RESUMO
Receptor-interacting protein kinase 1 (RIPK1) is a therapeutic target in treating neurodegenerative diseases and cancers. RIPK1 has three distinct functional domains, with the center domain containing a receptor-interacting protein homotypic interaction motif (RHIM), which mediates amyloid formation. The functional amyloid formed by RIPK1 and/or RIPK3 is a crucial intermediate in regulating cell necroptosis. In this study, the amyloid structure of mouse RIPK1, formed by an 82-residue sequence centered at RHIM, is presented. It reveals the "N"-shaped folding of the protein subunit in the fibril with four ß-strands. The folding pattern is shared by several amyloid structures formed by proteins with RHIM, with the central ß-strand formed by the most conserved tetrad sequence I/VQI/VG. However, the solid-state NMR results indicate a structural difference between mouse RIPK1 and mouse RIPK3. A change in the structural rigidity is also suggested by the observation of weakened signals for mouse RIPK3 upon mixing with RIPK1 to form the RIPK1/RIPK3 complex fibrils. Our results provide vital information to understand the interactions between different proteins with RHIM, which will help us further comprehend the regulation mechanism in cell necroptosis.
Assuntos
Amiloide , Proteína Serina-Treonina Quinases de Interação com Receptores , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/química , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Animais , Camundongos , Amiloide/metabolismo , Amiloide/química , Humanos , Necroptose , Sequência de Aminoácidos , Domínios Proteicos , Ligação Proteica , Modelos MolecularesRESUMO
d-Tagatose is a highly promising functional sweetener known for its various physiological functions. In this study, a novel tagatose 4-epimerase from Thermoprotei archaeon (Thar-T4Ease), with the ability to convert d-fructose to d-tagatose, was discovered through a combination of structure similarity search and sequence-based protein clustering. The recombinant Thar-T4Ease exhibited optimal activity at pH 8.5 and 85 °C, in the presence of 1 mM Ni2+. Its kcat and kcat/Km values toward d-fructose were measured to be 248.5 min-1 and 2.117 mM-1·min-1, respectively. Notably, Thar-T4Ease exhibited remarkable thermostability, with a t1/2 value of 198 h at 80 °C. Moreover, it achieved a conversion ratio of 18.9% using 100 g/L d-fructose as the substrate. Finally, based on sequence and structure analysis, crucial residues for the catalytic activity of Thar-T4Ease were identified by molecular docking and site-directed mutagenesis. This research expands the repertoire of enzymes with C4-epimerization activity and opens up new possibilities for the cost-effective production of d-tagatose from d-fructose.
Assuntos
Estabilidade Enzimática , Hexoses , Simulação de Acoplamento Molecular , Hexoses/química , Hexoses/metabolismo , Cinética , Proteínas Arqueais/genética , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Frutose/química , Frutose/metabolismo , Carboidratos Epimerases/genética , Carboidratos Epimerases/química , Carboidratos Epimerases/metabolismo , Concentração de Íons de Hidrogênio , Especificidade por Substrato , Temperatura Alta , Sequência de Aminoácidos , Racemases e Epimerases/genética , Racemases e Epimerases/química , Racemases e Epimerases/metabolismoRESUMO
Transcription Termination Factor 1 (TTF1) is a multifunctional mammalian protein with vital roles in various cellular processes, including Pol I-mediated transcription initiation and termination, pre-rRNA processing, chromatin remodelling, DNA damage repair, and polar replication fork arrest. It comprises two distinct functional regions; the N-terminal regulatory region (1-445 aa), and the C-terminal catalytic region (445-859 aa). The Myb domain located at the C-terminal region is a conserved DNA binding domain spanning from 550 to 732 aa (183 residues). Despite its critical role in various cellular processes, the physical structure of TTF1 remains unsolved. Attempts to purify the functional TTF1 protein have been unsuccessful till date. Therefore, we focused on characterizing the Myb domain of this essential protein. We started with predicting a 3-D model of the Myb domain using homology modelling, and ab-initio method. We then determined its stability through MD simulation in an explicit solvent. The model predicted is highly stable, which stabilizes at 200ns. To experimentally validate the computational model, we cloned and expressed the codon optimized Myb domain into a bacterial expression vector and purified the protein to homogeneity. Further, characterization of the protein shows that, Myb domain is predominantly helical (65%) and is alone sufficient to bind the Sal Box DNA. This is the first-ever study to report a complete in silico model of the Myb domain, which is physically characterized. The above study will pave the way towards solving the atomic structure of this essential mammalian protein.
