RESUMO
BACKGROUND: Carbon nanotubes (CNTs) show great promise as theranostic agents due to their drug delivery properties, intrinsic near-infrared radiation-responsiveness, and magnetic functionalization. However, temperature elevation caused by these external stimuli during drug delivery should be considered for the evaluation of CNT-based systems loaded with temperature-sensitive biomolecules. METHODS: We examine the thermal stability of a 33 nucleotides long hairpin miRNA encapsulated in (20,20) CNT using all-atom molecular dynamics simulations in explicit water. We systematically increase the temperature as 298, 310, 327, and 343 K, reaching the melting temperature of miRNA. To emphasize the effect of the aromatic confined space, we compare the dynamics of miRNA inside the CNT to its dynamics free in the solution at the same temperatures, reaching a total simulation time of 7.9 µs. RESULTS: miRNA hairpin mostly maintains its double-stranded structure in the confined CNT, even at elevated temperatures. Binding free energies and potential of mean force calculations also underline the strong π-π interactions between the biomolecule and the CNT for 298-343 K. CONCLUSION: The let-7 miRNA mimic, which represents a wide family of RNAi-based therapeutics, can be transported in the CNT under medically applied hyperthermic conditions. GENERAL SIGNIFICANCE: This study shows how the structure and dynamics of miRNA hairpin are affected when encapsulated in an aromatic tube, during a systematic increase of temperature. It also indicates the high potential of CNT-based systems for the delivery of oligonucleotide therapeutics while simultaneous imaging/magnetic field guiding to the target tissue is achieved.
Assuntos
MicroRNAs/química , Nanotubos de Carbono/química , Animais , Caenorhabditis elegans/química , Modelos Moleculares , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Estabilidade de RNA , TemperaturaRESUMO
Late embryogenesis abundant (LEA) proteins play a protective role during desiccation and oxidation stresses. LEA3 proteins are a major group characterized by a hydrophilic domain (HD) with a highly conserved repeating 11-amino acid motif. We compared four different HD orthologs from distant organisms: (i) DrHD from the extremophilic bacterium Deinococcus radiodurans; (ii) CeHD from the nematode Caenorhabditis elegans; (iii) YlHD from the yeast Yarrowia lipolytica; and (iv) BnHD from the plant Brassica napus. Circular dichroism spectroscopy showed that all four HDs were intrinsically disordered in phosphate buffer and then folded into α-helical structures with the addition of glycerol or trifluoroethanol. Heterologous HD expression conferred enhanced desiccation and oxidation tolerance to Escherichia coli. These four HDs protected the enzymatic activities of lactate dehydrogenase (LDH) by preventing its aggregation under desiccation stress. The HDs also interacted with LDH, which was intensified by the addition of hydrogen peroxide (H2 O2 ), suggesting a protective role in a chaperone-like manner. Based on these results, the HDs of LEA3 proteins show promise as protectants for desiccation and oxidation stresses, especially DrHD, which is a potential ideal stress-response element that can be applied in synthetic biology due to its extraordinary protection and stress resistance ability.
Assuntos
Proteínas de Bactérias/química , Proteínas de Caenorhabditis elegans/química , Proteínas Fúngicas/química , Proteínas de Plantas/química , Animais , Proteínas de Bactérias/isolamento & purificação , Brassica napus/química , Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/isolamento & purificação , Dicroísmo Circular , Clonagem Molecular , Desidratação , Deinococcus/química , Escherichia coli/genética , Escherichia coli/fisiologia , Proteínas Fúngicas/isolamento & purificação , Expressão Gênica , Viabilidade Microbiana , Estresse Oxidativo , Proteínas de Plantas/isolamento & purificação , Conformação Proteica , Dobramento de Proteína , Estresse Fisiológico , Yarrowia/químicaRESUMO
Anthelmintic resistance is widespread in gastrointestinal nematode populations, such that there is a consistent need to search for new anthelmintics. However, the cost of screening for new compounds is high and has a very low success rate. Using the knowledge of traditional healers from Borneo Rainforests (Sarawak, Malaysia), we have previously shown that some traditional medicinal plants are a rich source of potential new anthelmintic drug candidates. In this study, Picria fel-terrae Lour. plant extract, which has previously shown promising anthelmintic activities, was fractionated via the use of a solid phase extraction cartridge and each isolated fraction was then tested on free-living nematode Caenorhabditis elegans and the parasitic nematode Haemonchus contortus. We found that a single fraction was enriched for nematocidal activity, killing ≥90% of C. elegans adults and inhibiting the motility of exsheathed L3 of H. contortus, while having minimal cytotoxic activity in mammalian cell culture. Metabolic profiling and chemometric analysis of the effective fraction indicated medium chained fatty acids and phenolic acids were highly represented.
