Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 47
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
2.
Int J Mol Sci ; 25(7)2024 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-38612505

RESUMO

SARS-CoV-2 has accumulated many mutations since its emergence in late 2019. Nucleotide substitutions leading to amino acid replacements constitute the primary material for natural selection. Insertions, deletions, and substitutions appear to be critical for coronavirus's macro- and microevolution. Understanding the molecular mechanisms of mutations in the mutational hotspots (positions, loci with recurrent mutations, and nucleotide context) is important for disentangling roles of mutagenesis and selection. In the SARS-CoV-2 genome, deletions and insertions are frequently associated with repetitive sequences, whereas C>U substitutions are often surrounded by nucleotides resembling the APOBEC mutable motifs. We describe various approaches to mutation spectra analyses, including the context features of RNAs that are likely to be involved in the generation of recurrent mutations. We also discuss the interplay between mutations and natural selection as a complex evolutionary trend. The substantial variability and complexity of pipelines for the reconstruction of mutations and the huge number of genomic sequences are major problems for the analyses of mutations in the SARS-CoV-2 genome. As a solution, we advocate for the development of a centralized database of predicted mutations, which needs to be updated on a regular basis.


Assuntos
COVID-19 , Humanos , COVID-19/genética , SARS-CoV-2/genética , Mutagênese , Mutação , Nucleotídeos
3.
Adv Exp Med Biol ; 1415: 533-537, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37440083

RESUMO

The visual cycle is a complex biological process that involves the sequential action of proteins in the retinal pigment epithelial (RPE) cells and photoreceptors to modify and shuttle visual retinoids. A majority of the visual cycle proteins are membrane proteins, either integral or peripheral membrane proteins. Despite significant progress in understanding their physiological function, very limited structural information is available for the visual cycle proteins. Moreover, the mechanism of membrane interaction is not yet clear in all cases. Here, we demonstrate the presence of an amphipathic helix in selected RPE visual cycle proteins, using in silico tools, and highlight their role in membrane association and function.


Assuntos
Epitélio Pigmentado da Retina , Retinoides , Proteínas de Transporte/metabolismo , Proteínas do Olho/metabolismo , Proteínas de Membrana/metabolismo , cis-trans-Isomerases
4.
Int J Mol Sci ; 24(14)2023 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-37511618

RESUMO

Here, we present evidence that caveolae-mediated endocytosis using LDLR is the pathway for SARS-CoV-2 virus internalization in the ocular cell line ARPE-19. Firstly, we found that, while Angiotensin-converting enzyme 2 (ACE2) is expressed in these cells, blocking ACE2 by antibody treatment did not prevent infection by SARS-CoV-2 spike pseudovirions, nor did antibody blockade of extracellular vimentin and other cholesterol-rich lipid raft proteins. Next, we implicated the role of cholesterol homeostasis in infection by showing that incubating cells with different cyclodextrins and oxysterol 25-hydroxycholesterol (25-HC) inhibits pseudovirion infection of ARPE-19. However, the effect of 25-HC is likely not via cholesterol biosynthesis, as incubation with lovastatin did not appreciably affect infection. Additionally, is it not likely to be an agonistic effect of 25-HC on LXR receptors, as the LXR agonist GW3965 had no significant effect on infection of ARPE-19 cells at up to 5 µM GW3965. We probed the role of endocytic pathways but determined that clathrin-dependent and flotillin-dependent rafts were not involved. Furthermore, 20 µM chlorpromazine, an inhibitor of clathrin-mediated endocytosis (CME), also had little effect. In contrast, anti-dynamin I/II antibodies blocked the entry of SARS-CoV-2 spike pseudovirions, as did dynasore, a noncompetitive inhibitor of dynamin GTPase activity. Additionally, anti-caveolin-1 antibodies significantly blocked spike pseudotyped lentiviral infection of ARPE-19. However, nystatin, a classic inhibitor of caveolae-dependent endocytosis, did not affect infection while indomethacin inhibited only at 10 µM at the 48 h time point. Finally, we found that anti-LDLR antibodies block pseudovirion infection to a similar degree as anti-caveolin-1 and anti-dynamin I/II antibodies, while transfection with LDLR-specific siRNA led to a decrease in spike pseudotyped lentiviral infection, compared to scrambled control siRNAs. Thus, we conclude that SARS-CoV-2 spike pseudovirion infection in ARPE-19 cells is a dynamin-dependent process that is primarily mediated by LDLR.


