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1.
PLoS Comput Biol ; 17(8): e1009284, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34347784

RESUMO

Modeling the impact of amino acid mutations on protein-protein interaction plays a crucial role in protein engineering and drug design. In this study, we develop GeoPPI, a novel structure-based deep-learning framework to predict the change of binding affinity upon mutations. Based on the three-dimensional structure of a protein, GeoPPI first learns a geometric representation that encodes topology features of the protein structure via a self-supervised learning scheme. These representations are then used as features for training gradient-boosting trees to predict the changes of protein-protein binding affinity upon mutations. We find that GeoPPI is able to learn meaningful features that characterize interactions between atoms in protein structures. In addition, through extensive experiments, we show that GeoPPI achieves new state-of-the-art performance in predicting the binding affinity changes upon both single- and multi-point mutations on six benchmark datasets. Moreover, we show that GeoPPI can accurately estimate the difference of binding affinities between a few recently identified SARS-CoV-2 antibodies and the receptor-binding domain (RBD) of the S protein. These results demonstrate the potential of GeoPPI as a powerful and useful computational tool in protein design and engineering. Our code and datasets are available at: https://github.com/Liuxg16/GeoPPI.


Assuntos
Substituição de Aminoácidos , Modelos Químicos , Proteínas/metabolismo , Mutação Puntual , Ligação Proteica , Proteínas/química , Proteínas/genética
2.
J Hematol Oncol ; 14(1): 123, 2021 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-34399825

RESUMO

Thromboembolism is a frequent cause of severity and mortality in COVID-19. However, the etiology of this phenomenon is not well understood. A cohort of 1186 subjects, from the GEN-COVID consortium, infected by SARS-CoV-2 with different severity was stratified by sex and adjusted by age. Then, common coding variants from whole exome sequencing were mined by LASSO logistic regression. The homozygosity of the cell adhesion molecule P-selectin gene (SELP) rs6127 (c.1807G > A; p.Asp603Asn) which has been already associated with thrombotic risk is found to be associated with severity in the male subcohort of 513 subjects (odds ratio = 2.27, 95% Confidence Interval 1.54-3.36). As the SELP gene is downregulated by testosterone, the odd ratio is increased in males older than 50 (OR 2.42, 95% CI 1.53-3.82). Asn/Asn homozygotes have increased D-dimers values especially when associated with poly Q ≥ 23 in the androgen receptor (OR 3.26, 95% CI 1.41-7.52). These results provide a rationale for the repurposing of antibodies against P-selectin as adjuvant therapy in rs6127 male homozygotes especially if older than 50 or with an impaired androgen receptor.


Assuntos
COVID-19/genética , Selectina-P/genética , Trombose/genética , COVID-19/complicações , Regulação para Baixo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação Puntual , SARS-CoV-2/isolamento & purificação , Trombose/etiologia
3.
Molecules ; 26(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34443538

RESUMO

Cytochrome c 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 c 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.


Assuntos
Técnicas Biossensoriais , Elétrons , Proteínas/metabolismo , Eletroquímica , Oxirredução , Mutação Puntual/genética , Dobramento de Proteína , Proteínas/química , Proteínas/genética
4.
Microb Pathog ; 159: 105145, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34411653

RESUMO

Pasteurella multocida (P. multocida) is a Gram-negative bacterium which causes diseases in poultry, livestock, and humans, resulting in huge economic losses. P. multocida serovar A CQ6 (PmCQ6) is a naturally occurring attenuated strain with a thin capsule. Thus, we aimed to explore why this strain is less virulent and produces less capsule compared with P. multocida serovar A strain CQ2 (PmCQ2). Analysis of capsular polysaccharide synthesis genes in PmCQ6 revealed that, compared with PmCQ2, there was only a single point mutation in the initiation codon sequence of the hyaC gene. To test whether this point mutation caused capsular deficiency and reduced virulence, we rescued this hyaC mutation and observed a restoration of capsule production and higher virulence. Transcriptome analysis showed that the hyaC point mutation led to a downregulation of capsule synthesis and/or iron utilization related-genes. Taken together, the results indicate that the start codon mutation of hyaC is an important factor affecting the capsule synthesis and virulence of PmCQ6.