Assuntos
Anti-Helmínticos/farmacologia , Caenorhabditis elegans/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Haemonchus/efeitos dos fármacos , Metabolômica/métodos , Extratos Vegetais/farmacologia , Plantas Medicinais/química , Animais , Anti-Helmínticos/isolamento & purificação , Bioensaio/métodos , Bornéu , Caenorhabditis elegans/química , Haemonchus/química , Lamiales/química , Metaboloma , Extratos Vegetais/isolamento & purificação , Análise de SobrevidaRESUMO
The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.
Assuntos
Caenorhabditis elegans/química , Gorduras/análise , Gorduras/química , Coloração e Rotulagem/métodos , 2-Propanol/química , Animais , Cromatografia Gasosa-Espectrometria de Massas/métodos , Microscopia de Fluorescência/métodos , Oxazinas/química , Fosfolipídeos/análise , Triglicerídeos/análiseRESUMO
Protein aggregation is a common feature of late onset neurodegenerative disorders, including Alzheimer's disease. In Alzheimer's disease, misassembly of the Abeta peptide is genetically linked to proteotoxicity associated with disease etiology. A reduction in Abeta proteotoxicity is accomplished, in part, by the previously reported Abeta disaggregation and proteolysis activities-under partial control of heat shock factor 1, a transcription factor regulating proteostasis in the cytosol and negatively regulated by insulin growth factor signaling. Herein, we report an improved in vitro assay to quantify recombinant fibrillar Abeta disaggregation kinetics accomplished by the exogenous application of C.elegans extracts. With this assay we demonstrate that the Abeta disaggregation and proteolysis activities of C.elegans are separable. The disaggregation activity found in C.elegans preparations is more heat resistant than the proteolytic activity. Abeta disaggregation in the absence of proteolysis was found to be a reversible process. Future discovery of the molecular basis of the disaggregation and proteolysis activities offers the promise of delaying the age-onset proteotoxicity that leads to neurodegeneration in a spectrum of maladies.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/química , Fragmentos de Peptídeos/metabolismo , Multimerização Proteica , Doença de Alzheimer , Peptídeos beta-Amiloides/química , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/enzimologia , Proteínas de Caenorhabditis elegans/química , Modelos Animais de Doenças , Cinética , Microscopia de Força Atômica , Fragmentos de Peptídeos/química , Peptídeo Hidrolases/metabolismo , Temperatura , Extratos de Tecidos/metabolismoRESUMO
Disordered or unstructured regions of proteins, while often very important biologically, can pose significant challenges for resonance assignment and three-dimensional structure determination of the ordered regions of proteins by NMR methods. In this article, we demonstrate the application of (1)H/(2)H exchange mass spectrometry (DXMS) for the rapid identification of disordered segments of proteins and design of protein constructs that are more suitable for structural analysis by NMR. In this benchmark study, DXMS is applied to five NMR protein targets chosen from the Northeast Structural Genomics project. These data were then used to design optimized constructs for three partially disordered proteins. Truncated proteins obtained by deletion of disordered N- and C-terminal tails were evaluated using (1)H-(15)N HSQC and (1)H-(15)N heteronuclear NOE NMR experiments to assess their structural integrity. These constructs provide significantly improved NMR spectra, with minimal structural perturbations to the ordered regions of the protein structure. As a representative example, we compare the solution structures of the full length and DXMS-based truncated construct for a 77-residue partially disordered DUF896 family protein YnzC from Bacillus subtilis, where deletion of the disordered residues (ca. 40% of the protein) does not affect the native structure. In addition, we demonstrate that throughput of the DXMS process can be increased by analyzing mixtures of up to four proteins without reducing the sequence coverage for each protein. Our results demonstrate that DXMS can serve as a central component of a process for optimizing protein constructs for NMR structure determination.
Assuntos
Medição da Troca de Deutério , Espectroscopia de Ressonância Magnética/métodos , Espectrometria de Massas/métodos , Proteínas/química , Amidas/química , Sequência de Aminoácidos , Animais , Bactérias/química , Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/química , Proteínas de Escherichia coli/química , Espectroscopia de Ressonância Magnética/economia , Espectrometria de Massas/economia , Modelos Moleculares , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Conformação Proteica , Fatores de TempoRESUMO
To date, 9 FMRFamide-related peptides (FaRPs) have been structurally characterised from Caenorhabditis elegans. Radioimmunometrical screening of an ethanolic extract of C. elegans revealed the presence of two additional FaRPs that were purified by reverse-phase HPLC and subjected to Edman degradation analysis and gas-phase sequencing. Unequivocal primary structures for the two FaRPs were determined as Ala-Ala-Asp-Gly-Ala-Pro-Leu-Ile-Arg-Phe-NH(2) and Ser-Val-Pro-Gly-Val-Leu-Arg-Phe-NH(2). Using MALDI-TOF mass spectrometry, the molecular masses of the peptides were found to be 1032 Da (MH) and 875 Da (MH)(+), respectively. Two copies of AADGAPLIRFamide are predicted to be encoded on the precursor gene termed flp-13, while one copy of SVPGVLRFamide is located on flp-18. Synthetic replicates of the peptides were tested on Ascaris suum somatic muscle to assess bioactivity. ADDGAPLIRFamide had inhibitory effects on A. suum muscle strips, which occurred over a range of concentrations from a threshold for activity of 10 nM to 10 microM. SVPGVLRFamide was excitatory on A. suum somatic musculature from a threshold concentration for activity of 1 nM to 10 microM. The inhibitory and excitatory effects of AADGAPLIRFamide and SVPGVLRFamide, respectively, were the same for dorsal and ventral muscle strips as well as innervated and denervated preparations, suggesting that these physiological effects are not nerve cord dependent. Addition of ADDGAPLIRFamide (10 microM) to muscle strips preincubated in high-K(+) and -Ca(2+)-free medium resulted in a normal inhibitory response. Peptide addition to muscle strips preincubated in Cl(-)-free medium showed no inhibitory response, suggesting that the inhibitory response of the peptide may be chloride mediated. A normal excitatory response was noted following the addition of 10 microM SVPGVLRFamide to muscle strips preincubated in high-K(+), Ca(2+)- and Cl(-)-free media.