Assuntos
COVID-19 , Glicoproteína da Espícula de Coronavírus , Humanos , Enzima de Conversão de Angiotensina 2/farmacologia , Colesterol/metabolismo , Clatrina/metabolismo , Dinamina II , Lipoproteínas LDL/farmacologia , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/farmacologia , Internalização do Vírus
5.
Life Sci Alliance ; 6(1)2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36265895

RESUMO

RPE65 retinol isomerase is an indispensable player in the visual cycle between the vertebrate retina and RPE. Although membrane association is critical for RPE65 function, its mechanism is not clear. Residues 107-125 are believed to interact with membranes but are unresolved in all RPE65 crystal structures, whereas palmitoylation at C112 also plays a role. We report the mechanism of membrane recognition and binding by RPE65. Binding of aa107-125 synthetic peptide with membrane-mimicking micellar surfaces induces transition from unstructured loop to amphipathic α-helical (AH) structure but this transition is automatic in the C112-palmitoylated peptide. We demonstrate that the AH significantly affects palmitoylation level, membrane association, and isomerization activity of RPE65. Furthermore, aa107-125 functions as a membrane sensor and the AH as a membrane-targeting motif. Molecular dynamic simulations clearly show AH-membrane insertion, supporting our experimental findings. Collectively, these studies allow us to propose a working model for RPE65-membrane binding, and to provide a novel role for cysteine palmitoylation.


Assuntos
Cisteína , Proteínas do Olho , Proteínas de Transporte/metabolismo , Cisteína/metabolismo , Proteínas do Olho/química , Proteínas do Olho/metabolismo , Lipoilação , Conformação Proteica em alfa-Hélice , cis-trans-Isomerases
6.
Int J Mol Sci ; 22(12)2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34204305

RESUMO

The SARS-CoV-2 Spike glycoprotein (S protein) acquired a unique new 4 amino acid -PRRA- insertion sequence at amino acid residues (aa) 681-684 that forms a new furin cleavage site in S protein as well as several new glycosylation sites. We studied various statistical properties of the -PRRA- insertion at the RNA level (CCUCGGCGGGCA). The nucleotide composition and codon usage of this sequence are different from the rest of the SARS-CoV-2 genome. One of such features is two tandem CGG codons, although the CGG codon is the rarest codon in the SARS-CoV-2 genome. This suggests that the insertion sequence could cause ribosome pausing as the result of these rare codons. Due to population variants, the Nextstrain divergence measure of the CCU codon is extremely large. We cannot exclude that this divergence might affect host immune responses/effectiveness of SARS-CoV-2 vaccines, possibilities awaiting further investigation. Our experimental studies show that the expression level of original RNA sequence "wildtype" spike protein is much lower than for codon-optimized spike protein in all studied cell lines. Interestingly, the original spike sequence produces a higher titer of pseudoviral particles and a higher level of infection. Further mutagenesis experiments suggest that this dual-effect insert, comprised of a combination of overlapping translation pausing and furin sites, has allowed SARS-CoV-2 to infect its new host (human) more readily. This underlines the importance of ribosome pausing to allow efficient regulation of protein expression and also of cotranslational subdomain folding.


