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ACS Chemical Neuroscience Jun 2022Parkinson's Disease (PD) is characterized by the accumulation of Lewy bodies in dopaminergic neurons. The main protein component of Lewy bodies, α-synuclein (αS), is...
Parkinson's Disease (PD) is characterized by the accumulation of Lewy bodies in dopaminergic neurons. The main protein component of Lewy bodies, α-synuclein (αS), is also firmly linked to PD through the identification of a number of single point mutations that are autosomal dominant for early-onset disease. Consequently, the misfolding and subsequent aggregation of αS is thought to be a key stage in the development and progression of PD. Therefore, modulating the aggregation pathway of αS is an attractive therapeutic target. Owing to the fact that all but one of the familial mutations is located in the preNAC 45-54 region of αS, we previously designed a semi-rational library using this sequence as a design scaffold. The 45-54 peptide library was screened using a protein-fragment complementation assay approach, leading to the identification of the 4554W peptide. The peptide was subsequently found to be effective in inhibiting primary nucleation of αS, the earliest stage of the aggregation pathway. Here, we build upon this previous work by screening the same 45-54 library against five of the known αS single-point mutants that are associated with early-onset PD (A30P, E46K, H50Q, G51D, and A53T). These point mutations lead to a rapid acceleration of PD pathology by altering either the rate or type of aggregates formed. All ultimately lead to earlier disease onset and were therefore used to enforce increased assay stringency during the library screening process. The ultimate aim was to identify a peptide that is effective against not only the familial αS variant from which it has been selected but that is also effective against WT αS. Screening resulted in five peptides that shared common residues at some positions, while deviating at others. All reduced aggregation of the respective target, with several also identified to be effective at reducing aggregation when incubated with other variants. In addition, our results demonstrate that a previously optimized peptide, 4554W(N6A), is highly effective against not only WT αS but also several of the single-point mutant forms and hence is a suitable baseline for further work toward a PD therapeutic.
Topics: Humans; Mutation; Parkinson Disease; Peptides; Point Mutation; alpha-Synuclein
PubMed: 35613323
DOI: 10.1021/acschemneuro.2c00190 -
British Journal of Cancer Feb 2024Alterations of FLT3 are among the most common driver events in acute leukaemia with important clinical implications, since it allows patient classification into...
BACKGROUND
Alterations of FLT3 are among the most common driver events in acute leukaemia with important clinical implications, since it allows patient classification into prognostic groups and the possibility of personalising therapy thanks to the availability of FLT3 inhibitors. Most of the knowledge on FLT3 implications comes from the study of acute myeloid leukaemia and so far, few studies have been performed in other leukaemias.
METHODS
A comprehensive genomic (DNA-seq in 267 patients) and transcriptomic (RNA-seq in 160 patients) analysis of FLT3 in 342 childhood acute lymphoblastic leukaemia (ALL) patients was performed. Mutations were functionally characterised by in vitro experiments.
RESULTS
Point mutations (PM) and internal tandem duplications (ITD) were detected in 4.3% and 2.7% of the patients, respectively. A new activating mutation of the TKD, G846D, conferred oncogenic properties and sorafenib resistance. Moreover, a novel alteration involving the circularisation of read-through transcripts (rt-circRNAs) was observed in 10% of the cases. Patients presenting FLT3 alterations exhibited higher levels of the receptor. In addition, patients with ZNF384- and MLL/KMT2A-rearranged ALL, as well as hyperdiploid subtype, overexpressed FLT3.
DISCUSSION
Our results suggest that specific ALL subgroups may also benefit from a deeper understanding of the biology of FLT3 alterations and their clinical implications.