Assuntos
Caenorhabditis elegans/química , FMRFamida/química , Oligopeptídeos/química , Peptídeos/química , Peptídeos/isolamento & purificação , Animais , Ascaris suum , Cálcio/química , Cloretos/química , Cromatografia Líquida de Alta Pressão , Eletrofisiologia , Feminino , Espectrometria de Massas , Dados de Sequência Molecular , Músculos/química , Músculos/metabolismo , Oligopeptídeos/isolamento & purificação , Potássio/química , Análise de Sequência de Proteína , Fatores de TempoRESUMO
The complete genome sequence of the nematode Caenorhabditis elegans provides an excellent basis for studying the distribution and evolution of protein families in higher eukaryotes. Three fundamental questions are as follows: How many paralog clusters exist in one species, how many of these are shared with other species, and how many proteins can be assigned a functional counterpart in other species? We have addressed these questions in a detailed study of predicted membrane proteins in C. elegans and their mammalian homologs. All worm proteins predicted to contain at least two transmembrane segments were clustered on the basis of sequence similarity. This resulted in 189 groups with two or more sequences, containing, in total, 2647 worm proteins. Hidden Markov models (HMMs) were created for each family, and were used to retrieve mammalian homologs from the SWISSPROT, TREMBL, and VTS databases. About one-half of these clusters had mammalian homologs. Putative worm-mammalian orthologs were extracted by use of nine different phylogenetic methods and BLAST. Eight clusters initially thought to be worm-specific were assigned mammalian homologs after searching EST and genomic sequences. A compilation of 174 orthology assignments made with high confidence is presented.
Assuntos
Caenorhabditis elegans/química , Caenorhabditis elegans/genética , Proteínas de Membrana/química , Proteínas de Membrana/classificação , Família Multigênica/genética , Homologia de Sequência de Aminoácidos , Sequência de Aminoácidos , Animais , Biologia Computacional/métodos , Bases de Dados Factuais , Etiquetas de Sequências Expressas , Proteínas de Helminto/classificação , Proteínas de Helminto/genética , Humanos , Cadeias de Markov , Proteínas de Membrana/genética , Dados de Sequência Molecular , Filogenia , Alinhamento de SequênciaRESUMO
The predicted proteins of the genome of Caenorhabditis elegans were analysed by various sequence comparison methods to identify the repertoire of proteins that are members of the immunoglobulin superfamily (IgSF). The IgSF is one of the largest families of protein domain in this genome and likely to be one of the major families in other multicellular eukaryotes too. This is because members of the superfamily are involved in a variety of functions including cell-cell recognition, cell-surface receptors, muscle structure and, in higher organisms, the immune system. Sixty-four proteins with 488 I set IgSF domains were identified largely by using Hidden Markov models. The domain architectures of the protein products of these 64 genes are described. Twenty-one of these had been characterised previously. We show that another 25 are related to proteins of known function. The C. elegans IgSF proteins can be classified into five broad categories: muscle proteins, protein kinases and phosphatases, three categories of proteins involved in the development of the nervous system, leucine-rich repeat containing proteins and proteins without homologues of known function, of which there are 18. The 19 proteins involved in nervous system development that are not kinases or phosphatases are homologues of neuroglian, axonin, NCAM, wrapper, klingon, ICCR and nephrin or belong to the recently identified zig gene family. Out of the set of 64 genes, 22 are on the X chromosome. This study should be seen as an initial description of the IgSF repertoire in C. elegans, because the current gene definitions may contain a number of errors, especially in the case of long sequences, and there may be IgSF genes that have not yet been detected. However, the proteins described here do provide an overview of the bulk of the repertoire of immunoglobulin superfamily members in C. elegans, a framework for refinement and extension of the repertoire as gene and protein definitions improve, and the basis for investigations of their function and for comparisons with the repertoires of other organisms.