RESUMO
Misfolding of the prion protein is linked to multiple neurodegenerative diseases. A better understanding of the process requires the identification and structural characterization of intermediate conformations via which misfolding proceeds. In this study, three conserved aromatic residues (Tyr168, Phe174, and Tyr217) located in the C-terminal domain of mouse PrP (wt moPrP) were mutated to Ala. The resultant mutant protein, 3A moPrP, is shown to adopt a molten globule (MG)-like native conformation. Hydrogen-deuterium exchange studies coupled with mass spectrometry revealed that for 3A moPrP, the free energy gap between the MG-like native conformation and misfolding-prone partially unfolded forms is reduced. Consequently, 3A moPrP misfolds in native conditions even in the absence of salt, unlike wt moPrP, which requires the addition of salt to misfold. 3A moPrP misfolds to a ß-rich dimer in the absence of salt, which can rapidly form an oligomer upon the addition of salt. In the presence of salt, 3A moPrP misfolds to a ß-rich oligomer about a thousand-fold faster than wt moPrP. Importantly, the misfolded structure of the dimer is similar to that of the salt-induced oligomer. Misfolding to oligomer seems to be induced at the level of the dimeric unit by monomer-monomer association, and the oligomer grows by accretion of misfolded dimeric units. Additionally, it is shown that the conserved aromatic residues collectively stabilize not only monomeric protein, but also the structural core of the ß-rich oligomers. Finally, it is also shown that 3A moPrP misfolds much faster to amyloid-fibrils than does the wt protein.
Assuntos
Proteínas Priônicas , Dobramento de Proteína , Multimerização Proteica , Animais , Camundongos , Proteínas Priônicas/química , Proteínas Priônicas/genética , Proteínas Priônicas/metabolismo , Conformação Proteica , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , MutaçãoRESUMO
Uric acid is the end product of purine metabolism in humans due to inactivation of the uricase determined by the mutated uricase gene. Uricase catalyzes the conversion of uric acid into water-soluble allantoin that is easily excreted by the kidneys. Hyperuricemia occurs when the serum concentration of uric acid exceeds its solubility (7 mg/dL). However, modifications to improve the uricase activity is under development for treating the hyperuricemia. Here we designed 7 types of human-porcine chimeric uricase by multiple sequence comparisons and targeted mutagenesis. An optimal human-porcine chimeric uricase mutant (uricase-10) with both high activity (6.33 U/mg) and high homology (91.45 %) was determined by enzyme activity measurement. The engineering uricase was further modified with PEGylation to improve the stability of recombinant protein drugs and reduce immunogenicity, uricase-10 could be more suitable for the treatment of gout and hyperuricemia theoretically.
Assuntos
Polietilenoglicóis , Proteínas Recombinantes de Fusão , Urato Oxidase , Animais , Humanos , Hiperuricemia/tratamento farmacológico , Hiperuricemia/genética , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação , Polietilenoglicóis/química , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Solubilidade , Urato Oxidase/química , Urato Oxidase/genética , Urato Oxidase/metabolismo , Ácido Úrico/metabolismoRESUMO
Intellectual developmental disorder, X-linked 104 (XLID104), caused by the FRMPD4 gene variant, is a rare X-linked genetic disease that primarily manifests as intellectual disability (ID) and language delay, and may be accompanied by behavioural abnormalities. Currently, only 11 patients from four families have been reported to carry FRMPD4 gene variants. Here, we report a rare case of a Chinese patient with XLID104 who was presented with severe ID and language impairment. Genetic testing results showed that the patient had a novel hemizygous variant on FRMPD4 inherited from the heterozygous variant NM_001368397: c.1772A>C (p.Glu591Ala) carried by his mother. To our knowledge, this variant has not been reported previously. Western blot results for the recombinant plasmid constructed in vitro indicated that the expression of the mutant protein may be reduced. Using molecular dynamics simulations, we predicted that the mutant protein may affect the interaction of the FRMPD4 protein with DLG4. In this study, we expand the spectrum of FRMPD4 variants and suggest that the clinical awareness of the genetic diagnosis of nonsyndromic ID should be strengthened.
