RESUMO
Asparagine peptide lyase (APL) is among the seven groups of proteases, also known as proteolytic enzymes, which are classified according to their catalytic residue. APLs are synthesized as precursors or propeptides that undergo self-cleavage through autoproteolytic reaction. At present, APLs are grouped into 10 families belonging to six different clans of proteases. Recognizing their critical roles in many biological processes including virus maturation, and virulence, accurate identification and characterization of APLs is indispensable. Experimental identification and characterization of APLs is laborious and time-consuming. Here, we developed APLpred, a novel support vector machine (SVM) based predictor that can predict APLs from the primary sequences. APLpred was developed using Boruta-based optimal features derived from seven encodings and subsequently trained using five machine learning algorithms. After evaluating each model on an independent dataset, we selected APLpred (an SVM-based model) due to its consistent performance during cross-validation and independent evaluation. We anticipate APLpred will be an effective tool for identifying APLs. This could aid in designing inhibitors against these enzymes and exploring their functions. The APLpred web server is freely available at https://procarb.org/APLpred/.
Assuntos
Máquina de Vetores de Suporte , Aprendizado de Máquina , Biologia Computacional/métodos , Software , Sequência de Aminoácidos/genética , Bases de Dados de ProteínasRESUMO
SARS-CoV-2's global spread has instigated a critical health and economic emergency, impacting countless individuals. Understanding the virus's phosphorylation sites is vital to unravel the molecular intricacies of the infection and subsequent changes in host cellular processes. Several computational methods have been proposed to identify phosphorylation sites, typically focusing on specific residue (S/T) or Y phosphorylation sites. Unfortunately, current predictive tools perform best on these specific residues and may not extend their efficacy to other residues, emphasizing the urgent need for enhanced methodologies. In this study, we developed a novel predictor that integrated all the residues (STY) phosphorylation sites information. We extracted ten different feature descriptors, primarily derived from composition, evolutionary, and position-specific information, and assessed their discriminative power through five classifiers. Our results indicated that Light Gradient Boosting (LGB) showed superior performance, and five descriptors displayed excellent discriminative capabilities. Subsequently, we identified the top two integrated features have high discriminative capability and trained with LGB to develop the final prediction model, LGB-IPs. The proposed approach shows an excellent performance on 10-fold cross-validation with an ACC, MCC, and AUC values of 0.831, 0.662, 0.907, respectively. Notably, these performances are replicated in the independent evaluation. Consequently, our approach may provide valuable insights into the phosphorylation mechanisms in SARS-CoV-2 infection for biomedical researchers.
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COVID-19 , Biologia Computacional , SARS-CoV-2 , Fosforilação , SARS-CoV-2/metabolismo , Humanos , COVID-19/virologia , COVID-19/metabolismo , Biologia Computacional/métodosRESUMO
The histone variant H3.3 is encoded by two distinct genes, H3f3a and H3f3b, exhibiting identical amino-acid sequence. H3.3 is required for spermatogenesis, but the molecular mechanism of its spermatogenic function remains obscure. Here, we have studied the role of each one of H3.3A and H3.3B proteins in spermatogenesis. We have generated transgenic conditional knock-out/knock-in (cKO/KI) epitope-tagged FLAG-FLAG-HA-H3.3B (H3.3BHA) and FLAG-FLAG-HA-H3.3A (H3.3AHA) mouse lines. We show that H3.3B, but not H3.3A, is required for spermatogenesis and male fertility. Analysis of the molecular mechanism unveils that the absence of H3.3B led to alterations in the meiotic/post-meiotic transition. Genome-wide RNA-seq reveals that the depletion of H3.3B in meiotic cells is associated with increased expression of the whole sex X and Y chromosomes as well as of both RLTR10B and RLTR10B2 retrotransposons. In contrast, the absence of H3.3B resulted in down-regulation of the expression of piRNA clusters. ChIP-seq experiments uncover that RLTR10B and RLTR10B2 retrotransposons, the whole sex chromosomes and the piRNA clusters are markedly enriched of H3.3. Taken together, our data dissect the molecular mechanism of H3.3B functions during spermatogenesis and demonstrate that H3.3B, depending on its chromatin localization, is involved in either up-regulation or down-regulation of expression of defined large chromatin regions.