Assuntos
RNA Viral/metabolismo , Ribossomos/metabolismo , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Animais , Sequência de Bases , Células COS , COVID-19/patologia , COVID-19/virologia , Chlorocebus aethiops , Uso do Códon , Células HEK293 , Humanos , Mutagênese , SARS-CoV-2/isolamento & purificação , Alinhamento de Sequência , Glicoproteína da Espícula de Coronavírus/metabolismo
7.
Antioxidants (Basel) ; 10(3)2021 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-33803144

RESUMO

An extensive body of work has documented the antioxidant role of xanthophylls (lutein and zeaxanthin) in human health and specifically how they provide photoprotection in human vision. More recently, evidence is emerging for the transcriptional regulation of antioxidant response by lutein/lutein cleavage products, similar to the role of ß-carotene cleavage products in the modulation of retinoic acid receptors. Supplementation with xanthophylls also provides additional benefits for the prevention of age-related macular degeneration (AMD) and attenuation of Alzheimer's disease symptoms. Mammalian ß-carotene oxygenase 2 (BCO2) asymmetrically cleaves xanthophylls as well as ß-carotene in vitro. We recently demonstrated that mouse BCO2 (mBCO2) is a functionally palmitoylated enzyme and that it loses palmitoylation when cells are treated with ß-carotene. The mouse enzyme is the easiest model to study mammalian BCO2 because it has only one isoform, unlike human BCO2 with several major isoforms with various properties. Here, we used the same acyl-RAC methodology and confocal microscopy to elucidate palmitoylation and localization status of mBCO2 in the presence of xanthophylls. We created large unilamellar vesicle-based nanocarriers for the successful delivery of xanthophylls into cells. We demonstrate here that, upon treatment with low micromolar concentration of lutein (0.15 µM), mBCO2 is depalmitoylated and shows partial nuclear localization (38.00 ± 0.04%), while treatment with zeaxanthin (0.45 µM) and violaxanthin (0.6 µM) induces depalmitoylation and protein translocation from mitochondria to a lesser degree (20.00 ± 0.01% and 35.00 ± 0.02%, respectively). Such a difference in the behavior of mBCO2 toward various xanthophylls and its translocation into the nucleus in the presence of various xanthophylls suggests a possible mechanism for transport of lutein/lutein cleavage products to the nucleus to affect transcriptional regulation.

8.
Genetics ; 218(2)2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-33844024

RESUMO

Current eukaryotic replication models postulate that leading and lagging DNA strands are replicated predominantly by dedicated DNA polymerases. The catalytic subunit of the leading strand DNA polymerase ε, Pol2, consists of two halves made of two different ancestral B-family DNA polymerases. Counterintuitively, the catalytically active N-terminal half is dispensable, while the inactive C-terminal part is required for viability. Despite extensive studies of yeast Saccharomyces cerevisiae strains lacking the active N-terminal half, it is still unclear how these strains survive and recover. We designed a robust method for constructing mutants with only the C-terminal part of Pol2. Strains without the active polymerase part show severe growth defects, sensitivity to replication inhibitors, chromosomal instability, and elevated spontaneous mutagenesis. Intriguingly, the slow-growing mutant strains rapidly accumulate fast-growing clones. Analysis of genomic DNA sequences of these clones revealed that the adaptation to the loss of the catalytic N-terminal part of Pol2 occurs by a positive selection of mutants with improved growth. Elevated mutation rates help generate sufficient numbers of these variants. Single nucleotide changes in the cell cycle-dependent kinase gene, CDC28, improve the growth of strains lacking the N-terminal part of Pol2, and rescue their sensitivity to replication inhibitors and, in parallel, lower mutation rates. Our study predicts that changes in mammalian homologs of cyclin-dependent kinases may contribute to cellular responses to the leading strand polymerase defects.