Assuntos
Fatores de Transcrição , Humanos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/química , Domínios Proteicos , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Simulação de Dinâmica Molecular , DNA/metabolismo , Ligação Proteica , Animais , Sequência de Aminoácidos , Sítios de Ligação , Estabilidade ProteicaRESUMO
The current investigation focused on separating Cerastes cerastes venom to produce the first Kunitz-type peptide. Based on its anti-trypsin effect, Cerastokunin, a 7.75 kDa peptide, was purified until homogenity by three steps of chromatography. Cerastokunin was found to include 67 amino acid residues that were obtained by de novo sequencing using LC-MALDI-MSMS. Upon alignment with Kunitz-type peptides, there was a high degree of similarity. Cerastokunin's 3D structure had 12% α-helices and 21% ß-strands with pI 8.48. Cerastokunin showed a potent anticoagulant effect by inhibiting the protease activity of thrombin and trypsin as well as blocking the intrinsic and extrinsic coagulation pathways. In both PT and aPPT, Cerastokunin increased the blood clotting time in a dose-dependent way. Using Lys48 and Gln192 for direct binding, Cerastokunin inhibited thrombin, Factor Xa and trypsin as shown by molecular docking. Cerastokunin exhibited a dose-response blockade of PARs-dependent pathway platelet once stimulated by thrombin. An increased concentration of Cerastokunin resulted in a larger decrease of tail thrombus in the mice-carrageenan model in an in vivo investigation when compared to the effects of antithrombotic medications. At all Cerastokunin doses up to 6 mg/kg, no in vivo toxicity was seen in challenged mice over the trial's duration.
Assuntos
Plaquetas , Inibidores do Fator Xa , Trombina , Animais , Trombina/química , Trombina/metabolismo , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Inibidores do Fator Xa/farmacologia , Inibidores do Fator Xa/química , Simulação de Acoplamento Molecular , Humanos , Camundongos , Anticoagulantes/farmacologia , Anticoagulantes/química , Fator Xa/química , Fator Xa/metabolismo , Sequência de AminoácidosRESUMO
Sleep disturbances are common among children with neurodevelopmental disorders. Here, we report a syndrome characterized by prenatal microcephaly, intellectual disability and severe disruption of sleep-wake cycles in a consanguineous family. Exome sequencing revealed homozygous variants (c.5224G>A and c.6506G>T) leading to the missense mutations E1742K and G2169V in integrator complex subunit 1 (INTS1), the core subunit of the Integrator complex. Conservation and structural analyses suggest that G2169V has a minor impact on the structure and function of the complex, while E1742K significantly alters a negatively charged conserved patch on the surface of the protein. The severe sleep-wake cycles disruption in human carriers highlights a new aspect of Integrator complex impairment. To further study INTS1 pathogenicity, we generated Ints1-deficient zebrafish lines. Mutant zebrafish larvae displayed abnormal circadian rhythms of locomotor activity and sleep, as is the case with the affected humans. Furthermore, Ints1-deficent larvae exhibited elevated levels of dopamine ß-hydroxylase (dbh) mRNA in the locus coeruleus, a wakefulness-inducing brainstem center. Altogether, these findings suggest a significant, likely indirect, effect of INTS1 and the Integrator complex on maintaining circadian rhythms of locomotor activity and sleep homeostasis across vertebrates.