Assuntos
Infecções por Pasteurella , Pasteurella multocida , Uridina Difosfato Glucose Desidrogenase/genética , Humanos , Infecções por Pasteurella/veterinária , Pasteurella multocida/enzimologia , Pasteurella multocida/genética , Mutação Puntual , Sorogrupo , Virulência/genética
5.
Science ; 373(6558): 998-1004, 2021 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-34446601

RESUMO

In eukaryotic cells, half of all proteins function as subunits within multiprotein complexes. Imbalanced synthesis of subunits leads to unassembled intermediates that must be degraded to minimize cellular toxicity. Here, we found that excess PSMC5, a subunit of the proteasome base, was targeted for degradation by the HERC1 ubiquitin ligase in mammalian cells. HERC1 identified unassembled PSMC5 by its cognate assembly chaperone PAAF1. Because PAAF1 only dissociates after assembly, HERC1 could also engage later assembly intermediates such as the PSMC4-PSMC5-PAAF1 complex. A missense mutant of HERC1 that causes neurodegeneration in mice was impaired in the recognition and ubiquitination of the PSMC5-PAAF1 complex. Thus, proteasome assembly factors can serve as adaptors for ubiquitin ligases to facilitate elimination of unassembled intermediates and maintain protein homeostasis.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Calmodulina/metabolismo , Humanos , Células MCF-7 , Camundongos , Mutação , Mutação de Sentido Incorreto , Doenças Neurodegenerativas/genética , Mutação Puntual , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/metabolismo , Proteólise , Proteínas Proto-Oncogênicas/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
6.
Nat Commun ; 12(1): 4217, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244513

RESUMO

The functional consequences of genetic variants within 5' untranslated regions (UTRs) on a genome-wide scale are poorly understood in disease. Here we develop a high-throughput multi-layer functional genomics method called PLUMAGE (Pooled full-length UTR Multiplex Assay on Gene Expression) to quantify the molecular consequences of somatic 5' UTR mutations in human prostate cancer. We show that 5' UTR mutations can control transcript levels and mRNA translation rates through the creation of DNA binding elements or RNA-based cis-regulatory motifs. We discover that point mutations can simultaneously impact transcript and translation levels of the same gene. We provide evidence that functional 5' UTR mutations in the MAP kinase signaling pathway can upregulate pathway-specific gene expression and are associated with clinical outcomes. Our study reveals the diverse mechanisms by which the mutational landscape of 5' UTRs can co-opt gene expression and demonstrates that single nucleotide alterations within 5' UTRs are functional in cancer.


Assuntos
Regiões 5' não Traduzidas/genética , Análise Mutacional de DNA/métodos , Regulação Neoplásica da Expressão Gênica , Genômica/métodos , Neoplasias da Próstata/genética , Linhagem Celular Tumoral , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Masculino , Mutação Puntual , Próstata/patologia , Neoplasias da Próstata/patologia , Biossíntese de Proteínas/genética , RNA-Seq
7.
Anal Chem ; 93(30): 10538-10545, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34279918

RESUMO

Multiplexing of analyses is essential to reduce sample and reagent consumption in applications with large target panels. In applications such as cancer diagnostics, the required degree of multiplexing often exceeds the number of available fluorescence channels in polymerase chain reaction (PCR) devices. The combination of photobleaching-sensitive and photobleaching-resistant fluorophores of the same color can boost the degree of multiplexing by a factor of 2 per channel. The only additional hardware required to create virtual fluorescence color channels is a low-cost light-emitting diode (LED) setup for selective photobleaching. Here, we present an assay concept for fluorescence color multiplexing in up to 10 channels (five standard channels plus five virtual channels) using the mediator probe PCR with universal reporter (UR) fluorogenic oligonucleotides. We evaluate the photobleaching characteristic of 21 URs, which cover the whole spectral range from blue to crimson. This comprehensive UR data set is employed to demonstrate the use of three virtual channels in addition to the three standard channels of a commercial dPCR device (blue, green, and red) targeting cancer-associated point mutations (KRAS G12D and G12V). Moreover, a LOD (limit of detection) analysis of this assay confirms the high sensitivity of the multiplexing method (KRAS G12D: 16 DNA copies/reaction in the standard red channel and KRAS G12V: nine DNA copies/reaction in the virtual red channel). Based on the presented data set, optimal fluorogenic reporter combinations can be easily selected for the application-specific creation of virtual channels, enabling a high degree of multiplexing at low optical and technical effort.