Assuntos
Proteína Quinase CDC28 de Saccharomyces cerevisiae/genética , Proteína Quinase CDC28 de Saccharomyces cerevisiae/metabolismo , DNA Polimerase II/genética , Replicação do DNA , Saccharomyces cerevisiae/genética , DNA Polimerase II/metabolismo , DNA Fúngico , Genoma Fúngico , Mutagênese , Taxa de Mutação , Polimorfismo de Nucleotídeo Único , Saccharomyces cerevisiae/enzimologia , Seleção Genética
9.
Int J Mol Sci ; 22(4)2021 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-33672790

RESUMO

Nonsense mutations turn a coding (sense) codon into an in-frame stop codon that is assumed to result in a truncated protein product. Thus, nonsense substitutions are the hallmark of pseudogenes and are used to identify them. Here we show that in-frame stop codons within bacterial protein-coding genes are widespread. Their evolutionary conservation suggests that many of them are not pseudogenes, since they maintain dN/dS values (ratios of substitution rates at non-synonymous and synonymous sites) significantly lower than 1 (this is a signature of purifying selection in protein-coding regions). We also found that double substitutions in codons-where an intermediate step is a nonsense substitution-show a higher rate of evolution compared to null models, indicating that a stop codon was introduced and then changed back to sense via positive selection. This further supports the notion that nonsense substitutions in bacteria are relatively common and do not necessarily cause pseudogenization. In-frame stop codons may be an important mechanism of regulation: Such codons are likely to cause a substantial decrease of protein expression levels.


Assuntos
Códon sem Sentido , Códon de Terminação/genética , Fases de Leitura Aberta/genética , Células Procarióticas/metabolismo , Bactérias/classificação , Bactérias/genética , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Sequência de Bases , Evolução Molecular , Modelos Genéticos , Filogenia , Mutação Puntual , Pseudogenes/genética , Seleção Genética , Homologia de Sequência do Ácido Nucleico
10.
PLoS One ; 15(10): e0240659, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33057430

RESUMO

SR-BI binds various lipoproteins, including HDL, LDL as well as VLDL, and mediates selective cholesteryl ester (CE) uptake. HDL derived CE accumulates in cellular lipid droplets (LDs), which also store triacylglycerol (TAG). We hypothesized that SR-BI could significantly facilitate LD formation, in part, by directly transporting LDL derived neutral lipids (NL) such as CE and TAG into LDs without lipolysis and de novo lipid synthesis. SR-BI overexpression greatly increased LDL uptake and LD formation in stably transfected HeLa cells (SR-BI-HeLa). LDs isolated from SR-BI-HeLa contained 4- and 7-times more CE and TAG, respectively, than mock-transfected HeLa (Mock-HeLa). In contrast, LDL receptor overexpression in HeLa (LDLr-HeLa) greatly increased LDL uptake, degradation with moderate 1.5- and 2-fold increases of CE and TAG, respectively. Utilizing CE and TAG analogs, BODIPY-TAG (BP-TAG) and BODIPY-CE (BP-CE), for tracking LDL NL, we found that after initial binding of LDL to SR-BI-HeLa, apoB remained at the cell surface, while BP-CE and BP-TAG were sorted and simultaneously transported together to LDs. Both lipids demonstrated limited internalization to lysosomes or endoplasmic reticulum in SR-BI-HeLa. In LDLr-HeLa, NLs demonstrated clear lysosomal sequestration without their sorting to LDs. An inhibition of TAG and CE de novo synthesis by 90-95% only reduced TAG and CE LD content by 45-50%, and had little effect on BP-CE and BP-TAG transport to LDs in SR-BI HeLa. Furthermore, intravenous infusion of 1-2 mg of LDL increased liver LDs in normal (WT) but not in SR-BI KO mice. Mice transgenic for human SR-BI demonstrated higher liver LD accumulation than WT mice. Finally, Electro Spray Infusion Mass Spectrometry (ESI-MS) using deuterated d-CE found that LDs accumulated up to 40% of unmodified d-CE LDL. We conclude that SR-BI mediates LDL-induced LD formation in vitro and in vivo. In addition to cytosolic NL hydrolysis and de novo lipid synthesis, this process includes selective sorting and transport of LDL NL to LDs with limited lysosomal NL sequestration and the transport of LDL CE, and TAG directly to LDs independently of de novo synthesis.


