Search Results (61)

Because of the involvement of π-electron cyclic constituents in their side chains, the so-called aromatic residues give rise to a number of strong, narrow, and well-resolved lines spread over the middle wavenumber (1800–600 cm⁻¹) region of the Raman spectra of peptides and proteins. The number of characteristic aromatic markers increases with the structural complexity (Phe → Tyr → Trp), herein referred to as (F_i = 1, …, 6) in Phe, (Y_i = 1, …, 7) in Tyr, and (W_i = 1, …, 8) in Trp. Herein, we undertake an overview of these markers through the analysis of a representative data base gathered from the most structurally simple tripeptides, Gly-Xxx-Gly (where Xxx = Phe, Tyr, Trp). In this framework, off-resonance Raman spectra obtained from the aqueous samples of these tripeptides were jointly used with the structural and vibrational data collected from the density functional theory (DFT) calculations using the M062X hybrid functional and 6-311++G(d,p) atomic basis set. The conformation dependence of aromatic Raman markers was explored upon a representative set of 75 conformers, having five different backbone secondary structures (i.e., β-strand, polyproline-II, helix, classic, and inverse γ-turn), and plausible side chain rotamers. The hydration effects were considered upon using both implicit (polarizable solvent continuum) and explicit (minimal number of 5–7 water molecules) models. Raman spectra were calculated through a multiconformational approach based on the thermal (Boltzmann) average of the spectra arising from all calculated conformers. A subsequent discussion highlights the conformational landscape of conformers and the wavenumber dispersion of aromatic Raman markers. In particular, a new interpretation was proposed for the characteristic Raman doublets arising from Tyr (~850–830 cm⁻¹) and Trp (~1360–1340 cm⁻¹), definitely excluding the previously suggested Fermi-resonance-based assignment of these markers through the consideration of the interactions between the aromatic side chain and its adjacent peptide bonds. Full article

Auxin response factor (ARF) is a plant-specific transcription factor that responds to changes in auxin levels, regulating various biological processes in plants such as flower development, senescence, lateral root formation, stress response, and secondary metabolite accumulation. In this study, we identified the ARF gene family in Populus euphratica Oliv. using bioinformatics analysis, examining their conserved structural domains, gene structure, expression products, and evolutionary relationships. We found that the 34 PeARF genes were unevenly distributed on 19 chromosomes of P. euphratica. All 56 PeARF proteins were hydrophilic and unstable proteins localized in the nucleus, with secondary structures containing α-helices, extended strands, random coils, and β-turns but lacking transmembrane helices (TM-helices) and signal peptides. Evolutionary analysis divided the PeARF proteins into five subfamilies (A–E), with high conservation observed in the order and number of motifs, domains, gene structure, and other characteristics within each subfamily. Expression pattern analysis revealed that 17 PeARF genes were upregulated during cell growth and heterophylly development. This comprehensive analysis provides insights into the molecular mechanisms of ARF genes in P. euphratica growth, development, and stress response, serving as a basis for further studies on the auxin signaling pathway in P. euphratica. Full article

α-Synuclein (αS) interacts with lipid membranes in neurons to form amyloid fibrils that contribute to Parkinson’s disease, and its non-amyloid-β component domain is critical in the fibrillation. In this study, the salt (NaCl) effect on the membrane interaction and fibril formation of αS_57–102 peptide (containing the non-amyloid-β component domain) was characterized at the molecular level because the αS_57–102 fibrils exhibited structural polymorphism with two morphologies (thin and thick) in the presence of NaCl but showed one morphology (thin) in the absence of NaCl. The membrane-bound conformation (before fibrillation) of αS_57–102 had two helical regions (first and second) on the membrane regardless of salt, but the length of the first region largely shortened when NaCl was present, exposing its hydrophobic area to the solvent. The exposed region induced two distinct pathways of fibril nucleation, depending on the molar ratios of free and membrane-bound αS_57–102: one from the association of free αS_57–102 with membrane-bound αS_57–102 and the other from the assembly among membrane-bound αS_57–102. The differences mainly affected the β-strand orientation and helical content within the fibril conformations, probably contributing to the thickness degree, leading to structural polymorphism. Full article

B30.2 domains, sometimes referred to as PRY/SPRY domains, were originally identified by sequence profiling methods at the gene level. The B30.2 domain comprises a concanavalin A-like fold consisting of two twisted seven-stranded anti-parallel β-sheets. B30.2 domains are present in about 150 human and 700 eukaryotic proteins, usually fused to other domains. The B30.2 domain represents a scaffold, which, through six variable loops, binds different unrelated peptides or endogenous low-molecular-weight compounds. At the cellular level, B30.2 proteins engage in supramolecular assemblies with important signaling functions. In humans, B30.2 domains are often found in E3-ligases, such as tripartite motif (Trim) proteins, SPRY domain-containing SOCS box proteins, Ran binding protein 9 and −10, Ret-finger protein-like, and Ring-finger proteins. The B30.2 protein recognizes the target and recruits the E2-conjugase by means of the fused domains, often involving specific adaptor proteins. Further well-studied B30.2 proteins are the methyltransferase adaptor protein Ash2L, some butyrophilins, and Ryanodine Receptors. Although the affinity of an isolated B30.2 domain to its ligand might be weak, it can increase strongly due to avidity effects upon recognition of oligomeric targets or in the context of macromolecular machines. Full article