Assuntos
Ritmo Circadiano , Sono , Peixe-Zebra , Animais , Peixe-Zebra/genética , Humanos , Feminino , Masculino , Ritmo Circadiano/genética , Sono/genética , Sono/fisiologia , Vigília/fisiologia , Vigília/genética , Larva/genética , Linhagem , Sequência de Aminoácidos , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , CriançaRESUMO
Spotted-wing drosophila, Drosophila suzukii (Matsumura), is an invasive vinegar fly that is a major threat to the small fruits industries globally. Insect capa genes encode multiple neuropeptides, including CAPA-periviscerokinin (CAPA-PVK) peptides, that are specifically known to cause diuresis or anti-diuresis in various organisms. Here we identified and characterized a corresponding G protein-coupled receptor (GPCR) of the D. suzukii CAPA-PVK peptides: CAPA receptor (CAPA-R). To better characterize the behavior of D. suzukii CAPA-R, we used insect cell-based functional expression assays to evaluate responses of CAPA-R against D. suzukii CAPA-PVKs, CAPA-PVKs from five species in Insecta, one species from Mollusca, modified CAPA-PVK peptides, and some PRXamide family peptides: pyrokinin (PK), diapause hormone (DH), and ecdysis-triggering hormone (ETH). Functional studies revealed that the D. suzukii CAPA-R is strongly activated by both of its own natural D. suzukii CAPA-PVKs, and interestingly, it was strongly activated by other CAPA-PVK peptides from Frankliniella occidentallis (Thysanoptera), Solenopsis invicta (Hymenoptera), Helicoverpa zea (Lepidoptera) and Plutella xylostella (Lepidoptera). However, D. suzukii CAPA-R was not activated by Mollusca CAPA-PVK or the other PRXamide peptides. Gene expression analyses showed that the CAPA-R was highly expressed in the Malpighian tubules and moderately in hindgut compared to other digestive organs or the rest of body, supporting diuretic/antidiuretic functionality. When compared across life stages of D. suzukii, expression of CAPA-R was approximately 1.5x greater in the third instar than the other stages and minimally detected in the eggs, 4-day old pupae and 3-day old adults. Our results functionally characterized the D. suzukii CAPA-R and a few short peptides were identified as potential biological targets to exploit the CAPA-R for D. suzukii management.
Assuntos
Proteínas de Drosophila , Drosophila , Neuropeptídeos , Animais , Feminino , Sequência de Aminoácidos , Drosophila/metabolismo , Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Trato Gastrointestinal/metabolismo , Hormônios de Inseto/metabolismo , Larva/crescimento & desenvolvimento , Larva/metabolismo , Larva/genética , Neuropeptídeos/metabolismo , Neuropeptídeos/genética , Pupa/crescimento & desenvolvimento , Pupa/metabolismo , Pupa/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genéticaRESUMO
The identification of enzyme functions plays a crucial role in understanding the mechanisms of biological activities and advancing the development of life sciences. However, existing enzyme EC number prediction methods did not fully utilize protein sequence information and still had shortcomings in identification accuracy. To address this issue, we proposed an EC number prediction network using hierarchical features and global features (ECPN-HFGF). This method first utilized residual networks to extract generic features from protein sequences, and then employed hierarchical feature extraction modules and global feature extraction modules to further extract hierarchical and global features of protein sequences. Subsequently, the prediction results of both feature types were combined, and a multitask learning framework was utilized to achieve accurate prediction of enzyme EC numbers. Experimental results indicated that the ECPN-HFGF method performed best in the task of predicting EC numbers for protein sequences, achieving macro F1 and micro F1 scores of 95.5% and 99.0%, respectively. The ECPN-HFGF method effectively combined hierarchical and global features of protein sequences, allowing for rapid and accurate EC number prediction. Compared to current commonly used methods, this method offers significantly higher prediction accuracy, providing an efficient approach for the advancement of enzymology research and enzyme engineering applications.
Assuntos
Biologia Computacional , Biologia Computacional/métodos , Sequência de Aminoácidos , Proteínas/química , Algoritmos , Análise de Sequência de Proteína/métodos , Enzimas/química , Enzimas/metabolismoRESUMO
In sensory neurons that transmit pain signals, whether acute or chronic, voltage-gated sodium channels (VGSCs) are crucial for regulating excitability. NaV1.1, NaV1.3, NaV1.6, NaV1.7, NaV1.8, and NaV1.9 have been demonstrated and defined their functional roles in pain signaling based on their biophysical properties and distinct patterns of expression in each subtype of sensory neurons. Scorpions and spiders are traditional Chinese medicinal materials, belonging to the arachnid class. Most of the studied species of them have evolved venom peptides that exhibit a wide variety of knottins specifically targeting VGSCs with subtype selectivity and conformational specificity. This review provides an overview on the exquisite knottins from scorpion and spider venoms targeting pain-related NaV channels, describing the sequences and the structural features as well as molecular determinants that influence their selectivity on special subtype and at particular conformation, with an aim for the development of novel research tools on NaV channels and analgesics with minimal adverse effects.