Assuntos
Gotículas Lipídicas/metabolismo , Lipídeos/química , Lipoproteínas LDL/metabolismo , Receptores Depuradores Classe B/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Compostos de Boro/metabolismo , Ésteres do Colesterol/metabolismo , Coenzima A Ligases/antagonistas & inibidores , Coenzima A Ligases/metabolismo , Inibidores Enzimáticos/farmacologia , Células HeLa , Humanos , Gotículas Lipídicas/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de LDL/metabolismo , Triazenos/farmacologia , Triglicerídeos/metabolismo
11.
Molecules ; 25(8)2020 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-32331396

RESUMO

Abundant in nature, carotenoids are a class of fat-soluble pigments with a polyene tetraterpenoid structure. They possess antioxidant properties and their consumption leads to certain health benefits in humans. Carotenoid cleavage oxygenases (CCOs) are a superfamily of enzymes which oxidatively cleave carotenoids and they are present in all kingdoms of life. Complexity of CCO evolution is high. For example, in this study we serendipitously found a new family of eukaryotic CCOs, the apocarotenoid oxygenase-like (ACOL) family. This family has several members in animal genomes and lacks the animal-specific amino acid motif PDPCK. This motif is likely to be associated with palmitoylation of some animal CCOs. We recently demonstrated that two mammalian members of the carotenoid oxygenase family retinal pigment epithelial-specific 65 kDa protein (RPE65) and beta-carotene oxygenase 2 (BCO2) are palmitoylated proteins. Here we used the acyl-resin-assisted capture (acyl-RAC) method to demonstrate protein palmitoylation and immunochemistry to localize mouse BCO2 (mBCO2) in COS7 cell line in the absence and presence of its substrate ß-carotene. We demonstrate that mBCO2 palmitoylation depends on the evolutionarily conserved motif PDPCK and that metazoan family members lacking the motif (Lancelet beta-carotene oxygenase-like protein (BCOL) and Acropora ACOL) are not palmitoylated. Additionally, we observed that the palmitoylation status of mBCO2 and its membrane association depend on the presence of its substrate ß-carotene. Based on our results we conclude that most metazoan carotenoid oxygenases retain the evolutionarily conserved palmitoylation PDPCK motif to target proteins to internal membranes depending on substrate status. Exceptions are in the secreted BCOL subfamily and the strictly cytosolic ancient ACOL subfamily of carotenoid oxygenases.


Assuntos
Oxigenases/química , Animais , Carotenoides/química , Dioxigenases/metabolismo , Ácidos Graxos Monoinsaturados/química , Imunofluorescência , Humanos , Camundongos , Família Multigênica , Mutação , Oxigenases/genética , Filogenia , Transporte Proteico , Especificidade por Substrato
12.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1865(11): 158665, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32061750

RESUMO

The carotenoids are terpenoid fat-soluble pigments produced by plants, algae, and several bacteria and fungi. They are ubiquitous components of animal diets. Carotenoid cleavage oxygenase (CCO) superfamily members are involved in carotenoid metabolism and are present in all kingdoms of life. Throughout the animal kingdom, carotenoid oxygenases are widely distributed and they are completely absent only in two unicellular organisms, Monosiga and Leishmania. Mammals have three paralogs 15,15'-ß-carotene oxygenase (BCO1), 9',10'-ß-carotene oxygenase (BCO2) and RPE65. The first two enzymes are classical carotenoid oxygenases: they cleave carbon­carbon double bonds and incorporate two atoms of oxygen in the substrate at the site of cleavage. The third, RPE65, is an unusual family member, it is the retinoid isomerohydrolase in the visual cycle that converts all-trans-retinyl ester into 11-cis-retinol. Here we discuss evolutionary aspects of the carotenoid cleavage oxygenase superfamily and their enzymology to deduce what insight we can obtain from their evolutionary conservation.