Assuntos
Doenças Genéticas Ligadas ao Cromossomo X , Deficiência Intelectual , Criança , Humanos , Deficiência Intelectual/genética , Domínios FERM , Genes Ligados ao Cromossomo X , Doenças Genéticas Ligadas ao Cromossomo X/genética , Proteínas Mutantes/genéticaRESUMO
Huntington's disease (HD) arises from the abnormal expansion of CAG repeats in the huntingtin gene (HTT), resulting in the production of the mutant huntingtin protein (mHTT) with a polyglutamine stretch in its N-terminus. The pathogenic mechanisms underlying HD are complex and not yet fully elucidated. However, mHTT forms aggregates and accumulates abnormally in neuronal nuclei and processes, leading to disruptions in multiple cellular functions. Although there is currently no effective curative treatment for HD, significant progress has been made in developing various therapeutic strategies to treat HD. In addition to drugs targeting the neuronal toxicity of mHTT, gene therapy approaches that aim to reduce the expression of the mutant HTT gene hold great promise for effective HD therapy. This review provides an overview of current HD treatments, discusses different therapeutic strategies, and aims to facilitate future therapeutic advancements in the field.
Assuntos
Doença de Huntington , Humanos , Doença de Huntington/genética , Doença de Huntington/terapia , Terapia Genética , Proteínas MutantesRESUMO
α-Synuclein, a presynaptic neuronal protein encoded by the SNCA gene, is involved in the pathogenesis of Parkinson's disease. Point mutations and multiplications of α-synuclein (A30P and A53T) are correlated with early-onset Parkinson's disease characterized by rapid progression and poor prognosis. Currently, the clinical identification of SNCA variants, especially disease-related A30P and A53T mutants, remains challenging and also time consuming. This study aimed to develop a novel label-free detection method for distinguishing the SNCA mutants using transmission terahertz (THz) time-domain spectroscopy. The protein was spin-coated onto the quartz to form a thin film, which was measured using THz time-domain spectroscopy. The spectral characteristics of THz broadband pulse waves of α-synuclein protein variants (SNCA wild type, A30P, and A53T) at different frequencies were analyzed via Fourier transform. The amplitude A intensity (AWT, AA30P, and AA53T) and peak occurrence time in THz time-domain spectroscopy sensitively distinguished the three protein variants. The phase φ difference in THz frequency domain followed the trend of φWT > φA30P > φA53T. There was a significant difference in THz frequency amplitude A' corresponding to the frequency ranging from 0.4 to 0.66 THz (A'A53T > A'A30P > A'WT). At a frequency of 0.4-0.6 THz, the transmission T of THz waves distinguished three variants (TA53T > TA30P > TWT), whereas there was no difference in the transmission T at 0.66 THz. The SNCA wild-type protein and two mutant variants (A30P and A53T) had distinct characteristic fingerprint spectra on THz time-domain spectroscopy. This novel label-free detection method has great potential for the differential diagnosis of Parkinson's disease subtypes.
Assuntos
Mutação , Espectroscopia Terahertz , alfa-Sinucleína , alfa-Sinucleína/química , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Humanos , Proteínas Mutantes/química , Proteínas Mutantes/genéticaRESUMO
The substitution of leucine to proline at position 39 (p.P39L) in human αB-crystallin (αB-Cry) has been associated with conflicting interpretations of pathogenicity in cataracts and cardiomyopathy. This study aimed to investigate the effects of the p.P39L mutation on the structural and functional features of human αB-Cry. The mutant protein was expressed in Escherichia coli (E. coli) and purified using anion exchange chromatography. We employed a wide range of spectroscopic analyses, gel electrophoresis, transmission electron microscopy (TEM), and atomic force microscopy (AFM) techniques to investigate the structure, function, stability, and fibrillation propensity of the mutant protein. The p.P39L mutation caused significant changes in the secondary, tertiary, and quaternary structures of human αB-Cry and increased the thermal stability of the protein. The mutant αB-Cry exhibited an increased chaperone activity and an altered oligomeric size distribution, along with an increased propensity to form amyloid aggregates. It is worth mentioning, increased chaperone activity has important positive and negative effects on damaged cells related to cataracts and cardiomyopathy, particularly by interfering in the process of apoptosis. Despite the apparent positive nature of the increased chaperone activity, it is also linked to adverse consequences. This study provides important insights into the effect of proline substitution by leucine at the N-terminal region on the dual nature of chaperone activity in human αB-Cry, which can act as a double-edged sword.
