical Functional Importance

2006 ◽  
Vol 281 (42) ◽  
pp. 31832-31842
Author(s):  
Jeffrey Mercante ◽  
Kazushi Suzuki ◽  
Xiaodong Cheng ◽  
Paul Babitzke ◽  
Tony Romeo
Keyword(s):  
Escherichia Coli ◽  
Functional Importance ◽  
Alanine Scanning Mutagenesis ◽  
Alanine Scanning

Congenital Hypothyroidism Due to Truncating PAX8 Mutations: A Case Series and Molecular Function Studies

2020 ◽  
Vol 105 (11) ◽  
Author(s):  
Megumi Iwahashi-Odano ◽  
Keisuke Nagasaki ◽  
Maki Fukami ◽  
Junko Nishioka ◽  
Shuichi Yatsuga ◽  
...  
Keyword(s):  
Western Blotting ◽  
Congenital Hypothyroidism ◽  
Cultured Cells ◽  
Mutation Screening ◽  
Intracellular Localization ◽  
Functional Characterization ◽  
Case Series ◽  
Luciferase Reporter ◽  
Loss Of Function ◽  
Paired Domain

Abstract Context PAX8 is a transcription factor required for thyroid development, and its mutation causes congenital hypothyroidism (CH). More than 20 experimentally verified loss-of-function PAX8 mutations have been described, and all but one were located in the DNA-binding paired domain. Objective We report the identification and functional characterization of 3 novel truncating PAX8 mutations located outside the paired domain. Methods Three CH probands, diagnosed in the frame of newborn screening, had thyroid hypoplasia and were treated with levothyroxine. Next-generation sequencing-based mutation screening was performed. Functionality of the identified mutations were verified with Western blotting, intracellular localization assays, and transactivation assays with use of HeLa cells. Luciferase complementation assays were used to evaluate the effect of mutations on the interaction between PAX8 and its partner, NKX2-1. Results Each proband had novel truncating PAX8 mutations that were I160Sfs*52, Q213Efs*27, and F342Rfs*85. Western blotting showed destabilization of the I160fs-PAX8 protein. Q213fs-PAX8 and F342fs-PAX8 showed normal protein expression levels and normal nuclear localization, but showed loss of transactivation of the luciferase reporter. By luciferase complementation assays, we showed that PAX8-NKX2-1 interaction was defective in Q213fs-PAX8. We also characterized the recombinant PAX8 proteins, and found that the protein sequence corresponding to exon 10 (363-400 aa residues) was essential for the PAX8-NKX2-1 interaction. Conclusions Clinical and molecular findings of 3 novel truncating PAX8 mutations located outside the paired domain were reported. Experiments using cultured cells and recombinant proteins showed that the C-terminal portion (ie, 363-400 aa) of PAX8 is required for the PAX8-NKX2-1 interaction.

scholarly journals Translational Attenuation Mechanism of ErmB Induction by Erythromycin Is Dependent on Two Leader Peptides

2021 ◽  
Vol 12 ◽  
Author(s):  
Shasha Wang ◽  
Kai Jiang ◽  
Xinyue Du ◽  
Yanli Lu ◽  
Lijun Liao ◽  
...  
Keyword(s):  
Premature Termination ◽  
Regulatory Region ◽  
Terminal Region ◽  
Leader Peptide ◽  
Alanine Scanning Mutagenesis ◽  
Alanine Scanning ◽  
Major Mechanism ◽  
Attenuation Mechanism

Ribosome stalling on ermBL at the tenth codon (Asp) is believed to be a major mechanism of ermB induction by erythromycin (Ery). In this study, we demonstrated that the mechanism of ermB induction by Ery depends not only on ermBL expression but also on previously unreported ermBL2 expression. Introducing premature termination codons in ermBL, we proved that translation of the N-terminal region of ermBL is the key component for ermB induced by Ery, whereas translation of the C-terminal region of ermBL did not affect Ery-induced ermB. Mutation of the tenth codon (Asp10) of ermBL with other amino acids showed that the degree of induction in vivo was not completely consistent with the data from the in vitro toe printing assay. Alanine-scanning mutagenesis of ermBL demonstrated that both N-terminal residues (R7-K11) and the latter part of ermBL (K20-K27) are critical for Ery induction of ermB. The frameshifting reporter plasmid showed that a new leader peptide, ermBL2, exists in the ermB regulatory region. Further, introducing premature termination mutation and alanine-scanning mutagenesis of ermBL2 demonstrated that the N-terminus of ermBL2 is essential for induction by Ery. Therefore, the detailed function of ermBL2 requires further study.

scholarly journals Analysis of Mutations in the Yeast mRNA Decapping Enzyme

Genetics ◽  
1999 ◽  
Vol 151 (4) ◽  
pp. 1273-1285 ◽  
Author(s):  
Sundaresan Tharun ◽  
Roy Parker
Keyword(s):  
Specific Activity ◽  
Temperature Sensitive ◽  
Loss Of Function ◽  
Alanine Scanning Mutagenesis ◽  
Mrna Decapping ◽  
Nonsense Codons ◽  
Decapping Enzyme ◽  
Decapping Enzymes