Assuntos
Mutação Puntual , Proteínas Proto-Oncogênicas p21(ras) , Corantes Fluorescentes , Humanos , Fotodegradação , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas p21(ras)/genética
8.
Methods Mol Biol ; 2312: 225-233, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34228293

RESUMO

The CRISPR-Cas9 system offers targeted genome manipulation with simplicity. Combining the CRISPR-Cas9 with optogenetics technology, we have engineered photoactivatable Cas9 to precisely control the genome sequence in a spatiotemporal manner. Here we provide a detailed protocol for optogenetic genome editing experiments using photoactivatable Cas9, including that for the generation of guide RNA vectors, light-mediated Cas9 activation, and quantification of genome editing efficiency in mammalian cells.


Assuntos
Proteína 9 Associada à CRISPR/efeitos da radiação , Sistemas CRISPR-Cas/efeitos da radiação , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes , Regulação da Expressão Gênica/efeitos da radiação , Luz , Optogenética , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Técnicas de Cultura de Células , Reparo do DNA por Junção de Extremidades , Células HEK293 , Humanos , Mutação INDEL , Mutação Puntual , RNA Guia/genética , RNA Guia/metabolismo
9.
Int J Mol Sci ; 22(14)2021 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-34299222

RESUMO

FMS-like tyrosine kinase 3 (FLT3) gene mutations have been found in more than one-third of Acute Myeloid Leukemia (AML) cases. The most common point mutation in FLT3 occurs at the 835th residue (D835A/E/F/G/H/I/N/V/Y), in the activation loop region. The D835 residue is critical in maintaining FLT3 inactive conformation; these mutations might influence the interaction with clinically approved AML inhibitors used to treat the AML. The molecular mechanism of each of these mutations and their interactions with AML inhibitors at the atomic level is still unknown. In this manuscript, we have investigated the structural consequence of native and mutant FLT-3 proteins and their molecular mechanisms at the atomic level, using molecular dynamics simulations (MDS). In addition, we use the molecular docking method to investigate the binding pattern between the FLT-3 protein and AML inhibitors upon mutations. This study apparently elucidates that, due to mutations in the D835, the FLT-3 structure loses its conformation and becomes more flexible compared to the native FLT3 protein. These structural changes are suggested to contribute to the relapse and resistance responses to AML inhibitors. Identifying the effects of FLT3 at the molecular level will aid in developing a personalized therapeutic strategy for treating patients with FLT-3-associated AML.


Assuntos
Leucemia Mieloide Aguda/genética , Tirosina Quinase 3 Semelhante a fms/genética , Simulação por Computador , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Simulação de Acoplamento Molecular/métodos , Simulação de Dinâmica Molecular , Mutação/efeitos dos fármacos , Mutação/genética , Mutação Puntual/efeitos dos fármacos , Mutação Puntual/genética , Polimorfismo de Nucleotídeo Único/efeitos dos fármacos , Polimorfismo de Nucleotídeo Único/genética , Conformação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Tirosina Quinase 3 Semelhante a fms/metabolismo
10.
Sci Rep ; 11(1): 13705, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34210996

RESUMO

The D614G mutation in the Spike protein of the SARS-CoV-2 has effectively replaced the early pandemic-causing variant. Using pseudotyped lentivectors, we confirmed that the aspartate replacement by glycine in position 614 is markedly more infectious. Molecular modelling suggests that the G614 mutation facilitates transition towards an open state of the Spike protein. To explain the epidemiological success of D614G, we analysed the evolution of 27,086 high-quality SARS-CoV-2 genome sequences from GISAID. We observed striking coevolution of D614G with the P323L mutation in the viral polymerase. Importantly, the exclusive presence of G614 or L323 did not become epidemiologically relevant. In contrast, the combination of the two mutations gave rise to a viral G/L variant that has all but replaced the initial D/P variant. Our results suggest that the P323L mutation, located in the interface domain of the RNA-dependent RNA polymerase, is a necessary alteration that led to the epidemiological success of the present variant of SARS-CoV-2. However, we did not observe a significant correlation between reported COVID-19 mortality in different countries and the prevalence of the Wuhan versus G/L variant. Nevertheless, when comparing the speed of emergence and the ultimate predominance in individual countries, it is clear that the G/L variant displays major epidemiological supremacy over the original variant.