Assuntos
Dioxigenases/genética , Evolução Molecular , beta-Caroteno 15,15'-Mono-Oxigenase/genética , cis-trans-Isomerases/genética , Animais , Carotenoides/metabolismo , Metabolismo dos Lipídeos/genética , Mamíferos/genética , Oxigenases/classificação , Oxigenases/genética
13.
Adv Exp Med Biol ; 1185: 537-541, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31884667

RESUMO

RPE65, the retinal pigment epithelium (RPE) smooth endoplasmic reticulum (sER) membrane-associated retinoid isomerase, plays an indispensable role in sustaining visual function in vertebrates. An important aspect which has attracted considerable attention is the posttranslational modification by S-palmitoylation of RPE65. Some studies show that RPE65 is a palmitoylated protein, but others deny that conclusion. While it is considered to be mainly responsible for RPE65's membrane association, we still lack conclusive evidence about RPE65 palmitoylation. In this review, we provide an overview of the history and current understanding of RPE65 palmitoylation.


Assuntos
Proteínas do Olho/química , Lipídeos/química , Lipoilação , Processamento de Proteína Pós-Traducional , Epitélio Pigmentado da Retina/enzimologia , cis-trans-Isomerases/química , Animais , Retículo Endoplasmático , Humanos
14.
Front Genet ; 10: 634, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31379919

RESUMO

Currently, there is much interest in intronic sequence-containing long non-coding RNAs and the role of intronic transcription in regulation of cellular metabolism and fate. Several stable intronic sequence RNAs (sisRNAs) were recently implicated in regulation of parental genes. To investigate transcription from introns of the RPE65 gene, we analyzed RNA-seq and Nanopore sequencing data from different cell models of human retinal pigment epithelium (RPE) and native bovine RPE. We discovered putative stable poly-adenylated transcripts with sequences corresponding to intronic regions of the RPE65 gene in the cytoplasm of RPE cells. These stable intronic sequences could be important for RPE65 transcription, splicing or translation. We also analyzed alternative splicing events in RPE65. Frequent exon skipping events involving exons 2, 3, and 7 were detected. The rate of these events was much higher in human RPE cell cultures compared with native RPE , consistent with lack of translation of RPE65 mRNA in cell cultures.

15.
Genes (Basel) ; 10(3)2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30857256

RESUMO

Currently, several long non-coding RNAs (lncRNAs) (TUG1, MALAT1, MEG3 and others) have been discovered to regulate normal visual function and may potentially contribute to dysfunction of the retina. We decided to extend these analyses of lncRNA genes to the retinal pigment epithelium (RPE) to determine whether there is conservation of RPE-expressed lncRNA between human and bovine genomes. We reconstructed bovine RPE lncRNAs based on genome-guided assembly. Next, we predicted homologous human transcripts based on whole genome alignment. We found a small set of conserved lncRNAs that could be involved in signature RPE functions that are conserved across mammals. However, the fraction of conserved lncRNAs in the overall pool of lncRNA found in RPE appeared to be very small (less than 5%), perhaps reflecting a fast and flexible adaptation of the mammalian eye to various environmental conditions.


Assuntos
Evolução Molecular , RNA Longo não Codificante/genética , Epitélio Pigmentado da Retina/metabolismo , Animais , Bovinos , Linhagem Celular , Sequência Conservada , Humanos , Alinhamento de Sequência
16.
Sci Rep ; 9(1): 5218, 2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30914787