Assuntos
Histonas , RNA Interferente Pequeno/metabolismo , Retroelementos , Espermatogênese , Animais , Cromatina/genética , Histonas/genética , Histonas/metabolismo , Masculino , Camundongos , Cromossomos Sexuais/metabolismoRESUMO
Presented here are new insights into the marine monoraphid diatom genera Schizostauron and Astartiella, based on molecular and morphological data, including descriptions of new species. Although no unambiguous morphological synapomorphies between the two genera are currently recognized, they are closely related by DNA sequence data. Heterovalvate frustules of Schizostauron are characterized by a bifid stauros on the raphe-bearing valve and intricate areolate occlusions on the sternum valve. In Astartiella, the raphe-bearing valve is characterized by a process resembling a fistula by morphology, while the sternum valve presents a particular striation pattern. Observations by light and electron microscopy were made, along with a molecular phylogenetic analysis using a three-gene (SSU, rbcL, and psbC) concatenated dataset. Three new Schizostauron species are described (S. kajotkei, S. rawaii, S. papilliareae), and two new combinations proposed (S. citronella and S. trachyderma) for species that were previously included either in Achnanthes and Cocconeis, respectively. Likewise, six new species of Astartiella (A. almalikii, A. bornmanii, A. chunlianlii, A. marksii, A. persica, and A. wangii) are described. Molecular results exclude Schizostauron and Astartiella from three clades of exclusively monoraphid diatoms, the Achnanthaceae, Cocconeidaceae, and Achnanthidiaceae, instead placing them in the Stauroneidaceae. Morphological features of Schizostauron and Astartiella, such as the stauros, fistula, and coaxial internal proximal raphe endings, are found in other genera in this clade, whereas the only common feature with monoraphid diatoms as whole group is the heterovalvy of frustules.
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Diatomáceas , Fístula , Diatomáceas/genética , Microscopia Eletrônica , FilogeniaRESUMO
We provide for the first time the complete plastid and mitochondrial genomes of a monoraphid diatom: Schizostauron trachyderma. The mitogenome is 41,957 bp in size and displays two group II introns in the cox1 gene. The 187,029 bp plastid genome features the typical quadripartite architecture of diatom genomes. It contains a group II intron in the petB gene that overlaps the large single-copy and the inverted repeat region. There is also a group IB4 intron encoding a putative LAGLIDADG homing endonuclease in the rnl gene. The multigene phylogenies conducted provide more evidence of the proximity between S. trachyderma and fistula-bearing species of biraphid diatoms.
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Diatomáceas/genética , Genoma Mitocondrial , Genomas de Plastídeos , Diatomáceas/classificação , Diatomáceas/citologia , Evolução Molecular , Fases de Leitura Aberta/genética , Filogenia , Análise de Sequência de DNARESUMO
Chemosensory perception in insects involves a broad set of chemosensory proteins (CSPs) that identify the bouquet of chemical compounds present in the external environment and regulate specific behaviors. The current study is focused on the Spodoptera litura (Fabricius) chemosensory-related protein, SlitCSP3, a midgut-expressed CSP, which demonstrates differential gene expression upon different diet intake. There is an intriguing possibility that SlitCSP3 can perceive food-derived chemical signals and modulate insect feeding behavior. We predicted the three-dimensional structure of SlitCSP3 and subsequently performed an accelerated molecular dynamics (aMD) simulation of the best-modeled structure. SlitCSP3 structure has six α-helices arranged as a prism and a hydrophobic binding pocket predominated by leucine and isoleucine. We analyzed the interaction of selected host plant metabolites with the modeled structure of SlitCSP3. Out of two predicted binding pockets in SlitCSP3, the plant-derived defensive metabolites 2-b-D-glucopyranosyloxy-4-hydroxy-7-methoxy-1, 4-benzoxazin-3-one (DIMBOA), 6-Methoxy-2-benzoxazolinone (MBOA), and nicotine were found to interact preferably to the hydrophobic site 1, compared to site 2. The current study provides the potential role of CSPs in recognizing food-derived chemical signals, host-plant specialization, and adaptation to the varied ecosystem. Our work opens new perspectives in designing novel pest-management strategies. It can be further used in the development of CSP-based advanced biosensors.