Assuntos
COVID-19/virologia , RNA-Polimerase RNA-Dependente de Coronavírus/genética , Mutação Puntual , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , COVID-19/epidemiologia , RNA-Polimerase RNA-Dependente de Coronavírus/química , Humanos , Modelos Moleculares , Conformação Proteica , SARS-CoV-2/química , Glicoproteína da Espícula de Coronavírus/química
11.
BMC Neurol ; 21(1): 265, 2021 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-34225694

RESUMO

BACKGROUND: Oculopharyngeal muscular dystrophy (OPMD) is a late-onset muscular dystrophy characterised by slowly progressive ptosis, dysphagia, and proximal limb muscle weakness. A common cause of OPMD is the short expansion of a GCG or GCA trinucleotide repeat in PABPN1 gene. CASE PRESENTATION: A 78-year-old woman presented with ptosis and gradually progressive dysphagia. Her son had the same symptoms. A physical examination and muscle imaging (MRI and ultrasound) showed impairment of the tongue, proximal muscles of the upper limbs, and flexor muscles of the lower limbs. Needle-electromyography (EMG) of bulbar and facial muscles revealed a myopathic pattern. Based on the characteristic muscle involvement pattern and needle-EMG findings, we suspected that the patient had OPMD. Gene analysis revealed PABPN1 c.35G > C point mutation, which mimicked the effect of a common causative repeat expansion mutation of OPMD. CONCLUSION: We herein describe the first reported Japanese case of OPMD with PABPN1 point mutation, suggesting that this mutation is causative in Asians as well as in Europeans, in whom it was originally reported.


Assuntos
Distrofia Muscular Oculofaríngea , Proteína I de Ligação a Poli(A)/genética , Idoso , Feminino , Humanos , Masculino , Distrofia Muscular Oculofaríngea/diagnóstico , Distrofia Muscular Oculofaríngea/genética , Mutação Puntual
12.
Int J Mol Sci ; 22(14)2021 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-34299357

RESUMO

The airborne fungus Aspergillus fumigatus causes opportunistic infections in humans with high mortality rates in immunocompromised patients. Previous work established that the bZIP transcription factor HapX is essential for virulence via adaptation to iron limitation by repressing iron-consuming pathways and activating iron acquisition mechanisms. Moreover, HapX was shown to be essential for transcriptional activation of vacuolar iron storage and iron-dependent pathways in response to iron availability. Here, we demonstrate that HapX has a very short half-life during iron starvation, which is further decreased in response to iron, while siderophore biosynthetic enzymes are very stable. We identified Fbx22 and SumO as HapX interactors and, in agreement, HapX post-translational modifications including ubiquitination of lysine161, sumoylation of lysine242 and phosphorylation of threonine319. All three modifications were enriched in the immediate adaptation from iron-limiting to iron-replete conditions. Interfering with these post-translational modifications, either by point mutations or by inactivation, of Fbx22 or SumO, altered HapX degradation, heme biosynthesis and iron resistance to different extents. Consistent with the need to precisely regulate HapX protein levels, overexpression of hapX caused significant growth defects under iron sufficiency. Taken together, our results indicate that post-translational regulation of HapX is important to control iron homeostasis in A. fumigatus.


Assuntos
Aspergillus fumigatus/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Homeostase/genética , Ferro/metabolismo , Processamento de Proteína Pós-Traducional/genética , Adaptação Fisiológica/genética , Aspergillus fumigatus/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica/genética , Mutação Puntual/genética , Sideróforos/genética , Treonina/genética , Virulência/genética
13.
Biomolecules ; 11(7)2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34206274