Topics: Humans; Leukemia, Myeloid, Acute; Mutation; Transcription Factors; Point Mutation; Precursor Cell Lymphoblastic Leukemia-Lymphoma; fms-Like Tyrosine Kinase 3
PubMed: 38049555
DOI: 10.1038/s41416-023-02511-8 -
Viruses Apr 2022The spread of SARS-CoV-2 variants in the population depends on their ability to anchor the ACE2 receptor in the host cells. Differences in the electrostatic potentials... (Review)
Review
The spread of SARS-CoV-2 variants in the population depends on their ability to anchor the ACE2 receptor in the host cells. Differences in the electrostatic potentials of the spike protein RBD (electropositive/basic) and ACE2 receptor (electronegative/acidic) play a key role in both the rapprochement and the recognition of the coronavirus by the cell receptors. Accordingly, point mutations that result in an increase in electropositively charged residues, e.g., arginine and lysine, especially in the RBD of spike proteins in the SARS-CoV-2 variants, could contribute to their spreading capacity by favoring their recognition by the electronegatively charged ACE2 receptors. All SARS-CoV-2 variants that have been recognized as being highly transmissible, such as the kappa (κ), delta (δ) and omicron (o) variants, which display an enhanced electropositive character in their RBDs associated with a higher number of lysine- or arginine-generating point mutations. Lysine and arginine residues also participate in the enhanced RBD-ACE2 binding affinity of the omicron variant, by creating additional salt bridges with aspartic and glutamic acid residues from ACE2. However, the effects of lysine- and arginine-generating point mutations on infectivity is more contrasted, since the overall binding affinity of omicron RBD for ACE2 apparently results from some epistasis among the whole set of point mutations.
Topics: Angiotensin-Converting Enzyme 2; Arginine; COVID-19; Humans; Lysine; Mutation; Point Mutation; Protein Binding; SARS-CoV-2; Spike Glycoprotein, Coronavirus
PubMed: 35458513
DOI: 10.3390/v14040783 -
Disease Models & Mechanisms Aug 2023High-throughput DNA sequencing studies increasingly associate DNA variants with congenital heart disease (CHD). However, functional modeling is a crucial prerequisite...
High-throughput DNA sequencing studies increasingly associate DNA variants with congenital heart disease (CHD). However, functional modeling is a crucial prerequisite for translating genomic data into clinical care. We used CRISPR-Cas9-mediated targeting of 12 candidate genes in the vertebrate model medaka (Oryzias latipes), five of which displayed a novel cardiovascular phenotype spectrum in F0 (crispants): mapre2, smg7, cdc42bpab, ankrd11 and myrf, encoding a transcription factor recently linked to cardiac-urogenital syndrome. Our myrf mutant line showed particularly prominent embryonic cardiac defects recapitulating phenotypes of pediatric patients, including hypoplastic ventricle. Mimicking human mutations, we edited three sites to generate specific myrf single-nucleotide variants via cytosine and adenine base editors. The Glu749Lys missense mutation in the conserved intramolecular chaperon autocleavage domain fully recapitulated the characteristic myrf mutant phenotype with high penetrance, underlining the crucial function of this protein domain. The efficiency and scalability of base editing to model specific point mutations accelerate gene validation studies and the generation of human-relevant disease models.
Topics: Humans; Child; Gene Editing; Mutation; Point Mutation; Transcription Factors; Heart Defects, Congenital; CRISPR-Cas Systems
PubMed: 37584388
DOI: 10.1242/dmm.049811 -
Biomolecules Apr 2023The Lewy bodies and Lewy neurites are key pathological hallmarks of Parkinson's disease (PD). Single-point mutations associated with familial PD cause α-synuclein...