Assuntos
Interações Hospedeiro-Parasita , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Modelos Moleculares , Plantas/metabolismo , Plantas/parasitologia , Spodoptera/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Ligantes , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação ProteicaRESUMO
The emergence of resistance on exposure to pharmaceuticals among microorganisms has raised serious concern in the therapeutic approach against infectious diseases. Effluents discharge from hospitals, industries, and urban settlements containing pharmaceuticals and other toxic compounds into the aquatic ecosystem selects bacterial population against them; thereby promotes acquisition and dissemination of resistant traits among the inhabitant microbiota. The present study was aimed to determine the prevalence and multidrug resistance pattern of Extended Spectrum ß-lactamase (ESBL) producing and non-producing bacterial isolates from the heavily polluted Delhi stretch of river Yamuna, India. Additionally, the role of abiotic factors in the dissemination of conjugative plasmids harbouring resistance genes was also studied using E. coli J53 as recipient and resistant E. coli isolates as donor strains. Of the 227 non-duplicate bacterial isolates, 60% (136) were identified as ESBL+ and 40% (91) as ESBL. ESBL+ isolates were found highly resistant to ß-lactam and non-ß-lactam classes of antibiotics compared with the ESBL- isolates. 68% of ESBL+ and 24% of ESBL- isolates showed an MAR index of ≥0.5. Surprisingly, multidrug resistance (MDR), extensively drug resistance (XDR), and pandrug resistance (PDR) phenotype were observed for 78.6%, 16.9%, and 0.7% of ESBL+ and 90%, 3%, and none for PDR among ESBL- isolates. Conjugation under different conditions showed a higher mobilization rate at neutral pH (7-7.5) for ESBL+ isolates. Conjugation frequency was maximum at 40 °C for the isolate E. coli MRB6 (4.1 × 10-5) and E. coli MRE32 (4.89 × 10-4) and at 35 °C for E. coli MRA11 (4.89 × 10-5). The transconjugants obtained were found tolerating different concentrations of mercuric chloride (0.0002-0.2 mg/L). Increased biofilm formation for ESBL+ isolates was observed on supplementing media with HgCl2 (2 µg/mL) either singly or in combination with CTX (10 µg/mL). The present study demonstrates that anthropogenically influenced aquatic environments act as a reservoir of MDR, XDR, and even PDR strains; thereby posing a potent public health risk.
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BACKGROUND: Organelle genome studies of Fabaceae, an economically and ecologically important plant family, have been biased towards the plastid genome (plastome). Thus far, less than 15 mitochondrial genome (mitogenome) sequences of Fabaceae have been published, all but four of which belong to the subfamily Papilionoideae, limiting the understanding of size variation and content across the family. To address this, four mitogenomes were sequenced and assembled from three different subfamilies (Cercidoideae, Detarioideae and Caesalpinioideae). RESULTS: Phylogenetic analysis based on shared mitochondrial protein coding regions produced a fully resolved and well-supported phylogeny that was completely congruent with the plastome tree. Comparative analyses suggest that two kinds of mitogenome expansions have occurred in Fabaceae. Size expansion of four genera (Tamarindus, Libidibia, Haematoxylum, and Leucaena) in two subfamilies (Detarioideae and Caesalpinioideae) occurred in relatively deep nodes, and was mainly caused by intercellular gene transfer and/or interspecific horizontal gene transfer (HGT). The second, more recent expansion occurred in the Papilionoideae as a result of duplication of native mitochondrial sequences. Family-wide gene content analysis revealed 11 gene losses, four (rps2, 7, 11 and 13) of which occurred in the ancestor of Fabaceae. Losses of the remaining seven genes (cox2, rpl2, rpl10, rps1, rps19, sdh3, sdh4) were restricted to specific lineages or occurred independently in different clades. Introns of three genes (cox2, ccmFc and rps10) showed extensive lineage-specific length variation due to large sequence insertions and deletions. Shared DNA analysis among Fabaceae mitogenomes demonstrated a substantial decay of intergenic spacers and provided further insight into HGT between the mimosoid clade of Caesalpinioideae and the holoparasitic Lophophytum (Balanophoraceae). CONCLUSION: This study represents the most exhaustive analysis of Fabaceae mitogenomes so far, and extends the understanding the dynamic variation in size and gene/intron content. The four newly sequenced mitogenomes reported here expands the phylogenetic coverage to four subfamilies. The family has experienced multiple mitogenome size fluctuations in both ancient and recent times. The causes of these size variations are distinct in different lineages. Fabaceae mitogenomes experienced extensive size fluctuation by recruitment of exogenous DNA and duplication of native mitochondrial DNA.