RESUMO

SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) protein is the target for the antiviral drug Remdesivir (RDV). With RDV clinical trials on COVID-19 patients showing a reduced hospitalisation time. During the spread of the virus, the RdRp has developed several mutations, with the most frequent being A97V and P323L. The current study sought to investigate whether A97V and P323L mutations influence the binding of RDV to the RdRp of SARS-CoV-2 compared to wild-type (WT). The interaction of RDV with WT-, A97V-, and P323L-RdRp were measured using molecular dynamic (MD) simulations, and the free binding energies were extracted. Results showed that RDV that bound to WT- and A97V-RdRp had a similar dynamic motion and internal residue fluctuations, whereas RDV interaction with P323L-RdRp exhibited a tighter molecular conformation, with a high internal motion near the active site. This was further corroborated with RDV showing a higher binding affinity to P323L-RdRp (-24.1 kcal/mol) in comparison to WT-RdRp (-17.3 kcal/mol). This study provides insight into the potential significance of administering RDV to patients carrying the SARS-CoV-2 P323L-RdRp mutation, which may have a more favourable chance of alleviating the SARS-CoV-2 illness in comparison to WT-RdRp carriers, thereby suggesting further scientific consensus for the usage of Remdesivir as clinical candidate against COVID-19.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Alanina/análogos & derivados , Antivirais/farmacologia , COVID-19/tratamento farmacológico , RNA-Polimerase RNA-Dependente de Coronavírus/genética , Mutação Puntual , SARS-CoV-2/genética , Monofosfato de Adenosina/farmacologia , Alanina/farmacologia , Sítios de Ligação/efeitos dos fármacos , COVID-19/virologia , Domínio Catalítico/efeitos dos fármacos , Humanos , Simulação de Dinâmica Molecular , Mutação Puntual/efeitos dos fármacos , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia
14.
Nucleic Acids Res ; 49(13): 7644-7664, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34181727

RESUMO

Protein oligomerization is one mechanism by which homogenous solutions can separate into distinct liquid phases, enabling assembly of membraneless organelles. Survival Motor Neuron (SMN) is the eponymous component of a large macromolecular complex that chaperones biogenesis of eukaryotic ribonucleoproteins and localizes to distinct membraneless organelles in both the nucleus and cytoplasm. SMN forms the oligomeric core of this complex, and missense mutations within its YG box domain are known to cause Spinal Muscular Atrophy (SMA). The SMN YG box utilizes a unique variant of the glycine zipper motif to form dimers, but the mechanism of higher-order oligomerization remains unknown. Here, we use a combination of molecular genetic, phylogenetic, biophysical, biochemical and computational approaches to show that formation of higher-order SMN oligomers depends on a set of YG box residues that are not involved in dimerization. Mutation of key residues within this new structural motif restricts assembly of SMN to dimers and causes locomotor dysfunction and viability defects in animal models.


Assuntos
Proteínas do Complexo SMN/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sequência Conservada , Dimerização , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Humanos , Locomoção , Modelos Moleculares , Mutação , Mutação Puntual , Domínios Proteicos , Multimerização Proteica , Proteínas do Complexo SMN/genética , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética
15.
Commun Biol ; 4(1): 666, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34079053

RESUMO

Calcium dynamics control synaptic transmission. Calcium triggers synaptic vesicle fusion, determines release probability, modulates vesicle recycling, participates in long-term plasticity and regulates cellular metabolism. Mitochondria, the main source of cellular energy, serve as calcium signaling hubs. Mitochondrial calcium transients are primarily determined by the balance between calcium influx, mediated by the mitochondrial calcium uniporter (MCU), and calcium efflux through the sodium/lithium/calcium exchanger (NCLX). We identified a human recessive missense SLC8B1 variant that impairs NCLX activity and is associated with severe mental retardation. On this basis, we examined the effect of deleting NCLX in mice on mitochondrial and synaptic calcium homeostasis, synaptic activity, and plasticity. Neuronal mitochondria exhibited basal calcium overload, membrane depolarization, and a reduction in the amplitude and rate of calcium influx and efflux. We observed smaller cytoplasmic calcium transients in the presynaptic terminals of NCLX-KO neurons, leading to a lower probability of release and weaker transmission. In agreement, synaptic facilitation in NCLX-KO hippocampal slices was enhanced. Importantly, deletion of NCLX abolished long term potentiation of Schaffer collateral synapses. Our results show that NCLX controls presynaptic calcium transients that are crucial for defining synaptic strength as well as short- and long-term plasticity, key elements of learning and memory processes.