Assuntos
Dor , Venenos de Escorpião , Transdução de Sinais , Venenos de Aranha , Canais de Sódio Disparados por Voltagem , Animais , Venenos de Escorpião/química , Venenos de Escorpião/farmacologia , Venenos de Escorpião/metabolismo , Venenos de Aranha/farmacologia , Venenos de Aranha/química , Venenos de Aranha/metabolismo , Canais de Sódio Disparados por Voltagem/metabolismo , Canais de Sódio Disparados por Voltagem/efeitos dos fármacos , Canais de Sódio Disparados por Voltagem/química , Canais de Sódio Disparados por Voltagem/fisiologia , Dor/tratamento farmacológico , Dor/metabolismo , Humanos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Escorpiões/metabolismo , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/química , Sequência de Aminoácidos , Aranhas/metabolismoRESUMO
Tardigrades are unique micro-organisms with a high tolerance to desiccation. The protection of their cells against desiccation involves tardigrade-specific proteins, which include the so-called cytoplasmic abundant heat soluble (CAHS) proteins. As a first step towards the design of peptides capable of mimicking the cytoprotective properties of CAHS proteins, we have synthesized several model peptides with sequences selected from conserved CAHS motifs and investigated to what extent they exhibit the desiccation-induced structural changes of the full-length proteins. Using circular dichroism spectroscopy, two-dimensional infrared spectroscopy, and molecular dynamics simulations, we have found that the CAHS model peptides are mostly disordered, but adopt a more α $$ \alpha $$ -helical structure upon addition of 2,2,2-trifluoroethanol, which mimics desiccation. This structural behavior is similar to that of full-length CAHS proteins, which also adopt more ordered conformations upon desiccation. We also have investigated the surface activity of the peptides at the air/water interface, which also mimics partial desiccation. Interestingly, sum-frequency generation spectroscopy shows that all model peptides are surface active and adopt a helical structure at the air/water interface. Our results suggest that amino acids with high helix-forming propensities might contribute to the propensity of these peptides to adopt a helical structure when fully or partially dehydrated. Thus, the selected sequences retain part of the CAHS structural behavior upon desiccation, and might be used as a basis for the design of new synthetic peptide-based cryoprotective materials.
Assuntos
Simulação de Dinâmica Molecular , Peptídeos , Tardígrados , Tardígrados/química , Animais , Peptídeos/química , Estrutura Secundária de Proteína , Sequência de AminoácidosRESUMO
Voltage-gated sodium (Nav) and calcium (Cav) channels are responsible for the initiation of electrical signals. They have long been targeted for the treatment of various diseases. The mounting number of cryoelectron microscopy (cryo-EM) structures for diverse subtypes of Nav and Cav channels from multiple organisms necessitates a generic residue numbering system to establish the structure-function relationship and to aid rational drug design or optimization. Here we suggest a structure-based residue numbering scheme, centering around the most conserved residues on each of the functional segments. We elaborate the generic numbers through illustrative examples, focusing on representative drug-binding sites of eukaryotic Nav and Cav channels. We also extend the numbering scheme to compare common disease mutations among different Nav subtypes. Application of the generic residue numbering scheme affords immediate insights into hotspots for pathogenic mutations and critical loci for drug binding and will facilitate drug discovery targeting Nav and Cav channels.