Assuntos
Fabaceae/genética , Tamanho do Genoma , Genoma Mitocondrial/genética , Mitocôndrias/genéticaRESUMO
This study represents the most comprehensive plastome-wide comparison of nucleotide substitution rates across the three subfamilies of Fabaceae: Caesalpinioideae, Mimosoideae, and Papilionoideae. Caesalpinioid and mimosoid legumes have large, unrearranged plastomes compared with papilionoids, which exhibit varying levels of rearrangement including the loss of the inverted repeat (IR) in the IR-lacking clade (IRLC). Using 71 genes common to 39 legume taxa representing all the three subfamilies, we show that papilionoids consistently have higher nucleotide substitution rates than caesalpinioids and mimosoids, and rates in the IRLC papilionoids are generally higher than those in the IR-containing papilionoids. Unsurprisingly, this pattern was significantly correlated with growth habit as most papilionoids are herbaceous, whereas caesalpinioids and mimosoids are largely woody. Both nonsynonymous (dN) and synonymous (dS) substitution rates were also correlated with several biological features including plastome size and plastomic rearrangements such as the number of inversions and indels. In agreement with previous reports, we found that genes in the IR exhibit between three and fourfold reductions in the substitution rates relative to genes within the large single-copy or small single-copy regions. Furthermore, former IR genes in IR-lacking taxa exhibit accelerated rates compared with genes contained in the IR.
Assuntos
Fabaceae/genética , Genomas de Plastídeos/genética , Evolução Molecular , Genoma de Planta/genética , Sequências Repetidas Invertidas/genética , Mutação , Nucleotídeos/genética , FilogeniaRESUMO
BACKGROUND: Calotropis procera is a wild plant species in the family Apocynaceae that is able to grow in harsh, arid and heat stressed conditions. Understanding how this highly adapted plant persists in harsh environments should inform future efforts to improve the hardiness of crop and forage plant species. To study the plant response to droµght and osmotic stress, we treated plants with polyethylene glycol and NaCl and carried out transcriptomic and metabolomics measurements across a time-course of five days. RESULTS: We identified a highly dynamic transcriptional response across the time-course including dramatic changes in inositol signaling, stress response genes and cytokinins. The resulting metabolome changes also involved sharp increases of myo-inositol, a key signaling molecule and elevated amino acid metabolites at later times. CONCLUSIONS: The data generated here provide a first glimpse at the expressed genome of C. procera, a plant that is exceptionally well adapted to arid environments. We demonstrate, through transcriptome and metabolome analysis that myo-inositol signaling is strongly induced in response to drought and salt stress and that there is elevation of amino acid concentrations after prolonged osmotic stress. This work should lay the foundations of future studies in adaptation to arid environments.
Assuntos
Calotropis/metabolismo , Calotropis/genética , Genes de Plantas , Metaboloma , Estresse Oxidativo , Estresse Fisiológico , TranscriptomaRESUMO
PREMISE OF THE STUDY: There is a misinterpretation in the literature regarding the variable orientation of the small single copy region of plastid genomes (plastomes). The common phenomenon of small and large single copy inversion, hypothesized to occur through intramolecular recombination between inverted repeats (IR) in a circular, single unit-genome, in fact, more likely occurs through recombination-dependent replication (RDR) of linear plastome templates. If RDR can be primed through both intra- and intermolecular recombination, then this mechanism could not only create inversion isomers of so-called single copy regions, but also an array of alternative sequence arrangements. METHODS: We used Illumina paired-end and PacBio single-molecule real-time (SMRT) sequences to characterize repeat structure in the plastome of Monsonia emarginata (Geraniaceae). We used OrgConv and inspected nucleotide alignments to infer ancestral nucleotides and identify gene conversion among repeats and mapped long (>1 kb) SMRT reads against the unit-genome assembly to identify alternative sequence arrangements. RESULTS: Although M. emarginata lacks the canonical IR, we found that large repeats (>1 kilobase; kb) represent â¼22% of the plastome nucleotide content. Among the largest repeats (>2 kb), we identified GC-biased gene conversion and mapping filtered, long SMRT reads to the M. emarginata unit-genome assembly revealed alternative, substoichiometric sequence arrangements. CONCLUSION: We offer a model based on RDR and gene conversion between long repeated sequences in the M. emarginata plastome and provide support that both intra-and intermolecular recombination between large repeats, particularly in repeat-rich plastomes, varies unit-genome structure while homogenizing the nucleotide sequence of repeats.