Assuntos
Cardiomiopatias , Catarata , Cristalinas , Humanos , Catarata/genética , Cristalinas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Leucina , Chaperonas Moleculares/metabolismo , Proteínas Mutantes/metabolismo , Prolina/genética , Estrutura Secundária de ProteínaRESUMO
Sequencing-based, massively parallel genetic assays have enabled simultaneous characterization of the genotype-phenotype relationships for libraries encoding thousands of unique protein variants. Since plasmid transfection and lentiviral transduction have characteristics that limit multiplexing with pooled libraries, we developed a mammalian synthetic biology platform that harnesses the Bxb1 bacteriophage DNA recombinase to insert single promoterless plasmids encoding a transgene of interest into a pre-engineered "landing pad" site within the cell genome. The transgene is expressed behind a genomically integrated promoter, ensuring only one transgene is expressed per cell, preserving a strict genotype-phenotype link. Upon selecting cells based on a desired phenotype, the transgene can be sequenced to ascribe each variant a phenotypic score. We describe how to create and utilize landing pad cells for large-scale, library-based genetic experiments. Using the provided examples, the experimental template can be adapted to explore protein variants in diverse biological problems within mammalian cells.
Assuntos
Bacteriófagos , Genômica , Animais , Sequenciamento de Nucleotídeos em Larga Escala , Biblioteca Gênica , Bioensaio , Proteínas Mutantes , MamíferosRESUMO
Huntington's disease (HD) is an inherited neurodegenerative disorder caused by an expanded CAG repeat in the huntingtin (HTT) gene. The repeat-expanded HTT encodes a mutated HTT (mHTT), which is known to induce DNA double-strand breaks (DSBs), activation of the cGAS-STING pathway, and apoptosis in HD. However, the mechanism by which mHTT triggers these events is unknown. Here, we show that HTT interacts with both exonuclease 1 (Exo1) and MutLα (MLH1-PMS2), a negative regulator of Exo1. While the HTT-Exo1 interaction suppresses the Exo1-catalyzed DNA end resection during DSB repair, the HTT-MutLα interaction functions to stabilize MLH1. However, mHTT displays a significantly reduced interaction with Exo1 or MutLα, thereby losing the ability to regulate Exo1. Thus, cells expressing mHTT exhibit rapid MLH1 degradation and hyperactive DNA excision, which causes severe DNA damage and cytosolic DNA accumulation. This activates the cGAS-STING pathway to mediate apoptosis. Therefore, we have identified unique functions for both HTT and mHTT in modulating DNA repair and the cGAS-STING pathway-mediated apoptosis by interacting with MLH1. Our work elucidates the mechanism by which mHTT causes HD.
Assuntos
Doença de Huntington , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Proteínas Mutantes/genética , Doença de Huntington/genética , Doença de Huntington/metabolismo , Nucleotidiltransferases/genética , DNA , Apoptose/genética , Proteína 1 Homóloga a MutL/genéticaRESUMO
In preparation for a detailed exploration of the structural and functional aspects of the Ser2Ala mutant of human carbonic anhydrase II, we present here almost complete sequence-specific resonance assignments for 1H, 15N, and 13C. The mutation of serine to alanine at position 2, located in the N-terminal region of the enzyme, significantly alters the hydrophilic nature of the site, rendering it hydrophobic. Consequently, there is an underlying assumption that this mutation would repel water from the site. However, intriguingly, comparative analysis of the mutant structure with the wild type reveals minimal discernible differences. These assignments serve as the basis for in-depth studies on histidine dynamics, protonation states, and its intricate role in protein-water interactions and catalysis.
