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Molecules (Basel, Switzerland) Sep 2020The finding that bitter taste receptors are expressed in numerous tissues outside the oral cavity and fulfill important roles in metabolic regulation, innate immunity... (Review)
Review
The finding that bitter taste receptors are expressed in numerous tissues outside the oral cavity and fulfill important roles in metabolic regulation, innate immunity and respiratory control, have made these receptors important targets for drug discovery. Efficient drug discovery depends heavily on detailed knowledge on structure-function-relationships of the target receptors. Unfortunately, experimental structures of bitter taste receptors are still lacking, and hence, the field relies mostly on structures obtained by molecular modeling combined with functional experiments and point mutageneses. The present article summarizes the current knowledge on the structure-function relationships of human bitter taste receptors. Although these receptors are difficult to express in heterologous systems and their homology with other G protein-coupled receptors is very low, detailed information are available at least for some of these receptors.
Topics: Humans; Immunity, Innate; Models, Molecular; Point Mutation; Receptors, G-Protein-Coupled; Structure-Activity Relationship
PubMed: 32993119
DOI: 10.3390/molecules25194423 -
Molecular Therapy : the Journal of the... Sep 2020CRISPR-mediated DNA base editors, which include cytosine base editors (CBEs) and adenine base editors (ABEs), are promising tools that can induce point mutations at... (Review)
Review
CRISPR-mediated DNA base editors, which include cytosine base editors (CBEs) and adenine base editors (ABEs), are promising tools that can induce point mutations at desired sites in a targeted manner to correct or disrupt gene expression. Their high editing efficiency, coupled with their ability to generate a targeted mutation without generating a DNA double-strand break (DSB) or requiring a donor DNA template, suggests that DNA base editors will be useful for treating genetic diseases, among other applications. However, this hope has recently been challenged by the discovery of DNA base editor shortcomings, including off-target DNA editing, the generation of bystander mutations, and promiscuous deamination effects in both DNA and RNA, which arise from the main DNA base editor constituents, a Cas nuclease variant and a deaminase. In this review, we summarize information about the DNA base editors that have been developed to date, introduce their associated potential challenges, and describe current efforts to minimize or mitigate those issues of DNA base editors.
Topics: CRISPR-Associated Protein 9; CRISPR-Cas Systems; DNA; DNA Breaks, Double-Stranded; DNA Replication; Deamination; Gene Editing; Humans; Point Mutation; RNA, Guide, CRISPR-Cas Systems
PubMed: 32763143
DOI: 10.1016/j.ymthe.2020.07.021 -
Journal of Molecular Modeling Mar 2023Crimean-Congo haemorrhagic fever virus (CCHFV) has tripartite RNA genome and is endemic in various countries of Asia, Africa and Europe.
INTRODUCTION
Crimean-Congo haemorrhagic fever virus (CCHFV) has tripartite RNA genome and is endemic in various countries of Asia, Africa and Europe.
METHOD
The present study is focused on mutation profiling of CCHFV L segment and phylogenetic clustering of protein dataset into six CCHFV genotypes.
RESULTS
Phylogenetic tree rooted with NCBI reference sequence (YP_325663.1) indicated less divergence from genotype III and the sequences belonging to same genotypes have shown less divergence among each other. Mutation frequency at 729 mutated positions was calculated and 563, 49, 33, 46 and 38 amino acid positions were found to be mutated at mutation frequency intervals of 0-0.2, 0.21-0.4, 0.41-0.6, 0.61-0.8 and 0.81-1.0 respectively. Thirty-eight highly frequent mutations (0.81-1.0 interval) were found in all genotypes and mapping in L segment (encoded for RdRp) revealed four mutations (V2074I, I2134T/A, V2148A and Q2695H/R) in catalytic site domain and no mutation in OTU domain. Molecular dynamic simulation and in silico analysis showed that catalytic site domain displayed large deviation and fluctuation upon introduction of these point mutations.
CONCLUSION
Overall study provides strong evidence that OTU domain is highly conserved and less prone to mutation whereas point mutations recorded in catalytic domain have affected the stability of protein and were found to be persistent in the large population.
Topics: Catalytic Domain; Phylogeny; Mutation; Point Mutation; RNA
PubMed: 36877258
DOI: 10.1007/s00894-023-05487-7 -
Molecules (Basel, Switzerland) Aug 2021Cytochrome is a small globular protein whose main physiological role is to shuttle electrons within the mitochondrial electron transport chain. This protein has been... (Review)
Review
Cytochrome is a small globular protein whose main physiological role is to shuttle electrons within the mitochondrial electron transport chain. This protein has been widely investigated, especially as a paradigmatic system for understanding the fundamental aspects of biological electron transfer and protein folding. Nevertheless, cytochrome can also be endowed with a non-native catalytic activity and be immobilized on an electrode surface for the development of third generation biosensors. Here, an overview is offered of the most significant examples of such a functional transformation, carried out by either point mutation(s) or controlled unfolding. The latter can be induced chemically or upon protein immobilization on hydrophobic self-assembled monolayers. We critically discuss the potential held by these systems as core constituents of amperometric biosensors, along with the issues that need to be addressed to optimize their applicability and response.