Assuntos
Canais de Cálcio , Humanos , Canais de Cálcio/metabolismo , Canais de Cálcio/química , Canais de Cálcio/genética , Canais de Sódio Disparados por Voltagem/química , Canais de Sódio Disparados por Voltagem/metabolismo , Canais de Sódio Disparados por Voltagem/genética , Animais , Sítios de Ligação , Mutação , Microscopia Crioeletrônica , Modelos Moleculares , Sequência de AminoácidosRESUMO
Here, we characterized a novel mitovirus from the fungus Nigrospora oryzae, which was named "Nigrospora oryzae mitovirus 3" (NoMV3). The NoMV3 genome is 2,492 nt in length with a G + C content of 33%, containing a single large open reading frame (ORF) using the fungal mitochondrial genetic code. The ORF encodes an RNA-dependent RNA polymerase (RdRp) of 775 amino acids with a molecular mass of 88.75 kDa. BLASTp analysis revealed that the RdRp of NoMV3 had 68.6%, 50.6%, and 48.6% sequence identity to those of Nigrospora oryzae mitovirus 2, Suillus luteus mitovirus 6, and Fusarium proliferatum mitovirus 3, respectively, which belong to the genus Unuamitovirus within the family Mitoviridae. Phylogenetic analysis based on amino acid sequences supported the classification of NoMV3 as a member of a new species in the genus Unuamitovirus within the family Mitoviridae.
Assuntos
Ascomicetos , Micovírus , Genoma Viral , Fases de Leitura Aberta , Filogenia , Doenças das Plantas , Vírus de RNA , RNA Polimerase Dependente de RNA , Genoma Viral/genética , Micovírus/genética , Micovírus/classificação , Micovírus/isolamento & purificação , Vírus de RNA/genética , Vírus de RNA/classificação , Vírus de RNA/isolamento & purificação , RNA Polimerase Dependente de RNA/genética , Doenças das Plantas/microbiologia , Doenças das Plantas/virologia , Ascomicetos/virologia , Ascomicetos/genética , RNA Viral/genética , Proteínas Virais/genética , Composição de Bases , Sequência de AminoácidosRESUMO
Targeting heterologous multi-transmembrane domain (TMD) proteins to plant chloroplasts requires sequences in addition to the chloroplast transit peptide (cTP). The N-terminal domain (N-region), located C-terminal to the cTP in chloroplast inner envelope membrane proteins, is an essential region for import. However, it was unclear if the N-region functions solely as a spacer sequence to facilitate cTP access or if it plays an active role in the import process. This study addresses the N-region's role by using combinations of cTPs and N-regions from Arabidopsis chloroplast inner envelope membrane proteins to direct the cyanobacterial protein SbtA to the chloroplast. We find that the sequence context of the N-region affects the chloroplast import efficiency of SbtA, with particular sequences mis-targeting the protein to different cellular sub-compartments. Additionally, specific cTP and N-region pairs exhibit varying targeting efficiencies for different heterologous proteins. Substituting individual N-region motifs did not significantly alter the chloroplast targeting efficiency of a particular cTP and N-region pair. We conclude that the N-region exhibits contextual functioning and potentially functional redundancy in motifs.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Cloroplastos , Cloroplastos , Transporte Proteico , Cloroplastos/metabolismo , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Cloroplastos/metabolismo , Proteínas de Cloroplastos/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Sinais Direcionadores de Proteínas , Domínios Proteicos , Sequência de Aminoácidos , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genéticaRESUMO
BACKGROUND: Cathelicidins are vital antimicrobial peptides expressed in diverse vertebrates, crucial for immunity. Despite being a new field, amphibian cathelicidin research holds promise. RESULTS: We isolated the cDNA sequence of the cathelicidin (Ll-CATH) gene from the liver transcriptome of the Chong'an Moustache Toad (Leptobrachium liui). We confirmed the authenticity of the cDNA sequence by rapid amplification of cDNA ends and reverse transcription PCR, and obtained the Ll-CATH amino acid sequence using the Open Reading Frame Finder, an online bioinformatics tool. Its translated protein contained a cathelin domain, signal peptide, and mature peptide, confirmed by amino acid sequence. The comparative analysis showed that the mature peptides were variable between the amphibian species, while the cathelin domain was conserved. The concentration of Ll-CATH protein and the expression of its gene varied in the tissues, with the spleen showing the highest levels. The expression levels of Ll-CATH in different tissues of toads was significantly increased post infection with Aeromonas hydrophila. Chemically synthesized Ll-CATH effectively combated Proteus mirabilis, Staphylococcus epidermidis, Vibrio harveyi, V. parahaemolyticus, and V. vulnificus; disrupted the membrane of V. harveyi, hydrolyzed its DNA. Ll-CATH induced chemotaxis and modulated the expression of pro-inflammatory cytokine genes in RAW264.7 macrophages. CONCLUSIONS: This study unveiled the antibacterial and immunomodulatory potential of amphibian cathelicidin, implying its efficacy against infections. Ll-CATH characterization expands our knowledge, emphasizing its in a bacterial infection therapy.