Assuntos
Anidrase Carbônica II , Mutação , Ressonância Magnética Nuclear Biomolecular , Humanos , Isótopos de Carbono , Anidrase Carbônica II/química , Proteínas Mutantes/química , Isótopos de NitrogênioRESUMO
BACKGROUND: PKD1, which has a relatively high mutation rate, is highly polymorphic, and the role of PKD1 is incompletely defined. In the current study, in order to determine the molecular etiology of a family with autosomal dominant polycystic kidney disease, the pathogenicity of an frameshift mutation in the PKD1 gene, c.9484delC, was evaluated. METHODS: The family clinical data were collected. Whole exome sequencing analysis determined the level of this mutation in the proband's PKD1, and Sanger sequencing and bioinformatics analysis were performed. SIFT, Polyphen2, and MutationTaster were used to evaluate the conservation of the gene and pathogenicity of the identified mutations. SWISS-MODEL was used to predict and map the protein structure of PKD1 and mutant neonate proteins. RESULTS: A novel c.9484delC (p.Arg3162Alafs*154) mutation of the PKD1 gene was identified by whole exome sequencing in the proband, which was confirmed by Sanger sequencing in his sister (II7). The same mutation was not detected in the healthy pedigree members. Random screening of 100 normal and end-stage renal disease patients did not identify the c.9484delC mutation. Bioinformatics analysis suggested that the mutation caused the 3162 nd amino acid substitution of arginine by alanine and a shift in the termination codon. As a result, the protein sequence was shortened from 4302 amino acids to 3314 amino acids, the protein structure was greatly changed, and the PLAT/LH2 domain was destroyed. Clustal analysis indicated that the altered amino acids were highly conserved in mammals. CONCLUSION: A novel mutation in the PKD1 gene has been identified in an affected Chinese family. The mutation is probably responsible for a range of clinical manifestations for which reliable prenatal diagnosis and genetic counseling may be provided.
Assuntos
Rim Policístico Autossômico Dominante , Humanos , Recém-Nascido , Alanina , China , Proteínas Mutantes , Mutação , Linhagem , Rim Policístico Autossômico Dominante/genética , Canais de Cátion TRPP/genéticaRESUMO
Noise control, together with other regulatory functions facilitated by microRNAs (miRNAs), is believed to have played important roles in the evolution of multicellular eukaryotic organisms. miRNAs can dampen protein fluctuations via enhanced degradation of messenger RNA (mRNA), but this requires compensation by increased mRNA transcription to maintain the same expression levels. The overall mechanism is metabolically expensive, leading to questions about how it might have evolved in the first place. We develop a stochastic model of miRNA noise regulation, coupled with a detailed analysis of the associated metabolic costs. Additionally, we calculate binding free energies for a range of miRNA seeds, the short sequences which govern target recognition. We argue that natural selection may have fine-tuned the Michaelis-Menten constant [Formula: see text] describing miRNA-mRNA affinity and show supporting evidence from analysis of experimental data. [Formula: see text] is constrained by seed length, and optimal noise control (minimum protein variance at a given energy cost) is achievable for seeds of 6 to 7 nucleotides in length, the most commonly observed types. Moreover, at optimality, the degree of noise reduction approaches the theoretical bound set by the Wiener-Kolmogorov linear filter. The results illustrate how selective pressure toward energy efficiency has potentially shaped a crucial regulatory pathway in eukaryotes.
Assuntos
Eucariotos , MicroRNAs , MicroRNAs/genética , Proteínas Mutantes , RNA Mensageiro , Metabolismo Energético/genéticaRESUMO
Subacute sclerosing panencephalitis (SSPE) is a rare but fatal late neurological complication of measles, caused by persistent measles virus (MeV) infection of the central nervous system. There are no drugs approved for the treatment of SSPE. Here, we followed the clinical progression of a 5-year-old SSPE patient after treatment with the nucleoside analog remdesivir, conducted a post-mortem evaluation of the patient's brain, and characterized the MeV detected in the brain. The quality of life of the patient transiently improved after the first two courses of remdesivir, but a third course had no further clinical effect, and the patient eventually succumbed to his condition. Post-mortem evaluation of the brain displayed histopathological changes including loss of neurons and demyelination paired with abundant presence of MeV RNA-positive cells throughout the brain. Next-generation sequencing of RNA isolated from the brain revealed a complete MeV genome with mutations that are typically detected in SSPE, characterized by a hypermutated M gene. Additional mutations were detected in the polymerase (L) gene, which were not associated with resistance to remdesivir. Functional characterization showed that mutations in the F gene led to a hyperfusogenic phenotype predominantly mediated by N465I. Additionally, recombinant wild-type-based MeV with the SSPE-F gene or the F gene with the N465I mutation was no longer lymphotropic but instead efficiently disseminated in neural cultures. Altogether, this case encourages further investigation of remdesivir as a potential treatment of SSPE and highlights the necessity to functionally understand SSPE-causing MeV.IMPORTANCEMeasles virus (MeV) causes acute, systemic disease and remains an important cause of morbidity and mortality in humans. Despite the lack of known entry receptors in the brain, MeV can persistently infect the brain causing the rare but fatal neurological disorder subacute sclerosing panencephalitis (SSPE). SSPE-causing MeVs are characterized by a hypermutated genome and a hyperfusogenic F protein that facilitates the rapid spread of MeV throughout the brain. No treatment against SSPE is available, but the nucleoside analog remdesivir was recently demonstrated to be effective against MeV in vitro. We show that treatment of an SSPE patient with remdesivir led to transient clinical improvement and did not induce viral escape mutants, encouraging the future use of remdesivir in SSPE patients. Functional characterization of the viral proteins sheds light on the shared properties of SSPE-causing MeVs and further contributes to understanding how those viruses cause disease.