Assuntos
Deficiência Intelectual/genética , Deficiência Intelectual/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Trocador de Sódio e Cálcio/genética , Trocador de Sódio e Cálcio/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sinalização do Cálcio , Cardiomiopatias/genética , Cardiomiopatias/metabolismo , Feminino , Hipocampo/metabolismo , Humanos , Técnicas In Vitro , Potenciação de Longa Duração , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Mitocondriais/química , Proteínas Mitocondriais/deficiência , Plasticidade Neuronal , Neurônios/metabolismo , Linhagem , Mutação Puntual , Terminações Pré-Sinápticas/metabolismo , Trocador de Sódio e Cálcio/química , Transmissão Sináptica/genética , Transmissão Sináptica/fisiologia
16.
Commun Biol ; 4(1): 665, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34079061

RESUMO

In the malaria vector Anopheles gambiae, two point mutations in the acetylcholinesterase (ace-1R) and the sodium channel (kdrR) genes confer resistance to organophosphate/carbamate and pyrethroid insecticides, respectively. The mechanisms of compensation that recover the functional alterations associated with these mutations and their role in the modulation of insecticide efficacy are unknown. Using multidisciplinary approaches adapted to neurons isolated from resistant Anopheles gambiae AcerKis and KdrKis strains together with larval bioassays, we demonstrate that nAChRs, and the intracellular calcium concentration represent the key components of an adaptation strategy ensuring neuronal functions maintenance. In AcerKis neurons, the increased effect of acetylcholine related to the reduced acetylcholinesterase activity is compensated by expressing higher density of nAChRs permeable to calcium. In KdrKis neurons, changes in the biophysical properties of the L1014F mutant sodium channel, leading to enhance overlap between activation and inactivation relationships, diminish the resting membrane potential and reduce the fraction of calcium channels available involved in acetylcholine release. Together with the lower intracellular basal calcium concentration observed, these factors increase nAChRs sensitivity to maintain the effect of low concentration of acetylcholine. These results explain the opposite effects of the insecticide clothianidin observed in AcerKis and KdrKis neurons in vitro and in vivo.


Assuntos
Anopheles/efeitos dos fármacos , Anopheles/genética , Inseticidas/farmacologia , Mosquitos Vetores/efeitos dos fármacos , Mosquitos Vetores/genética , Acetilcolina/farmacologia , Acetilcolinesterase/genética , Animais , Anopheles/metabolismo , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Feminino , Genes de Insetos , Humanos , Técnicas In Vitro , Resistência a Inseticidas/genética , Malária/transmissão , Controle de Mosquitos/métodos , Mosquitos Vetores/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Técnicas de Patch-Clamp , Mutação Puntual , Receptores Nicotínicos/metabolismo , Canais de Sódio/genética
17.
J Biol Chem ; 297(1): 100867, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34118236

RESUMO

Molnupiravir, a prodrug of the nucleoside derivative ß-D-N4-hydroxycytidine (NHC), is currently in clinical trials for COVID-19 therapy. However, the biochemical mechanisms involved in molnupiravir-induced mutagenesis had not been explored. In a recent study, Gordon et al. demonstrated that NHC can be incorporated into viral RNA and subsequently extended and used as template for RNA-dependent RNA synthesis, proposing a mutagenesis model consistent with available virological evidence. Their study uncovers molecular mechanisms by which molnupiravir drives SARS-CoV-2 into error catastrophe.


Assuntos
Antivirais/farmacologia , COVID-19/virologia , Citidina/análogos & derivados , Hidroxilaminas/farmacologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/genética , Citidina/farmacologia , Humanos , Mutação Puntual/efeitos dos fármacos , RNA Viral/genética , SARS-CoV-2/metabolismo
18.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 50(2): 229-238, 2021 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-34137224

RESUMO

To establish a rabbit model of proprotein convertase subtilisin/kexin type9 () point mutation with CRISPR/Cas9 gene editing technique. According to the PubMed gene protein data, the PCSK9 protein functional regions of human and rabbit were analyzed by Blast. The 386S (Ser) amino acid functional region of human gene was homologous to the 485S of rabbit gene. Three small guide RNAs and one single-stranded donor oligonucleotide were designed according to the 485S base substitution position and sequence analysis of rabbit gene. The synthetic small guide RNAs, Cas9 mRNA and single-stranded donor oligonucleotide were co-injected into the cytoplasm of rabbit fertilized eggs and the embryos were transferred into the pregnant rabbits. PCR, TA cloning and off-target analysis were performed on the F0 rabbits to identify whether the PCSK9 mutation was successful. Fifteen F0 rabbits were obtained. The sequencing results showed that one of them was PCSK9 point mutation homozygote and two of them were PCSK9 point mutation heterozygotes, and the mutation could be stably inherited. The rabbit model of PCSK9 point mutation was successfully constructed by CRISPR/Cas9 technique, which provides an animal model for exploring the molecular mechanism of impaired PCSK9 function and developing reliable and effective diagnosis and treatment measures.