Assuntos
Conversão Gênica/genética , Genomas de Plastídeos/genética , Recombinação Genética/genética , Replicação do DNA/genética , Genoma de Planta/genética , Geraniaceae/genética , Sequências Repetitivas de Ácido Nucleico/genética , Alinhamento de SequênciaRESUMO
Next-generation sequencing (NGS) provides a powerful tool for the discovery of important genes and alleles in crop plants and their wild relatives. Despite great advances in NGS technologies, whole-genome shotgun sequencing is cost-prohibitive for species with complex genomes. An attractive option is to reduce genome complexity to a single chromosome prior to sequencing. This work describes a strategy for studying the genomes of distant wild relatives of wheat by isolating single chromosomes from addition or substitution lines, followed by chromosome sorting using flow cytometry and sequencing of chromosomal DNA by NGS technology. We flow-sorted chromosome 5M(g) from a wheat/Aegilops geniculata disomic substitution line [DS5M(g) (5D)] and sequenced it using an Illumina HiSeq 2000 system at approximately 50 × coverage. Paired-end sequences were assembled and used for structural and functional annotation. A total of 4236 genes were annotated on 5M(g) , in close agreement with the predicted number of genes on wheat chromosome 5D (4286). Single-gene FISH indicated no major chromosomal rearrangements between chromosomes 5M(g) and 5D. Comparing chromosome 5M(g) with model grass genomes identified synteny blocks in Brachypodium distachyon, rice (Oryza sativa), sorghum (Sorghum bicolor) and barley (Hordeum vulgare). Chromosome 5M(g) -specific SNPs and cytogenetic probe-based resources were developed and validated. Deletion bin-mapped and ordered 5M(g) SNP markers will be useful to track 5M-specific introgressions and translocations. This study provides a detailed sequence-based analysis of the composition of a chromosome from a distant wild relative of bread wheat, and opens up opportunities to develop genomic resources for wild germplasm to facilitate crop improvement.
Assuntos
Cromossomos de Plantas/genética , Genes de Plantas/genética , Genoma de Planta/genética , Poaceae/genética , Brachypodium/genética , Mapeamento Cromossômico , Evolução Molecular , Ordem dos Genes , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Hordeum/genética , Hibridização in Situ Fluorescente , Oryza/genética , Poaceae/classificação , Polimorfismo de Nucleotídeo Único , Sorghum/genética , Triticum/genéticaRESUMO
BACKGROUND: Climate change is predicted to be a serious threat to agriculture due to the need for crops to be able to tolerate increased heat stress. Desert plants have already adapted to high levels of heat stress so they make excellent systems for identifying genes involved in thermotolerance. Rhazya stricta is an evergreen shrub that is native to extremely hot regions across Western and South Asia, making it an excellent system for examining plant responses to heat stress. Transcriptomes of apical and mature leaves of R. stricta were analyzed at different temperatures during several time points of the day to detect heat response mechanisms that might confer thermotolerance and protection of the plant photosynthetic apparatus. RESULTS: Biological pathways that were crosstalking during the day involved the biosynthesis of several heat stress-related compounds, including soluble sugars, polyols, secondary metabolites, phenolics and methionine. Highly downregulated leaf transcripts at the hottest time of the day (40-42.4 °C) included genes encoding cyclin, cytochrome p450/secologanin synthase and U-box containing proteins, while upregulated, abundant transcripts included genes encoding heat shock proteins (HSPs), chaperones, UDP-glycosyltransferase, aquaporins and protein transparent testa 12. The upregulation of transcripts encoding HSPs, chaperones and UDP-glucosyltransferase and downregulation of transcripts encoding U-box containing proteins likely contributed to thermotolerance in R. stricta leaf by correcting protein folding and preventing protein degradation. Transcription factors that may regulate expression of genes encoding HSPs and chaperones under heat stress included HSFA2 to 4, AP2-EREBP and WRKY27. CONCLUSION: This study contributed new insights into the regulatory mechanisms of thermotolerance in the wild plant species R. stricta, an arid land, perennial evergreen shrub common in the Arabian Peninsula and Indian subcontinent. Enzymes from several pathways are interacting in the biosynthesis of soluble sugars, polyols, secondary metabolites, phenolics and methionine and are the primary contributors to thermotolerance in this species.