Assuntos
Monofosfato de Adenosina , Alanina , Vírus do Sarampo , Sarampo , Panencefalite Esclerosante Subaguda , Proteínas Virais , Pré-Escolar , Humanos , Monofosfato de Adenosina/administração & dosagem , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/uso terapêutico , Alanina/administração & dosagem , Alanina/análogos & derivados , Alanina/uso terapêutico , Autopsia , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/virologia , Progressão da Doença , Evolução Fatal , Genoma Viral/genética , Sequenciamento de Nucleotídeos em Larga Escala , Sarampo/complicações , Sarampo/tratamento farmacológico , Sarampo/virologia , Vírus do Sarampo/efeitos dos fármacos , Vírus do Sarampo/genética , Vírus do Sarampo/metabolismo , Proteínas Mutantes/análise , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Qualidade de Vida , RNA Viral/análise , RNA Viral/genética , Panencefalite Esclerosante Subaguda/tratamento farmacológico , Panencefalite Esclerosante Subaguda/etiologia , Panencefalite Esclerosante Subaguda/virologia , Proteínas Virais/análise , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
αB-crystallin, a member of the small heat shock protein (sHSP) family, is expressed in diverse tissues, including the eyes, brain, muscles, and heart. This protein plays a crucial role in maintaining eye lens transparency and exhibits holdase chaperone and anti-apoptotic activities. Therefore, structural and functional changes caused by genetic mutations in this protein may contribute to the development of disorders like cataract and cardiomyopathy. Recently, the substitution of arginine 123 with tryptophan (p.R123W mutation) in human αB-crystallin has been reported to trigger cardiomyopathy. In this study, human αB-crystallin was expressed in Escherichia coli (E. coli), and the missense mutation p.R123W was created using site-directed mutagenesis. Following purification via anion exchange chromatography, the structural and functional properties of both proteins were investigated and compared using a wide range of spectroscopic and microscopic methods. The p.R123W mutation induced significant alterations in the secondary, tertiary, and quaternary structures of human αB-crystallin. This pathogenic mutation resulted in an increased ß-sheet structure and formation of protein oligomers with larger sizes compared to the wild-type protein. The mutant protein also exhibited reduced chaperone activity and lower thermal stability. Atomic force microscopy (AFM) and transmission electron microscopy (TEM) demonstrated that the p.R123W mutant protein is more prone to forming amyloid aggregates. The structural and functional changes observed in the p.R123W mutant protein, along with its increased propensity for aggregation, could impact its proper functional interaction with the target proteins in the cardiac muscle, such as calcineurin. Our results provide an explanation for the pathogenic intervention of p.R123W mutant protein in the occurrence of hypertrophic cardiomyopathy (HCM).
Assuntos
Cardiomiopatias , Escherichia coli , Humanos , Cadeia B de alfa-Cristalina/genética , Cadeia B de alfa-Cristalina/metabolismo , Cardiomiopatias/genética , Escherichia coli/metabolismo , Proteínas Mutantes/química , MutaçãoRESUMO
Proteogenomics studies generate hypotheses on protein function and provide genetic evidence for drug target prioritization. Most previous work has been conducted using affinity-based proteomics approaches. These technologies face challenges, such as uncertainty regarding target identity, non-specific binding, and handling of variants that affect epitope affinity binding. Mass spectrometry-based proteomics can overcome some of these challenges. Here we report a pQTL study using the Proteograph™ Product Suite workflow (Seer, Inc.) where we quantify over 18,000 unique peptides from nearly 3000 proteins in more than 320 blood samples from a multi-ethnic cohort in a bottom-up, peptide-centric, mass spectrometry-based proteomics approach. We identify 184 protein-altering variants in 137 genes that are significantly associated with their corresponding variant peptides, confirming target specificity of co-associated affinity binders, identifying putatively causal cis-encoded proteins and providing experimental evidence for their presence in blood, including proteins that may be inaccessible to affinity-based proteomics.