RESUMO

Association with the endoplasmic reticulum (ER) membrane is a critical requirement for the catalytic function of RPE65. Several studies have investigated the nature of the RPE65-membrane interaction; however, complete understanding of its mode of membrane binding is still lacking. Previous biochemical studies suggest the membrane interaction can be partly attributed to S-palmitoylation, but the existence of RPE65 palmitoylation remains a matter of debate. Here, we re-examined RPE65 palmitoylation, and its functional consequence in the visual cycle. We clearly demonstrate that RPE65 is post-translationally modified by a palmitoyl moiety, but this is not universal (about 25% of RPE65). By extensive mutational studies we mapped the S-palmitoylation sites to residues C112 and C146. Inhibition of palmitoylation using 2-bromopalmitate and 2-fluoropalmitate completely abolish its membrane association. Furthermore, palmitoylation-deficient C112 mutants are significantly impeded in membrane association. Finally, we show that RPE65 palmitoylation level is highly regulated by lecithin:retinol acyltransferase (LRAT) enzyme. In the presence of all-trans retinol, LRAT substrate, there is a significant decrease in the level of palmitoylation of RPE65. In conclusion, our findings suggest that RPE65 is indeed a dynamically-regulated palmitoylated protein and that palmitoylation is necessary for regulating its membrane binding, and to perform its normal visual cycle function.


Assuntos
Membrana Celular/metabolismo , Lipoilação , Visão Ocular , cis-trans-Isomerases/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Animais , Bovinos , Membrana Celular/genética , Células HEK293 , Humanos , Mutação , cis-trans-Isomerases/genética
17.
Hum Mutat ; 40(4): 426-443, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30628748

RESUMO

Human RPE65 mutations cause a spectrum of retinal dystrophies that result in blindness. While RPE65 mutations have been almost invariably recessively inherited, a c.1430A>G (p.(D477G)) mutation has been reported to cause autosomal dominant retinitis pigmentosa (adRP). To study the pathogenesis of this human mutation, we have replicated the mutation in a knock-in (KI) mouse model using CRISPR/Cas9-mediated genome editing. Significantly, in contrast to human patients, heterozygous KI mice do not exhibit any phenotypes in visual function tests. When raised in regular vivarium conditions, homozygous KI mice display relatively undisturbed visual functions with minimal retinal structural changes. However, KI/KI mouse retinae are more sensitive to light exposure and exhibit signs of degenerative features when subjected to light stress. We find that instead of merely producing a missense mutant protein, the A>G nucleotide substitution greatly affects appropriate splicing of Rpe65 mRNA by generating an ectopic splice site in comparable context to the canonical one, thereby disrupting RPE65 protein expression. Similar splicing defects were also confirmed for the human RPE65 c.1430G mutant in an in vitro Exontrap assay. Our data demonstrate that a splicing defect is associated with c.1430G pathogenesis, and therefore provide insights in the therapeutic strategy for human patients.


Assuntos
Alelos , Predisposição Genética para Doença , Mutação , Splicing de RNA , cis-trans-Isomerases/genética , Animais , Biomarcadores , Modelos Animais de Doenças , Expressão Gênica , Perfilação da Expressão Gênica , Estudos de Associação Genética , Genótipo , Humanos , Camundongos , Camundongos Transgênicos , Fenótipo , Sítios de Splice de RNA , Retina/metabolismo , Retina/patologia
18.
Genome Biol Evol ; 10(8): 1902-1919, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29986017

RESUMO

We previously proposed that changes in the efficiency of protein translation are associated with autism spectrum disorders (ASDs). This hypothesis connects environmental factors and genetic factors because each can alter translation efficiency. For genetic factors, we previously tested our hypothesis using a small set of ASD-associated genes, a small set of ASD-associated variants, and a statistic to quantify by how much a single nucleotide variant (SNV) in a protein coding region changes translation speed. In this study, we confirm and extend our hypothesis using a published set of 1,800 autism quartets (parents, one affected child and one unaffected child) and genome-wide variants. Then, we extend the test statistic to combine translation efficiency with other possibly relevant variables: ribosome profiling data, presence/absence of CpG dinucleotides, and phylogenetic conservation. The inclusion of ribosome profiling abundances strengthens our results for male-male sibling pairs. The inclusion of CpG information strengthens our results for female-female pairs, giving an insight into the significant gender differences in autism incidence. By combining the single-variant test statistic for all variants in a gene, we obtain a single gene score to evaluate how well a gene distinguishes between affected and unaffected siblings. Using statistical methods, we compute gene sets that have some power to distinguish between affected and unaffected siblings by translation efficiency of gene variants. Pathway and enrichment analysis of those gene sets suggest the importance of Wnt signaling pathways, some other pathways related to cancer, ATP binding, and ATP-ase pathways in the etiology of ASDs.