The Lewy bodies and Lewy neurites are key pathological hallmarks of Parkinson's disease (PD). Single-point mutations associated with familial PD cause α-synuclein (α-Syn) aggregation, leading to the formation of Lewy bodies and Lewy neurites. Recent studies suggest α-Syn nucleates through liquid-liquid phase separation (LLPS) to form amyloid aggregates in a condensate pathway. How PD-associated mutations affect α-Syn LLPS and its correlation with amyloid aggregation remains incompletely understood. Here, we examined the effects of five mutations identified in PD, A30P, E46K, H50Q, A53T, and A53E, on the phase separation of α-Syn. All other α-Syn mutants behave LLPS similarly to wild-type (WT) α-Syn, except that the E46K mutation substantially promotes the formation of α-Syn condensates. The mutant α-Syn droplets fuse to WT α-Syn droplets and recruit α-Syn monomers into their droplets. Our studies showed that α-Syn A30P, E46K, H50Q, and A53T mutations accelerated the formation of amyloid aggregates in the condensates. In contrast, the α-Syn A53E mutant retarded the aggregation during the liquid-to-solid phase transition. Finally, we observed that WT and mutant α-Syn formed condensates in the cells, whereas the E46K mutation apparently promoted the formation of condensates. These findings reveal that familial PD-associated mutations have divergent effects on α-Syn LLPS and amyloid aggregation in the phase-separated condensates, providing new insights into the pathogenesis of PD-associated α-Syn mutations.
Topics: Humans; Parkinson Disease; alpha-Synuclein; Mutation; Lewy Bodies; Point Mutation; Amyloid; Amyloidogenic Proteins
PubMed: 37238596
DOI: 10.3390/biom13050726 -
Microbiology Spectrum Feb 2022Rapid onsite whole-genome sequencing of two suspected severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) N gene diagnostic escape samples revealed a previously...
Rapid onsite whole-genome sequencing of two suspected severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) N gene diagnostic escape samples revealed a previously unreported N gene point mutation at genome position 29195. Because the G29195T mutation occurs within a region probed by a commonly referenced U.S. CDC N gene reverse transcription (RT)-PCR assay, we hypothesize that the G29195T mutation rendered the N gene target of a proprietary commercial assay undetectable. The putative diagnostic escape G29195T mutation demonstrates the need for nearly real-time surveillance, as emergence of a novel SARS-CoV-2 variant with the potential to escape diagnostic tests continues to be a threat. Accurate diagnostic detection of SARS-CoV-2 currently depends on the large-scale deployment of RT-PCR assays. SARS-CoV-2 RT-PCR assays target predetermined regions in the viral genomes by complementary binding of primers and probes to nucleic acid sequences in the clinical samples. Potential diagnostic escapes, such as those of clinical samples harboring the G29195T mutation, may result in false-negative SARS-CoV-2 RT-PCR results. The rapid detection and sharing of potential diagnostic escapes are essential for diagnostic laboratories and manufacturers around the world, to optimize their assays as SARS-CoV-2 continues to evolve.
Topics: COVID-19; Point Mutation; Reverse Transcriptase Polymerase Chain Reaction; SARS-CoV-2
PubMed: 35019683
DOI: 10.1128/spectrum.02223-21 -
Stem Cells Translational Medicine May 2020
Review
Topics: Anemia, Sickle Cell; Humans; Point Mutation
PubMed: 32329241
DOI: 10.1002/sctm.20-0135 -
International Journal of Molecular... Dec 2023spp. are ascomycete fungi and cause anthracnose disease in numerous crops of economic significance. The genomes of these fungi are distributed among ten core... (Review)
Review
spp. are ascomycete fungi and cause anthracnose disease in numerous crops of economic significance. The genomes of these fungi are distributed among ten core chromosomes and two to three minichromosomes. While the core chromosomes regulate fungal growth, development and virulence, the extent to which the minichromosomes are involved in these processes is still uncertain. Here, we discuss the minichromosomes of three hemibiotrophic pathogens, i.e., , and . These minichromosomes are typically less than one megabase in length, characterized by containing higher repetitive DNA elements, lower GC content, higher frequency of repeat-induced point mutations (RIPMs) and sparse gene distribution. Molecular genetics and functional analyses have revealed that these pathogens harbor one conditionally dispensable minichromosome, which is dispensable for fungal growth and development but indispensable for fungal virulence on hosts. They appear to be strain-specific innovations and are highly compartmentalized into AT-rich and GC-rich blocks, resulting from RIPMs, which may help protect the conditionally dispensable minichromosomes from erosion of already scarce genes, thereby helping the pathogens maintain adaptability on hosts. Overall, understanding the mechanisms underlying the conditional dispensability of these minichromosomes could lead to new strategies for controlling anthracnose disease in crops.