Assuntos
Proteogenômica , Proteômica , Humanos , Proteômica/métodos , Espectrometria de Massas/métodos , Proteínas/análise , Peptídeos/análise , Proteogenômica/métodos , Proteínas MutantesRESUMO
Protein structure prediction is a longstanding issue crucial for identifying new drug targets and providing a mechanistic understanding of protein functions. To enhance the progress in this field, a spectrum of computational methodologies has been cultivated. AlphaFold2 has exhibited exceptional precision in predicting wild-type protein structures, with performance exceeding that of other methods. However, predicting the structures of missense mutant proteins using AlphaFold2 remains challenging due to the intricate and substantial structural alterations caused by minor sequence variations in the mutant proteins. Molecular dynamics (MD) has been validated for precisely capturing changes in amino acid interactions attributed to protein mutations. Therefore, for the first time, a strategy entitled 'MoDAFold' was proposed to improve the accuracy and reliability of missense mutant protein structure prediction by combining AlphaFold2 with MD. Multiple case studies have confirmed the superior performance of MoDAFold compared to other methods, particularly AlphaFold2.
Assuntos
Aminoácidos , Simulação de Dinâmica Molecular , Proteínas Mutantes , Reprodutibilidade dos Testes , Mutação , Conformação ProteicaRESUMO
Diffuse intrinsic pontine gliomas (DIPGs) are deadly pediatric brain tumors, non-resectable due to brainstem localization and diffusive growth. Over 80% of DIPGs harbor a mutation in histone 3 (H3.3 or H3.1) resulting in a lysine-to-methionine substitution (H3K27M). Patients with DIPG have a dismal prognosis with no effective therapy. We show that histone deacetylase (HDAC) inhibitors lead to a significant reduction in the H3.3K27M protein (up to 80%) in multiple glioma cell lines. We discover that the SB939-mediated H3.3K27M loss is partially blocked by a lysosomal inhibitor, chloroquine. The H3.3K27M loss is facilitated by co-occurrence of H2A.Z, as evidenced by the knockdown of H2A.Z isoforms. Chromatin immunoprecipitation sequencing (ChIP-seq) analysis confirms the occupancy of H3.3K27M and H2A.Z at the same SB939-inducible genes. We discover a mechanism showing that HDAC inhibition in DIPG leads to pharmacological modulation of the oncogenic H3.3K27M protein levels. These findings show the possibility of directly targeting the H3.3K27M oncohistone.
Assuntos
Neoplasias Encefálicas , Glioma Pontino Intrínseco Difuso , Glioma , Humanos , Criança , Histonas , Proteínas Mutantes , Glioma/genética , Neoplasias Encefálicas/genética , Inibidores de Histona Desacetilases/farmacologiaRESUMO
The morphology of cells is dynamic and mediated by genetic and environmental factors. Characterizing how genetic variation impacts cell morphology can provide an important link between disease association and cellular function. Here, we combine genomic sequencing and high-content imaging approaches on iPSCs from 297 unique donors to investigate the relationship between genetic variants and cellular morphology to map what we term cell morphological quantitative trait loci (cmQTLs). We identify novel associations between rare protein altering variants in WASF2, TSPAN15, and PRLR with several morphological traits related to cell shape, nucleic granularity, and mitochondrial distribution. Knockdown of these genes by CRISPRi confirms their role in cell morphology. Analysis of common variants yields one significant association and nominate over 300 variants with suggestive evidence (P < 10-6) of association with one or more morphology traits. We then use these data to make predictions about sample size requirements for increasing discovery in cellular genetic studies. We conclude that, similar to molecular phenotypes, morphological profiling can yield insight about the function of genes and variants.