Abstract A major mechanism of mRNA decay in yeast is initiated by deadenylation, followed by mRNA decapping, which exposes the transcript to 5′ to 3′ exonucleolytic degradation. The decapping enzyme that removes the 5′ cap structure is encoded by the DCP1 gene. To understand the function of the decapping enzyme, we used alanine scanning mutagenesis to create 31 mutant versions of the enzyme, and we examined the effects of the mutations both in vivo and in vitro. Two types of mutations that affected mRNA decapping in vivo were identified, including a temperature-sensitive allele. First, two mutants produced decapping enzymes that were defective for decapping in vitro, suggesting that these mutated residues are required for enzymatic activity. In contrast, several mutants that moderately affected mRNA decapping in vivo yielded decapping enzymes that had at least the same specific activity as the wild-type enzyme in vitro. Combination of alleles within this group yielded decapping enzymes that showed a strong loss of function in vivo, but that still produced fully active enzymes in vitro. This suggested that interactions of the decapping enzyme with other factors may be required for efficient decapping in vivo, and that these particular mutations may be disrupting such interactions. Interestingly, partial loss of decapping activity in vivo led to a defect in normal deadenylation-dependent decapping, but it did not affect the rapid deadenylation-independent decapping triggered by early nonsense codons. This observation suggested that these two types of mRNA decapping differ in their requirements for the decapping enzyme.

scholarly journals An in vitro and computational validation of a novel loss-of-functional mutation in PAX9 associated with non-syndromic tooth agenesis

2022 ◽  
Author(s):  
Tanmoy Sarkara ◽  
Prashant Ranjan ◽  
Smitha Kanathur ◽  
Ankush Gupta ◽  
PARIMAL DAS
Keyword(s):  
Amino Acid Position ◽  
Binding Activity ◽  
Mutant Protein ◽  
Tooth Agenesis ◽  
Craniofacial Anomalies ◽  
Loss Of Function ◽  
Wild Type ◽  
Paired Domain ◽  
Computational Validation

Congenital tooth agenesis (CTA) is one of the most common craniofacial anomalies. Its frequency varies among different population depending upon the genetic heterogeneity. CTA could be of familial or sporadic and syndromic or non-syndromic. Five major genes are found to be associated with non-syndromic CTA namely, PAX9, MSX1, EDA1, AXIN2 and WNT10A. In this study, an India family with CTA was investigated and a novel c.336C>G variation was identified in the exon 3 of PAX9, leading to substitution of evolutionary conserved Cys with Trp at 112 amino acid position located at the functionally significant DNA binding paired domain region. Functional analysis revealed that p.Cys112Trp mutation did not prevent the nuclear localization although mutant protein had higher cytoplasmic retention. EMSA using e5 probe revealed that mutant protein was unable to bind with the paired-domain binding site. Subsequently, GST pull-down assay revealed lower binding activity of the mutant protein with its known interactor MSX1. Further RNA-sequencing of PAX9 over-expressed HEK293, identified two potential novel targets, WNT4 and WNT7b those are up-regulated by wild-type PAX9 but not by mutant. These in vitro results were consistent with the computational results. The in vitro and computational observations altogether suggest that c.336C>G (p.Cys112Trp) variation leads to loss-of-function of PAX9 leading to CTA in this family.

scholarly journals ABS–Scan: In silico alanine scanning mutagenesis for binding site residues in protein–ligand complex

F1000Research ◽  
2014 ◽  
Vol 3 ◽  
pp. 214 ◽  
Author(s):  
Praveen Anand ◽  
Deepesh Nagarajan ◽  
Sumanta Mukherjee ◽  
Nagasuma Chandra
Keyword(s):  
Binding Site ◽  
In Silico ◽  
Ligand Recognition ◽  
Loss Of Function ◽  
Ligand Complex ◽  
Alanine Scanning Mutagenesis ◽  
Ligand Interaction ◽  
Alanine Scanning ◽  
Protein Ligand Interactions ◽  
Ligand Interactions

Most physiological processes in living systems are fundamentally regulated by protein–ligand interactions. Understanding the process of ligand recognition by proteins is a vital activity in molecular biology and biochemistry. It is well known that the residues present at the binding site of the protein form pockets that provide a conducive environment for recognition of specific ligands. In many cases, the boundaries of these sites are not well defined. Here, we provide a web-server to systematically evaluate important residues in the binding site of the protein that contribute towards the ligand recognition through in silico alanine-scanning mutagenesis experiments. Each of the residues present at the binding site is computationally mutated to alanine. The ligand interaction energy is computed for each mutant and the corresponding ΔΔG values are computed by comparing it to the wild type protein, thus evaluating individual residue contributions towards ligand interaction. The server will thus provide clues to researchers about residues to obtain loss-of-function mutations and to understand drug resistant mutations. This web-tool can be freely accessed through the following address: http://proline.biochem.iisc.ernet.in/abscan/.