Assuntos
Apocynaceae/genética , Proteínas de Plantas/genética , Transcrição Gênica , Apocynaceae/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Temperatura Alta , Proteínas de Plantas/fisiologia , Estresse Fisiológico , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , TranscriptomaRESUMO
BACKGROUND: Rhazya stricta is native to arid regions in South Asia and the Middle East and is used extensively in folk medicine to treat a wide range of diseases. In addition to generating genomic resources for this medicinally important plant, analyses of the complete plastid and mitochondrial genomes and a nuclear transcriptome from Rhazya provide insights into inter-compartmental transfers between genomes and the patterns of evolution among eight asterid mitochondrial genomes. RESULTS: The 154,841 bp plastid genome is highly conserved with gene content and order identical to the ancestral organization of angiosperms. The 548,608 bp mitochondrial genome exhibits a number of phenomena including the presence of recombinogenic repeats that generate a multipartite organization, transferred DNA from the plastid and nuclear genomes, and bidirectional DNA transfers between the mitochondrion and the nucleus. The mitochondrial genes sdh3 and rps14 have been transferred to the nucleus and have acquired targeting presequences. In the case of rps14, two copies are present in the nucleus; only one has a mitochondrial targeting presequence and may be functional. Phylogenetic analyses of both nuclear and mitochondrial copies of rps14 across angiosperms suggests Rhazya has experienced a single transfer of this gene to the nucleus, followed by a duplication event. Furthermore, the phylogenetic distribution of gene losses and the high level of sequence divergence in targeting presequences suggest multiple, independent transfers of both sdh3 and rps14 across asterids. Comparative analyses of mitochondrial genomes of eight sequenced asterids indicates a complicated evolutionary history in this large angiosperm clade with considerable diversity in genome organization and size, repeat, gene and intron content, and amount of foreign DNA from the plastid and nuclear genomes. CONCLUSIONS: Organelle genomes of Rhazya stricta provide valuable information for improving the understanding of mitochondrial genome evolution among angiosperms. The genomic data have enabled a rigorous examination of the gene transfer events. Rhazya is unique among the eight sequenced asterids in the types of events that have shaped the evolution of its mitochondrial genome. Furthermore, the organelle genomes of R. stricta provide valuable genomic resources for utilizing this important medicinal plant in biotechnology applications.
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Apocynaceae/genética , Genoma Mitocondrial , Genoma de Planta , Apocynaceae/classificação , Sequência de Bases , Evolução Biológica , Elementos de DNA Transponíveis , Transferência Genética Horizontal , Genomas de Plastídeos , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Filogenia , Proteínas de Plantas/classificação , Proteínas de Plantas/genética , Plantas Medicinais/genética , Plastídeos/genética , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Lycopene is an effective antioxidant proposed as a possible treatment for some cancers and other degenerative human conditions. This study aims at generation of a yeast strain (Saccharomyces cerevisiae) of efficient productivity of lycopene by overexpressing synthetic genes derived from crtE, crtB and crtI genes of Erwinia uredovora. These synthetic genes were constructed in accordance with the preferred codon usage in S. cerevisiae but with no changes in amino acid sequences of the gene products. S. cerevisiae cells were transformed with these synthetic crt genes, whose expression was regulated by the ADH2 promoter, which is de-repressed upon glucose depletion. The RT-PCR and Western blotting analyses indicated that the synthetic crt genes were efficiently transcribed and translated in crt-transformed S. cerevisiae cells. The highest level of lycopene in one of the transformed lines was 3.3mglycopene/g dry cell weight, which is higher than the previously reported levels of lycopene in other microorganisms transformed with the three genes. These results suggest the excellence of using the synthetic crt genes and the ADH2 promoter in generation of recombinant S. cerevisiae that produces a high level of lycopene. The level of ergosterol was reversely correlated to that of lycopene in crt-transformed S. cerevisiae cells, suggesting that two pathways for lycopene and ergosterol syntheses compete for the use of farnesyl diphosphate.