Topics: Colletotrichum; Virulence; Crops, Agricultural; Point Mutation; Uncertainty
PubMed: 38203369
DOI: 10.3390/ijms25010198 -
Nucleic Acids Research Jan 2021The majority of naturally occurring proteins have evolved to function under mild conditions inside the living organisms. One of the critical obstacles for the use of...
The majority of naturally occurring proteins have evolved to function under mild conditions inside the living organisms. One of the critical obstacles for the use of proteins in biotechnological applications is their insufficient stability at elevated temperatures or in the presence of salts. Since experimental screening for stabilizing mutations is typically laborious and expensive, in silico predictors are often used for narrowing down the mutational landscape. The recent advances in machine learning and artificial intelligence further facilitate the development of such computational tools. However, the accuracy of these predictors strongly depends on the quality and amount of data used for training and testing, which have often been reported as the current bottleneck of the approach. To address this problem, we present a novel database of experimental thermostability data for single-point mutants FireProtDB. The database combines the published datasets, data extracted manually from the recent literature, and the data collected in our laboratory. Its user interface is designed to facilitate both types of the expected use: (i) the interactive explorations of individual entries on the level of a protein or mutation and (ii) the construction of highly customized and machine learning-friendly datasets using advanced searching and filtering. The database is freely available at https://loschmidt.chemi.muni.cz/fireprotdb.
Topics: Computational Biology; Databases, Protein; Datasets as Topic; Internet; Machine Learning; Models, Molecular; Molecular Sequence Annotation; Point Mutation; Protein Stability; Proteins; Software
PubMed: 33166383
DOI: 10.1093/nar/gkaa981 -
Cellular and Molecular Neurobiology Oct 2023The BAF (BRG1/BRM-associated factor) chromatin remodelling complex is essential for the regulation of DNA accessibility and gene expression during neuronal...
The BAF (BRG1/BRM-associated factor) chromatin remodelling complex is essential for the regulation of DNA accessibility and gene expression during neuronal differentiation. Mutations of its core subunit SMARCB1 result in a broad spectrum of pathologies, including aggressive rhabdoid tumours or neurodevelopmental disorders. Other mouse models have addressed the influence of a homo- or heterozygous loss of Smarcb1, yet the impact of specific non-truncating mutations remains poorly understood. Here, we have established a new mouse model for the carboxy-terminal Smarcb1 c.1148del point mutation, which leads to the synthesis of elongated SMARCB1 proteins. We have investigated its impact on brain development in mice using magnetic resonance imaging, histology, and single-cell RNA sequencing. During adolescence, Smarcb1 mice demonstrated rather slow weight gain and frequently developed hydrocephalus including enlarged lateral ventricles. In embryonic and neonatal stages, mutant brains did not differ anatomically and histologically from wild-type controls. Single-cell RNA sequencing of brains from newborn mutant mice revealed that a complete brain including all cell types of a physiologic mouse brain is formed despite the SMARCB1 mutation. However, neuronal signalling appeared disturbed in newborn mice, since genes of the AP-1 transcription factor family and neurite outgrowth-related transcripts were downregulated. These findings support the important role of SMARCB1 in neurodevelopment and extend the knowledge of different Smarcb1 mutations and their associated phenotypes.
Topics: Animals; Mice; Hydrocephalus; Mutation; Point Mutation; Signal Transduction; Transcription Factor AP-1
PubMed: 37219662
DOI: 10.1007/s10571-023-01361-5