Assuntos
Transtorno do Espectro Autista/genética , Genoma Humano , Biossíntese de Proteínas/genética , Algoritmos , Ilhas de CpG/genética , Metilação de DNA/genética , Evolução Molecular , Feminino , Redes Reguladoras de Genes , Humanos , Masculino , Modelos Genéticos , Pais , Polimorfismo de Nucleotídeo Único/genética , Análise de Regressão , Ribossomos/metabolismo , Seleção Genética , Irmãos
19.
Brief Bioinform ; 19(6): 1085-1101, 2018 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-28498882

RESUMO

Cancer is a genetic disorder, meaning that a plethora of different mutations, whether somatic or germ line, underlie the etiology of the 'Emperor of Maladies'. Point mutations, chromosomal rearrangements and copy number changes, whether they have occurred spontaneously in predisposed individuals or have been induced by intrinsic or extrinsic (environmental) mutagens, lead to the activation of oncogenes and inactivation of tumor suppressor genes, thereby promoting malignancy. This scenario has now been recognized and experimentally confirmed in a wide range of different contexts. Over the past decade, a surge in available sequencing technologies has allowed the sequencing of whole genomes from liquid malignancies and solid tumors belonging to different types and stages of cancer, giving birth to the new field of cancer genomics. One of the most striking discoveries has been that cancer genomes are highly enriched with mutations of specific kinds. It has been suggested that these mutations can be classified into 'families' based on their mutational signatures. A mutational signature may be regarded as a type of base substitution (e.g. C:G to T:A) within a particular context of neighboring nucleotide sequence (the bases upstream and/or downstream of the mutation). These mutational signatures, supplemented by mutable motifs (a wider mutational context), promise to help us to understand the nature of the mutational processes that operate during tumor evolution because they represent the footprints of interactions between DNA, mutagens and the enzymes of the repair/replication/modification pathways.


Assuntos
Genômica , Mutação , Neoplasias/genética , DNA/genética , Metilação de DNA , Evolução Molecular , Expressão Gênica , Predisposição Genética para Doença , Humanos , Modelos Genéticos , Mutagênicos/farmacologia , Oncogenes , Seleção Genética
20.
Sci Rep ; 7(1): 13192, 2017 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-29038443

RESUMO

Here we describe a new family of carotenoid cleavage oxygenases (CCOs) in metazoans, the BCO2-like (BCOL) clade, which contains lancelet, nematode, and molluscan carotenoid oxygenase sequences. Phylogenetic analysis of CCOs in all kingdoms of life confirmed that the BCOL enzymes are an independent clade of ancient origin. One of the predicted lancelet BCOL proteins, cloned and analyzed for carotenoid cleavage activity in a bacterial carotenoid expression system, had activity similar to lancelet BCO2 proteins, although with a preference for cis isomers. Our docking predictions correlated well with the cis-favored activity. The extensive expansions of the new animal BCOL family in some species (e.g., lancelet) suggests that the carotenoid cleavage oxygenase superfamily has evolved in the "extremely high turnover" fashion: numerous losses and duplications of this family are likely to reflect complex regulation processes during development, and interactions with the environment. These findings also serve to provide a rationale for the evolution of the BCO-related outlier RPE65 retinol isomerase, an enzyme that does not utilize carotenoids as substrate or perform double-bond cleavage.


Assuntos
Oxigenases/genética , Animais , Carotenoides , Oxigenases/classificação , Oxigenases/metabolismo , Filogenia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...