Assuntos
Carotenoides/biossíntese , Farnesiltranstransferase/genética , Geranil-Geranildifosfato Geranil-Geraniltransferase/genética , Oxirredutases/genética , Saccharomyces cerevisiae/genética , Clonagem Molecular , Ergosterol/biossíntese , Erwinia/enzimologia , Erwinia/genética , Farnesiltranstransferase/biossíntese , Expressão Gênica , Genes Bacterianos , Geranil-Geranildifosfato Geranil-Geraniltransferase/biossíntese , Licopeno , Organismos Geneticamente Modificados/genética , Organismos Geneticamente Modificados/metabolismo , Oxirredutases/biossíntese , Plasmídeos , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Transformação GenéticaRESUMO
The circadian clock generates daily rhythms in mammalian liver processes, such as glucose and lipid homeostasis, xenobiotic metabolism, and regeneration. The mechanisms governing these rhythms are not well understood, particularly the distinct contributions of the cell-autonomous clock and central pacemaker to rhythmic liver physiology. Through microarray expression profiling in Met murine hepatocytes (MMH)-D3, we identified over 1,000 transcripts that exhibit circadian oscillations, demonstrating that the cell-autonomous clock can drive many rhythms, and that MMH-D3 is a valid circadian model system. The genes represented by these circadian transcripts displayed both cophasic and antiphasic organization within a protein-protein interaction network, suggesting the existence of competition for binding sites or partners by genes of disparate transcriptional phases. Multiple pathways displayed enrichment in MMH-D3 circadian transcripts, including the polyamine synthesis module of the glutathione metabolic pathway. The polyamine synthesis module, which is highly associated with cell proliferation and whose products are required for initiation of liver regeneration, includes enzymes whose transcripts exhibit circadian oscillations, such as ornithine decarboxylase and spermidine synthase. Metabolic profiling revealed that the enzymatic product of spermidine synthase, spermidine, cycles as well. Thus, the cell-autonomous hepatocyte clock can drive a significant amount of transcriptional rhythms and orchestrate physiologically relevant modules such as polyamine synthesis.
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Poliaminas Biogênicas/biossíntese , Ritmo Circadiano , Hepatócitos/citologia , Transcrição Gênica , Animais , CamundongosRESUMO
The prevalence of obesity, characterized by an excessive accumulation of adipose tissue and adipocyte hypertrophy, presents a major public health challenge. This study investigates the therapeutic potential of two probiotic strains, Lactobacillus sakei Probio65 and Lactobacillus plantarum Probio-093, in the context of obesity. Utilizing 3T3-L1 cell-derived human adipocytes, we assessed Probio65's and Probio-093's capacity to mitigate triglyceride accumulation and influence adipocytokine production in vitro. Subsequently, an in vivo trial with male C57BL/6J mice examined the effects of both probiotic strains on adipose tissue characteristics, body weight, fat mass, and obesity-related gene expression. This study employed both live and ethanol-extracted bacterial cells. The results demonstrated significant reductions in the triglyceride deposition, body weight, and adipose tissue mass in the treated groups (p < 0.05). Furthermore, both strains modulated adipokine profiles by downregulating proinflammatory markers such as PAI-1, leptin, TNF-α, STAMP2, F4/80, resistin, and MCP-1, and upregulating the insulin-sensitive transporter GLUT4 and the anti-inflammatory adiponectin (p < 0.05). Our findings suggest that Lactobacillus sakei Probio65 and Lactobacillus plantarum Probio-093 are promising agents for microbiome-targeted anti-obesity therapies, offering the effective mitigation of obesity and improvement in adipocyte function in a murine model.
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HIRIP3 is a mammalian protein homologous to the yeast H2A.Z deposition chaperone Chz1. However, the structural basis underlying Chz's binding preference for H2A.Z over H2A, as well as the mechanism through which Chz1 modulates histone deposition or replacement, remains enigmatic. In this study, we aimed to characterize the function of HIRIP3 and to identify its interacting partners in HeLa cells. Our findings reveal that HIRIP3 is specifically associated in vivo with H2A-H2B dimers and CK2 kinase. While bacterially expressed HIRIP3 exhibited a similar binding affinity towards H2A and H2A.Z, the associated CK2 kinase showed a notable preference for H2A phosphorylation at serine 1. The recombinant HIRIP3 physically interacted with the H2A αC helix through an extended CHZ domain and played a crucial role in depositing the canonical core histones onto naked DNA. Our results demonstrate that mammalian HIRIP3 acts as an H2A histone chaperone, assisting in its selective phosphorylation by Ck2 kinase at serine 1 and facilitating its deposition onto chromatin.