Topics: Biosensing Techniques; Electrochemistry; Electrons; Oxidation-Reduction; Point Mutation; Protein Folding; Proteins
PubMed: 34443538
DOI: 10.3390/molecules26164950 -
Journal of Neurology Jan 2020ATP8A2-related disorders are autosomal recessive conditions that associate encephalopathy with or without hypotonia, psychomotor delay, abnormal movements, chorea,...
ATP8A2-related disorders are autosomal recessive conditions that associate encephalopathy with or without hypotonia, psychomotor delay, abnormal movements, chorea, tremor, optic atrophy and cerebellar atrophy (CARMQ4). Through a multi-centric collaboration, we identified six point mutations (one splice site and five missense mutations) involving ATP8A2 in six individuals from five families. Two patients from one family with the homozygous p.Gly585Val mutation had a milder presentation without encephalopathy. Expression and functional studies of the missense mutations demonstrated that protein levels of four of the five missense variants were very low and lacked phosphatidylserine-activated ATPase activity. One variant p.Ile215Leu, however, expressed at normal levels and displayed phospholipid-activated ATPase activity similar to the non-mutated protein. We therefore expand for the first time the phenotype related to ATP8A2 mutations to less severe forms characterized by cerebellar ataxia without encephalopathy and suggest that ATP8A2 should be analyzed for all cases of syndromic or non-syndromic recessive or sporadic ataxia.
Topics: Adenosine Triphosphatases; Adult; Cerebellar Ataxia; Child; Child, Preschool; Consanguinity; Female; Genes, Recessive; Humans; Infant; Male; Mutation, Missense; Pedigree; Phenotype; Phospholipid Transfer Proteins; Point Mutation
PubMed: 31612321
DOI: 10.1007/s00415-019-09579-4 -
The Tohoku Journal of Experimental... Aug 2022Virus genome mutation profiles with insertion, deletion, and point mutations have recently been revealed to differ remarkably between viruses. In RNA viruses like human...
Virus genome mutation profiles with insertion, deletion, and point mutations have recently been revealed to differ remarkably between viruses. In RNA viruses like human coronaviruses or influenza viruses, genome samples collected over two to three decades usually show point mutations in 10-20% of the bases, while the rate of insertion and/or deletion mutations (indels) largely depends on the virus. This study evaluates the mutation profiles of DNA viruses by comparing a recently sampled genome of human adenovirus species C type 2 (isolate SG06/HAdvC2/2016) with a genome of the same species sampled in the 1970s. It was found insertions of 23 bases at seven sites and deletions of 22 bases at nine sites. The longest indels were 6-base insertions in E2B and L4. All indels in the coding regions were in-frame mutations with base lengths in multiples of three. In the non-coding regions, the lengths of the indels ranged from 1-4 consecutive bases. Long indels with more than 10 consecutive bases, which comprise nearly half of indels in the SARS-CoV-2 genome, were absent. In other sites, the point mutation rate was approximately 0.3%, which was significantly lower than in RNA viruses. In summary, the estimated point mutation rate in human adenovirus species C type 2 (HAdvC-2) was over 10 times lower than in RNA viruses. Unlike the relatively long indels in the SARS-CoV-2 genome, the indels in HAdvC-2 were short, with 6 or fewer consecutive bases.
Topics: Adenoviruses, Human; Genome, Viral; INDEL Mutation; Point Mutation; SARS-CoV-2
PubMed: 35705320
DOI: 10.1620/tjem.2022.J049 -
Cells Dec 2023We report the first correction from prime editing a mutation in the gene, paving the way to gene therapies for RYR1-related myopathies. The gene codes for a calcium...
We report the first correction from prime editing a mutation in the gene, paving the way to gene therapies for RYR1-related myopathies. The gene codes for a calcium channel named Ryanodine receptor 1, which is expressed in skeletal muscle fibers. The failure of this channel causes muscle weakness in patients, which leads to motor disabilities. Currently, there are no effective treatments for these diseases, which are mainly caused by point mutations. Prime editing allows for the modification of precise nucleotides in the DNA. Our results showed a 59% correction rate of the T4709M mutation in the gene in human myoblasts by RNA delivery of the prime editing components. It is to be noted that T4709M is recessive and, thus, persons having a heterozygous mutation are healthy. These results are the first demonstration that correcting mutations in the RYR1 gene is possible.
Topics: Humans; Muscle Weakness; Muscular Diseases; Mutation; Point Mutation; Ryanodine Receptor Calcium Release Channel
PubMed: 38201236
DOI: 10.3390/cells13010031 -
G3 (Bethesda, Md.) Aug 2020The mutation rate and mutations' effects on fitness are crucial to evolution. Mutation rates are under selection due to linkage between mutation rate modifiers and...