scholarly journals Residues Required for Bacillus subtilis PhoP DNA Binding or RNA Polymerase Interaction: Alanine Scanning of PhoP Effector Domain Transactivation Loop and α Helix 3

2004 ◽  
Vol 186 (5) ◽  
pp. 1493-1502 ◽  
Author(s):  
Yinghua Chen ◽  
Wael R. Abdel-Fattah ◽  
F. Marion Hulett
Keyword(s):  
Bacillus Subtilis ◽  
Dna Binding ◽  
Rna Polymerase ◽  
Transcription Activation ◽  
Phosphate Deficiency ◽  
Alanine Scanning Mutagenesis ◽  
Alanine Scanning ◽  
Α Helix

ABSTRACT Bacillus subtilis PhoP is a member of the OmpR family of response regulators that activates or represses genes of the Pho regulon upon phosphorylation by PhoR in response to phosphate deficiency. Because PhoP binds DNA and is a dimer in solution independent of its phosphorylation state, phosphorylation of PhoP may optimize DNA binding or the interaction with RNA polymerase. We describe alanine scanning mutagenesis of the PhoP α loop and α helix 3 region of PhoPC (Val190 to E214) and functional analysis of the mutated proteins. Eight residues important for DNA binding were clustered between Val202 and Arg210. Using in vivo and in vitro functional analyses, we identified three classes of mutated proteins. Class I proteins (PhoPI206A, PhoPR210A, PhoPL209A, and PhoPH208A) were phosphorylation proficient and could dimerize but could not bind DNA or activate transcription in vivo or in vitro. Class II proteins (PhoPH205A and PhoPV204A) were phosphorylation proficient and could dimerize but could not bind DNA prior to phosphorylation. Members of this class had higher transcription activation in vitro than in vivo. The class III mutants, PhoPV202A and PhoPD203A, had a reduced rate of phosphotransfer and could dimerize but could not bind DNA or activate transcription in vivo or in vitro. Seven alanine substitutions in PhoP (PhoPV190A, PhoPW191A, PhoPY193A, PhoPF195A, PhoPG197A, PhoPT199A, and PhoPR200A) that specifically affected transcription activation were broadly distributed throughout the transactivation loop extending from Val190 to as far toward the C terminus as Arg200. PhoPW191A and PhoPR200A could not activate transcription, while the other five mutant proteins showed decreased transcription activation in vivo or in vitro or both. The mutagenesis studies may indicate that PhoP has a long transactivation loop and a short α helix 3, more similar to OmpR than to PhoB of Escherichia coli.

Inactivation of a Frameshift TSH Receptor Variant Val711Phefs*18 is Due to Acquisition of a Hydrophobic Degron

2020 ◽  
Vol 106 (1) ◽  
pp. e265-e272
Author(s):  
Chiho Sugisawa ◽  
Makoto Ono ◽  
Kenichi Kashimada ◽  
Tomonobu Hasegawa ◽  
Satoshi Narumi
Keyword(s):  
Amino Acid ◽  
Congenital Hypothyroidism ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Tsh Receptor ◽  
Hek293 Cells ◽  
Luciferase Reporter ◽  
Loss Of Function ◽  
Tail Region

Abstract Context Inactivating variants of thyrotropin (thyroid-stimulating hormone; TSH) receptor (TSHR) cause congenital hypothyroidism. More than 60 such variants have been reported so far, most of which were located in the extracellular or transmembrane domain. Objective We report the identification and characterization of a frameshift TSHR variant in the intracytoplasmic C-tail region. Methods Sequencing of TSHR was performed in a patient with congenital hypothyroidism. The functionality of the identified variants was assessed by expressing TSHR in HEK293 cells and measuring TSH-dependent activation of the cAMP–response element-luciferase reporter. A series of systematic mutagenesis experiments were performed to characterize the frameshifted amino acid sequence. Results The proband was heterozygous for a known TSHR variant (p.Arg519His) and a novel frameshift TSHR variant (p.Val711Phefs*18), which removed 54 C-terminal residues and added a 17–amino acid frameshifted sequence. The loss of function of Val711Phefs*18-TSHR was confirmed in vitro, but the function of Val711*-TSHR was found to be normal. Western blotting showed the low protein expression of Val711Phefs*18-TSHR. Fusion of the frameshift sequence to green fluorescent protein or luciferase induced inactivation of them, indicating that the sequence acted as a degron. A systematic mutagenesis study revealed that the density of hydrophobic residues in the frameshift sequence determined the stability. Eight additional frameshift TSHR variants that covered all possible shifted frames in C-tail were created, and another frameshift variant (Thr748Profs*27) with similar effect was found. Conclusions We characterized a naturally occurring frameshift TSHR variant located in C-tail, and provided a unique evidence that hydrophobicity in the C-terminal region of the receptor affects protein stability.

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