Assuntos
Chaperonas de Histonas , Histonas , Animais , Humanos , Células HeLa , Chaperonas de Histonas/genética , Histonas/metabolismo , Mamíferos/metabolismo , Chaperonas Moleculares/metabolismo , Saccharomyces cerevisiae/metabolismo , Serina , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismoRESUMO
DNA methylation is one of the most important epigenetic mark involved in many physiologic cellular processes and pathologies. During mitosis, the transmission of DNA methylation patterns from a mother to the daughter cells is ensured through the action of the Ubiquitin-like, containing PHD and RING domains, 1/DNA methyltransferase 1 (UHRF1/DNMT1) tandem. UHRF1 is involved in the silencing of many tumor suppressor genes (TSGs) via mechanisms that remain largely to be deciphered. The present study investigated the role and the regulation of UHRF1 poly-ubiquitination induced by thymoquinone, a natural anti-cancer drug, known to enhance or re-activate the expression of TSGs. We found that the auto-ubiquitination of UHRF1, induced by TQ, is mediated by reactive oxygen species, and occurs following DNA damage. We demonstrated that the poly-ubiquitinated form of UHRF1 is K63-linked and can still silence the tumor suppressor gene p16INK4A/CDKN2A. We further showed that TQ-induced auto-ubiquitination is mediated via the activity of Tip60. Since this latter is known as a nuclear receptor co-factor, we investigated if the glucocorticoid receptor (GR) might be involved in the regulation of UHRF1 ubiquitination. Activation of the GR, with dexamethasone, did not influence auto-ubiquitination of UHRF1. However, we could observe that TQ induced a K48-linked poly-ubiquitination of GR, probably involved in the proteosomal degradation pathway. Mass-spectrometry analysis of FLAG-HA-tagged UHRF1 identified UHRF1 partners involved in DNA repair and showed that TQ increased their association with UHRF1, suggesting that poly-ubiquitination of UHRF1 is involved in the DNA repair process. We propose that poly-ubiquitination of UHRF1 serves as a scaffold to recruit the DNA repair machinery at DNA damage sites.
Assuntos
Benzoquinonas , Proteínas Estimuladoras de Ligação a CCAAT , Reparo do DNA , Ubiquitina-Proteína Ligases , Ubiquitinação , Humanos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Ubiquitinação/efeitos dos fármacos , Benzoquinonas/farmacologia , Reparo do DNA/efeitos dos fármacos , Antineoplásicos/farmacologia , Dano ao DNA/efeitos dos fármacosRESUMO
Antimicrobial peptides (AMPs) are promising novel agents for targeting a wide range of pathogens. In this study, microalgal peptides derived from native microalgae were incorporated into polycaprolactone (PCL) with Æ-Carrageenan (Æ-C) forming nanofibers using the electrospinning method. The peptides incorporated in the nanofibers were characterized by fourier infrared spectroscopy, thermogravimetric analysis, scanning electron microscopy (SEM), and contact angle measurement. The results showed that peptides with molecular weights < 10 kDa, when loaded into nanofibers, exhibited lower wettability. The SEM analysis revealed a thin, smooth, interconnected bead-like structures. The antimicrobial activity of the electrospun nanofibers was evaluated through disc diffusion, and minimum inhibitory activity against Escherichia coli (MTTC 443), and Staphylococcus aureus (MTTC 96), resulting in zones of inhibition of 24 ± 0.5 mm and 14 ± 0.5 mm, respectively. The in vitro biocompatibility of the synthesized nanofibers was confirmed using in HEK 293 cell lines with an increased cell viability. Interestingly, the fibers also exhibited a significant wound-healing properties when used in vitro scratch assays. In conclusion, algal peptides incorporated with PCL/ Æ-C were found to exhibit antimicrobial and biocompatible biomaterials for wound healing applications.