Assuntos
Sistemas CRISPR-Cas , Pró-Proteína Convertase 9 , Animais , Humanos , Mutação Puntual , Pró-Proteína Convertase 9/genética , Coelhos
19.
Genes (Basel) ; 12(5)2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-34064921

RESUMO

In tomato (Solanum lycopersicum), there are at least three SlMLO (Mildew resistance Locus O) genes acting as susceptibility genes for the powdery mildew disease caused by Oidium neolycopersici, namely SlMLO1, SlMLO5 and SlMLO8. Of the three homologs, the SlMLO1 gene plays a major role since a natural mutant allele called ol-2 can almost completely prevent fungal penetration by formation of papillae. The ol-2 allele contains a 19-bp deletion in the coding sequence of the SlMLO1 gene, resulting in a premature stop codon within the second cytoplasmic loop of the predicted protein. In this study, we have developed a new genetic resource (M200) in the tomato cv. Micro-Tom genetic background by means of ethyl methane sulfonate (EMS) mutagenesis. The mutant M200 containing a novel allele (the m200 allele) of the tomato SlMLO1 gene showed profound resistance against powdery mildew with no fungal sporulation. Compared to the coding sequence of the SlMLO1 gene, the m200 allele carries a point mutation at T65A. The SNP results in a premature stop codon L22* located in the first transmembrane domain of the complete SlMLO1 protein. The length of the predicted protein is 21 amino acids, while the SlMLO1 full-length protein is 513 amino acids. A high-resolution melting (HRM) marker was developed to distinguish the mutated m200 allele from the SlMLO1 allele in backcross populations. The mutant allele conferred recessive resistance that was associated with papillae formation at fungal penetration sites of plant epidermal cells. A comprehensive list of known mlo mutations found in natural and artificial mutants is presented, which serves as a particularly valuable resource for powdery mildew resistance breeding.


Assuntos
Resistência à Doença , Lycopersicon esculentum/genética , Proteínas de Membrana/genética , Proteínas de Plantas/genética , Ascomicetos/patogenicidade , Metanossulfonato de Etila/toxicidade , Lycopersicon esculentum/microbiologia , Mutagênese , Mutagênicos/toxicidade , Mutação Puntual , Polimorfismo de Nucleotídeo Único
20.
Front Immunol ; 12: 635326, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34122405

RESUMO

Membranous nephropathy (MN), an autoimmune glomerular disease, is one of the most common causes of nephrotic syndrome in adults. In current clinical practice, the diagnosis is dependent on renal tissue biopsy. A new method for diagnosis and prognosis surveillance is urgently needed for patients. In the present study, we recruited 66 MN patients before any treatment and 11 healthy control (HC) and analyzed multiple aspects of the immunoglobulin heavy chain (IGH) repertoire of these samples using high-throughput sequencing. We found that the abnormalities of CDR-H3 length, hydrophobicity, somatic hypermutation (SHM), and germ line index were progressively more prominent in patients with MN, and the frequency of IGHV3-66 in post-therapy patients was significantly lower than that in pre-therapy patients. Moreover, we found that the IGHV3-38 gene was significantly related to PLA2R, which is the most commonly used biomarker. The most important discovery was that several IGHV, IGHD transcripts, CDR-H3 length, and SHM rate in pre-therapy patients had the potential to predict the therapeutic effect. Our study further demonstrated that the IGH repertoire could be a potential biomarker for prognosis prediction of MN. The landscape of circulating B-lymphocyte repertoires sheds new light on the detection and surveillance of MN.


Assuntos
Linfócitos B/imunologia , Regiões Determinantes de Complementaridade , Análise Mutacional de DNA , Genes de Cadeia Pesada de Imunoglobulina , Glomerulonefrite Membranosa/diagnóstico , Mutação Puntual , Adolescente , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Glomerulonefrite Membranosa/genética , Glomerulonefrite Membranosa/imunologia , Glomerulonefrite Membranosa/terapia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prognóstico , Adulto Jovem
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