Assuntos
Antibacterianos , Anuros , Catelicidinas , Animais , Antibacterianos/farmacologia , Sequência de Aminoácidos , Fatores Imunológicos/farmacologia , Camundongos , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/farmacologiaRESUMO
Oil seeds now make up the world's second-largest food source after cereals. In recent years, the medicinal- oil plant Camelina sativa has attracted much attention for its high levels of unsaturated fatty acids and low levels of saturated fatty acids as well as its resistance to abiotic stresses. Improvement of oil quality is considered an important trait in this plant. Erucic acid is one of the fatty acids affecting the quality of camelina oil. Altering the fatty acid composition in camelina oil through genetic manipulation requires the identification, isolation, and cloning of genes involved in fatty acid biosynthesis. The Fatty Acid Elongase 1 (FAE1) gene encodes the enzyme ß-ketoacyl CoA synthase (KCS), a crucial enzyme in the biosynthesis of erucic acid. In this study, the isolation and cloning of the FAE1 gene from Camelina sativa were conducted to construct an antisense structure. The molecular homology modeling of DFAE1 proteins using the SWISS-MODEL server on ExPASy led to the generation of the 3D structures of FAE1 and DFAE1 proteins. The GMQE values of 0.44 for FAE1 and 0.08 for DFAE1 suggest high accuracy in the structural estimation of these genes. The fragments were isolated from the DNA source of the genomic Soheil cultivar with an erucic acid content of about 3% (in matured seeds) using PCR. After cloning the FAE1 gene into the Bluescript II SK+ vector and sequencing, the resulting fragments were utilized to construct the antisense structure in the pBI121 plant expression vector. The approved antisense structure was introduced into the Camelina plant using the Agrobacterium-mediated method, with optimization of tissue culture and gene transfer conditions. This approach holds potential to advance our knowledge of fat biosynthesis, leading to potential improvements in oil quality in Camelina sativa.
Assuntos
Brassicaceae , Clonagem Molecular , Ácidos Erúcicos , Elongases de Ácidos Graxos , Brassicaceae/genética , Brassicaceae/metabolismo , Clonagem Molecular/métodos , Ácidos Erúcicos/metabolismo , Elongases de Ácidos Graxos/genética , Elongases de Ácidos Graxos/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Sequência de Aminoácidos , Sementes/genética , Sementes/metabolismo , Modelos Moleculares , Regulação da Expressão Gênica de Plantas , Acetiltransferases/genética , Acetiltransferases/metabolismo , Genes de PlantasRESUMO
Improving crop plants using biotechnological implications is a promising and modern approach compared to traditional methods. High-temperature exposure to the reproductive stage induces flower abortion and declines grain filling performance, leading to smaller grain production and low yield in lentil and other legumes. Thus, cloning effective candidate genes and their implication in temperature stress tolerance in lentil (Lens culinaris Medik.) using biotechnological tools is highly demandable. The 12-oxophytodienoic acid reductases (OPRs) are flavin mononucleotide-dependent oxidoreductases with vital roles in plants. They are members of the old yellow enzyme (OYE) family. These enzymes are involved in the octadecanoid pathway, which contributes to jasmonic acid biosynthesis and is essential in plant stress responses. Lentil is one of the vital legume crops affected by the temperature fluctuations caused by global warming. Therefore, in this study, the LcOPR1 gene was successfully cloned and isolated from lentils using RT-PCR to evaluate its functional responses in lentil under heat stress. The bioinformatics analysis revealed that the full-length cDNA of LcOPR1 was 1303 bp, containing an 1134 bp open reading frames (ORFs), encoding 377 amino acids with a predicted molecular weight of 41.63 and a theoretical isoelectric point of 5.61. Bioinformatics analyses revealed that the deduced LcOPR1 possesses considerable homology with other plant 12-oxophytodienoic acid reductases (OPRs). Phylogenetic tree analysis showed that LcOPR1 has an evolutionary relationship with other OPRs in different plant species of subgroup I, containing enzymes that are not required for jasmonic acid biosynthesis. The expression analysis of LcOPR1 indicated that this gene is upregulated in response to the heat-stress condition and during recovery in lentil. This study finding might be helpful to plant breeders and biotechnologists in LcOPR1 engineering and/or plant breeding programs in revealing the biological functions of LcOPR1 in lentils and the possibility of enhancing heat stress tolerance by overexpressing LcOPR1 in lentil and other legume plants under high temperature.