Assuntos
Células-Tronco Pluripotentes Induzidas , Locos de Características Quantitativas , Mapeamento Cromossômico , Locos de Características Quantitativas/genética , Núcleo Celular , Forma Celular , Proteínas MutantesRESUMO
BACKGROUND: Primary autosomal recessive microcephaly (MCPH) is a rare developmental disorder characterized by cognitive impairment, delayed neurodevelopment, and reduced brain size. It is a genetically heterogeneous condition, and several genes have been identified as associated with MCPH. METHODS AND RESULTS: In this study, we utilized whole-exome sequencing (WES) to identify disease-causing variations in two brothers from an Iranian family affected by MCPH, who had consanguineous parents. In the patients, we detected a novel homozygous missense mutation (c.806A > G, p.Gln269Arg) in the TEDC1 gene in one of the patients. Co-segregation analysis using Sanger sequencing confirmed that this variant was inherited from parents. The identified variant was evaluated for its pathogenicity and novelty using various databases. Additionally, bioinformatics tools were employed to predict the three-dimensional structure of the mutant TEDC1 protein. CONCLUSIONS: This study presents the second documented report of a mutation in the TEDC1 gene associated with MCPH. The identification of this novel biallelic mutation as a causative factor for MCPH in the proband further underscores the utility of genetic testing techniques, such as WES, as reliable diagnostic tools for individuals with this condition.
Assuntos
Disfunção Cognitiva , Microcefalia , Masculino , Humanos , Microcefalia/genética , Irã (Geográfico) , Consanguinidade , Proteínas Mutantes , Mutação/genéticaRESUMO
BACKGROUND: Müllerian duct anomalies (MDAs) are congenital developmental disorders exhibiting as a variety of malformations of female reproductive tract. The identified etiology of MDAs is limited. The present study aimed to unravel the underlying genetic causes of MDAs. METHODS: Rare variants in androgen receptor (AR) were called from the cohort consists of patients with MDAs and underwent whole exome sequencing (WES) at Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing, China. Sanger sequencing was used to confirm the causative genetic mutations. In silico analysis were used to classify the pathogenicity of each variant. Molecular modeling and simulations were conducted to investigate the conformational changes between the wild-type (WT) and mutant proteins. RESULTS: A total of 3 rare heterozygous variants in AR from the MDAs cohort in our institution were identified, with unknown effects. All variants were missense mutations, including c.173A > T, c.558C > A and c.1208C > T, and were absent or rare in East Asian populations in Genome Aggregation Database and the Exome Aggregation Consortium Database. According to the American College of Medical Genetics and Genomics guidelines, c.1208C > T variant was classified as likely pathogenic, while the other two were variants of uncertain significance. During molecular dynamics simulations, WT and mutant proteins all reached stable status according to root-mean-square variance. Values of radius of gyration showed that Q58L and S186R protein would be more compact than WT, while the structure of A403V became looser. Despite, in comparison with WT, the number of hydrogen bonds increased in Q58L, while decreased in the other two variants. Furthermore, the solvent-accessible surface area diminished in Q58L and A403V while enlarged in S186R proteins, when compared with WT. CONCLUSIONS: To our knowledge, this is the first report regarding the association of AR mutation and MDAs. The identification of these variants, predicted to damage the structure and function of AR protein, not only expanded the mutational spectrum of causative genes of MDAs, but provide novel molecular genetic reference for future studies.
Assuntos
Ductos Paramesonéfricos , Receptores Androgênicos , Gravidez , Humanos , Feminino , Ductos Paramesonéfricos/anormalidades , Receptores Androgênicos/genética , Mutação , Mutação de Sentido Incorreto , Proteínas MutantesRESUMO
Biomolecule misfolding and aggregation play a major role in human disease, spanning from neurodegeneration to cancer. Inhibition of these processes is of considerable interest, and due to the multifactorial nature of these diseases, the development of drugs that act on multiple pathways simultaneously is a promising approach. This Feature Article focuses on the development of multifunctional molecules designed to inhibit the misfolding and aggregation of the amyloid-ß (Aß) peptide in Alzheimer's disease (AD), and the mutant p53 protein in cancer. While for the former, the goal is to accelerate the removal of the Aß peptide and associated aggregates, for the latter, the goal is reactivation via stabilization of the active folded form of mutant p53 protein and/or aggregation inhibition. Due to the similar aggregation pathway of the Aß peptide and mutant p53 protein, a common therapeutic approach may be applicable.