The mutation rate and mutations' effects on fitness are crucial to evolution. Mutation rates are under selection due to linkage between mutation rate modifiers and mutations' effects on fitness. The linkage between a higher mutation rate and more beneficial mutations selects for higher mutation rates, while the linkage between a higher mutation rate and more deleterious mutations selects for lower mutation rates. The net direction of selection on mutations rates depends on the fitness landscape, and a great deal of work has elucidated the fitness landscapes of mutations. However, tests of the effect of varying a mutation rate on evolution in a single organism in a single environment have been difficult. This has been studied using strains of antimutators and mutators, but these strains may differ in additional ways and typically do not allow for continuous variation of the mutation rate. To help investigate the effects of the mutation rate on evolution, we have genetically engineered a strain of with a point mutation rate that can be smoothly varied over two orders of magnitude. We did this by engineering a strain with inducible control of the mismatch repair proteins MutH and MutL. We used this strain in an approximately 350 generation evolution experiment with controlled variation of the mutation rate. We confirmed the construct and the mutation rate were stable over this time. Sequencing evolved strains revealed a higher number of single nucleotide polymorphisms at higher mutations rates, likely due to either the beneficial effects of these mutations or their linkage to beneficial mutations.
Topics: Escherichia coli; Escherichia coli Proteins; Mutation; Mutation Rate; Point Mutation; Selection, Genetic
PubMed: 32503807
DOI: 10.1534/g3.120.401124 -
CRISPR/Cas9-Mediated Point Mutation in Prolongs Protein Half-Life and Reverses Effects Allelic Loss.Cancer Research Nov 2020is the most commonly deleted gene in prostate cancer and is a gatekeeper suppressor. is haploinsufficient, and pathogenic reduction in protein levels may result from...
is the most commonly deleted gene in prostate cancer and is a gatekeeper suppressor. is haploinsufficient, and pathogenic reduction in protein levels may result from genetic loss, decreased transcription, and increased protein degradation caused by inflammation or PTEN loss. NKX3.1 acts by retarding proliferation, activating antioxidants, and enhancing DNA repair. DYRK1B-mediated phosphorylation at serine 185 of NKX3.1 leads to its polyubiquitination and proteasomal degradation. Because NKX3.1 protein levels are reduced, but never entirely lost, in prostate adenocarcinoma, enhancement of NKX3.1 protein levels represents a potential therapeutic strategy. As a proof of principle, we used CRISPR/Cas9-mediated editing to engineer a point mutation in murine to code for a serine to alanine missense at amino acid 186, the target for Dyrk1b phosphorylation. , and mice were analyzed over one year to determine the levels of Nkx3.1 expression and effects of the mutant protein on the prostate. Allelic loss of caused reduced levels of Nkx3.1 protein, increased proliferation, and prostate hyperplasia and dysplasia, whereas mouse prostates had increased levels of Nkx3.1 protein, reduced prostate size, normal histology, reduced proliferation, and increased DNA end labeling. At 2 months of age, when all mice had normal prostate histology, mice demonstrated indices of metabolic activation, DNA damage response, and stress response. These data suggest that modulation of Nkx3.1 levels alone can exert long-term control over premalignant changes and susceptibility to DNA damage in the prostate. SIGNIFICANCE: These findings show that prolonging the half-life of Nkx3.1 reduces proliferation, enhances DNA end-labeling, and protects from DNA damage, ultimately blocking the proneoplastic effects of allelic loss.
Topics: Animals; CRISPR-Cas Systems; Gene Editing; Homeodomain Proteins; Male; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Point Mutation; Prostatic Neoplasms; Transcription Factors
PubMed: 32943441
DOI: 10.1158/0008-5472.CAN-20-1742 -
The Journal of Molecular Diagnostics :... Jun 2021Mitochondria harbor multiple copies of a maternally inherited nonnuclear genome. Point mutations, deletions, or depletion of the mitochondrial DNA (mtDNA) are associated...
Mitochondria harbor multiple copies of a maternally inherited nonnuclear genome. Point mutations, deletions, or depletion of the mitochondrial DNA (mtDNA) are associated with various human diseases. mtDNA defects are currently studied using Sanger sequencing, Southern blot, and long and quantitative PCR. However, these technologies are expensive and are limited in speed, throughput, and sensitivity. Recently, next-generation sequencing (NGS) has been used to study mtDNA defects; however, its potential applications still need to be fully validated. We analyzed mtDNA from 16 control samples and 33 affected samples, which were previously investigated by traditional techniques. Different NGS approaches were tested, using classic library preparation based on PCR amplifications and an innovative PCR-free protocol, defining their suitability and utility for: i) generating full accurate mtDNA sequence, ii) assessing heteroplasmy for single point mutations with high accuracy, and iii) detecting break positions and heteroplasmy of single large deletions. This study confirmed that PCR-based library preparations are appropriate for the first two points and showed that a new PCR-free method gave the best results for the third aim. This study tested different approaches and describes an innovative PCR-free protocol, suitable for detection and heteroplasmy quantification of mtDNA single large deletions. NGS may become the method of choice for genetic analysis on mtDNA.
Topics: DNA, Mitochondrial; Genetic Testing; High-Throughput Nucleotide Sequencing; Humans; Point Mutation; Sequence Deletion
PubMed: 33781964
DOI: 10.1016/j.jmoldx.2021.03.002