Assuntos
Clonagem Molecular , Regulação da Expressão Gênica de Plantas , Lens (Planta) , Filogenia , Lens (Planta)/genética , Lens (Planta)/enzimologia , Clonagem Molecular/métodos , Regulação da Expressão Gênica de Plantas/genética , Oxirredutases/genética , Oxirredutases/metabolismo , Sequência de Aminoácidos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Temperatura Alta , Genes de Plantas , Resposta ao Choque Térmico/genética , Oxilipinas/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CHRESUMO
OBJECTIVE: Proteasomes are conserved proteases crucial for proteostasis in eukaryotes and are promising drug targets for protozoan parasites. Yet, the proteasomes of Entamoeba histolytica remain understudied. The study's objective was to analyse the differences in the substrate binding pockets of amoeba proteasomes from those of host, and computational modelling of ß5 catalytic subunit, with the goal of finding selective inhibitors. RESULTS: Comparative sequence analysis revealed differences in substrate binding sites of E. histolytica proteasomes, especially in the S1 and S3 pockets of the catalytic beta subunits, implying differences in substrate preference and susceptibility to inhibitors from host proteasomes. This was strongly supported by significantly lower sensitivity to MG132 mediated inhibition of amoebic proteasome ß5 subunit's chymotryptic activity compared to human proteasomes, also reflected in lower sensitivity of E. histolytica to MG132 for inhibition of proliferation. Computational models of ß4 and ß5 subunits, and a docked ß4-ß5 model revealed a binding pocket between ß4-ß5, similar to that of Leishmania tarentolae. Selective inhibitors for visceral leishmaniasis, LXE408 and compound 8, docked well to this pocket. This functional and sequence-based analysis predicts differences between amoebic and host proteasomes that can be utilized to develop rationally designed, selective inhibitors against E. histolytica.
Assuntos
Entamoeba histolytica , Complexo de Endopeptidases do Proteassoma , Entamoeba histolytica/enzimologia , Entamoeba histolytica/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Humanos , Sítios de Ligação , Leupeptinas/farmacologia , Especificidade por Substrato , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Inibidores de Proteassoma/farmacologia , Simulação de Acoplamento Molecular , Sequência de Aminoácidos , Domínio Catalítico , Ligação Proteica , Modelos MolecularesRESUMO
Iron-binding proteins, known as ferritins, play pivotal roles in immunological response, detoxification, and iron storage. Despite their significance to organisms, little is known about how they affect the immunological system of the red swamp crayfish (Procambarus clarkii). In our previous research, one ferritin subunit was completely discovered as an H-like subunit (PcFeH) from P. clarkii. The full-length cDNA of PcFerH is 1779 bp, including a 5'-UTR (untranslated region, UTR) of 89 bp, 3'-UTR (untranslated region, UTR) of 1180 bp and an ORF (open reading frame, ORF) of 510 bp encoding a polypeptide of 169 amino acids that contains a signal peptide and a Ferritin domain. The deduced PcFerH protein sequence has highly identity with other crayfish. PcFerH protein's estimated tertiary structure is quite comparable to animal structure. The PcFerH is close to Cherax quadricarinatus, according to phylogenetic analysis. All the organs examined showed widespread expression of PcFerH mRNA, with the ovary exhibiting the highest levels of expression. Additionally, in crayfish muscles, intestines, and gills, the mRNA transcript of PcFerH was noticeably up-regulated, after LPS and Poly I:C challenge. The expression of downstream genes in the immunological signaling system was suppressed when the PcFerH gene was knocked down. All of these findings suggested that PcFerH played a vital role in regulating the expression of downstream effectors in the immunological signaling pathway of crayfish.
Assuntos
Astacoidea , Imunidade Inata , Filogenia , Animais , Astacoidea/imunologia , Astacoidea/genética , Sequência de Aminoácidos , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Proteínas de Artrópodes/metabolismoRESUMO
Interleukin-1α is a suggested dual-function cytokine that diverged from interleukin-1ß in mammals potentially by acquiring additional biological roles that relate to highly conserved regions in the pro-domain of interleukin-1α, including a nuclear localisation sequence and histone acetyltransferase-binding domains. Why evolution modified pro-interleukin-1α's subcellular location and protein interactome, and how this shaped interleukin-1α's intracellular role, is unknown. Here we show that TurboID proximity labelling with pro-interleukin-1α suggests a nuclear role for pro-interleukin-1α that involves interaction with histone acetyltransferases, including EP300. We also identify and validate inactivating mutations in the pro-interleukin-1α nuclear localisation sequence of multiple mammalian species, including toothed whales, castorimorpha and marsupials. However, histone acetyltransferase-binding domains are conserved in those species that have lost pro-interleukin-1α nuclear localisation. Together, these data suggest that histone acetyltransferase binding and nuclear localisation occurred together, and that while some species lost the nuclear localisation sequence in their pro-interleukin-1α, histone acetyltransferase binding ability was maintained. The nuclear localisation sequence was lost from several distinct species at different evolutionary times, suggesting convergent evolution, and that the loss of the nuclear localisation sequence confers some important biological outcome.
Assuntos
Núcleo Celular , Evolução Molecular , Interleucina-1alfa , Interleucina-1alfa/metabolismo , Interleucina-1alfa/genética , Animais , Núcleo Celular/metabolismo , Humanos , Proteína p300 Associada a E1A/metabolismo , Proteína p300 Associada a E1A/genética , Histona Acetiltransferases/metabolismo , Histona Acetiltransferases/genética , Sinais de Localização Nuclear/genética , Sinais de Localização Nuclear/metabolismo , Ligação Proteica , Sequência de AminoácidosRESUMO
Lipopeptides can self-assemble into diverse nanostructures which can be programmed to incorporate peptide sequences to achieve a remarkable range of bioactivities. Here, the influence of peptide sequence and chirality on micelle structure and interactions is investigated in a series of lipopeptides bearing two lysine or D-lysine residues and tyrosine or tryptophan residues, attached to a hexadecyl lipid chain. All molecules self-assemble into micelles above a critical micelle concentration (CMC). Small-angle x-ray scattering (SAXS) is used to probe micelle shape and structure from the form factor and to probe inter-micellar interactions via analysis of structure factor. The CMC is obtained consistently from surface tension and electrical conductivity measurements. We introduce a method to obtain the zeta potential from the SAXS structure factor which is in good agreement with directly measured values. Atomistic molecular dynamics simulations provide insights into molecular packing and conformation within the lipopeptide micelles which constitute model self-assembling colloidal systems and biomaterials.
Assuntos
Coloides , Lipopeptídeos , Lisina , Micelas , Simulação de Dinâmica Molecular , Espalhamento a Baixo Ângulo , Tensoativos , Difração de Raios X , Lipopeptídeos/química , Tensoativos/química , Coloides/química , Lisina/química , Sequência de Aminoácidos , Tensão SuperficialRESUMO
Post-translational modifications (PTMs) are pivotal in modulating protein functions and influencing cellular processes like signaling, localization, and degradation. The complexity of these biological interactions necessitates efficient predictive methodologies. In this work, we introduce PTMGPT2, an interpretable protein language model that utilizes prompt-based fine-tuning to improve its accuracy in precisely predicting PTMs. Drawing inspiration from recent advancements in GPT-based architectures, PTMGPT2 adopts unsupervised learning to identify PTMs. It utilizes a custom prompt to guide the model through the subtle linguistic patterns encoded in amino acid sequences, generating tokens indicative of PTM sites. To provide interpretability, we visualize attention profiles from the model's final decoder layer to elucidate sequence motifs essential for molecular recognition and analyze the effects of mutations at or near PTM sites to offer deeper insights into protein functionality. Comparative assessments reveal that PTMGPT2 outperforms existing methods across 19 PTM types, underscoring its potential in identifying disease associations and drug targets.