Host defense antimicrobial peptides (AMPs) are promising lead molecules with which to develop antibiotics against drug-resistant bacterial pathogens. Thanatin, an inducible antimicrobial peptide involved in the host defense of Podisus maculiventris insects, is gaining considerable attention in the generation of novel classes of antibiotics. Thanatin or thanatin-based analog peptides are extremely potent in killing bacterial pathogens in the Enterobacteriaceae family, including drug-resistant strains of Escherichia coli and Klebsiella pneumoniae. A single disulfide bond that covalently links two anti-parallel β-strands in thanatin could be pivotal to its selective antibacterial activity and mode of action. However, potential correlations of the disulfide covalent bond with structure, activity and target binding in thanatin peptides are currently unclear to. Here, we examined a 16-residue designed thanatin peptide, namely disulfide-bonded VF16QK, and its Cys to Ser substituted variant, VF16QK^Ser, to delineate their structure–activity relationships. Bacterial growth inhibitory activity was only detected for the disulfide-bonded VF16QK peptide. Mechanistically, both peptides vastly differ in their bacterial cell permeabilizations, atomic-resolution structures, interactions with the LPS-outer membrane and target periplasmic protein LptA_m binding. In particular, analysis of the 3-D structures of the two peptides revealed an altered folded conformation for the VF16QK^Ser peptide that was correlated with diminished LPS-outer membrane permeabilization and target interactions. Analysis of docked complexes of LPS–thanatin peptides indicated potential structural requirements and conformational adaptation for antimicrobial activity. Collectively, these observations contrast with those for the disulfide-bonded β-hairpin antimicrobial protegrin and tachyplesin peptides, where disulfide bonds are dispensable for activity. We surmise that the atomistic structures and associated molecular interactions presented in this work can be utilized to design novel thanatin-based antibiotics. Full article

β-amyloid (Aβ) peptides form self-organizing fibrils in Alzheimer’s disease. The biologically active, toxic Aβ25–35 fragment of the full-length Aβ-peptide forms a stable, oriented filament network on the mica surface with an epitaxial mechanism at the timescale of seconds. While many of the structural and dynamic features of the oriented Aβ25–35 fibrils have been investigated before, the β-strand arrangement of the fibrils and their exact orientation with respect to the mica lattice remained unknown. By using high-resolution atomic force microscopy, here, we show that the Aβ25–35 fibrils are oriented along the long diagonal of the oxygen hexagon of mica. To test the structure and stability of the oriented fibrils further, we carried out molecular dynamics simulations on model β-sheets. The models included the mica surface and a single fibril motif built from β-strands. We show that a sheet with parallel β-strands binds to the mica surface with its positively charged groups, but the C-terminals of the strands orient upward. In contrast, the model with antiparallel strands preserves its parallel orientation with the surface in the molecular dynamics simulation, suggesting that this model describes the first β-sheet layer of the mica-bound Aβ25–35 fibrils well. These results pave the way toward nanotechnological construction and applications for the designed amyloid peptides. Full article

Neurodegenerative diseases (NDDs) are a diverse group of neurological disorders characterized by alterations in the structure and function of the central nervous system. Alzheimer’s disease (AD), characterized by impaired memory and cognitive abilities, is the most prevalent type of senile dementia. Loss of synapses, intracellular aggregation of hyperphosphorylated tau protein, and extracellular amyloid-β peptide (Aβ) plaques are the hallmarks of AD. MicroRNAs (miRNAs/miRs) are single-stranded ribonucleic acid (RNA) molecules that bind to the 3′ and 5′ untranslated regions of target genes to cause post-transcriptional gene silencing. The brain expresses over 70% of all experimentally detected miRNAs, and these miRNAs are crucial for synaptic function and particular signals during memory formation. Increasing evidence suggests that miRNAs play a role in AD pathogenesis and we provide an overview of the role of miRNAs in synapse formation, Aβ synthesis, tau protein accumulation, and brain-derived neurotrophic factor-associated AD pathogenesis. We further summarize and discuss the role of miRNAs as potential therapeutic targets and biomarkers for AD detection and differentiation between early- and late-stage AD, based on recent research. In conclusion, altered expression of miRNAs in the brain and peripheral circulation demonstrates their potential as biomarkers and therapeutic targets in AD. Full article

Inhibiting MDM2-p53 interaction is considered an efficient mode of cancer treatment. In our current study, Gaussian-accelerated molecular dynamics (GaMD), deep learning (DL), and binding free energy calculations were combined together to probe the binding mechanism of non-peptide inhibitors K23 and 0Y7 and peptide ones PDI6W and PDI to MDM2. The GaMD trajectory-based DL approach successfully identified significant functional domains, predominantly located at the helixes α2 and α2’, as well as the β-strands and loops between α2 and α2’. The post-processing analysis of the GaMD simulations indicated that inhibitor binding highly influences the structural flexibility and collective motions of MDM2. Calculations of molecular mechanics–generalized Born surface area (MM-GBSA) and solvated interaction energy (SIE) not only suggest that the ranking of the calculated binding free energies is in agreement with that of the experimental results, but also verify that van der Walls interactions are the primary forces responsible for inhibitor–MDM2 binding. Our findings also indicate that peptide inhibitors yield more interaction contacts with MDM2 compared to non-peptide inhibitors. Principal component analysis (PCA) and free energy landscape (FEL) analysis indicated that the piperidinone inhibitor 0Y7 shows the most pronounced impact on the free energy profiles of MDM2, with the piperidinone inhibitor demonstrating higher fluctuation amplitudes along primary eigenvectors. The hot spots of MDM2 revealed by residue-based free energy estimation provide target sites for drug design toward MDM2. This study is expected to provide useful theoretical aid for the development of selective inhibitors of MDM2 family members. Full article

The Drosophila downstream receptor kinase (Drk), a homologue of human GRB2, participates in the signal transduction from the extracellular to the intracellular environment. Drk receives signals through the interaction of its Src homology 2 (SH2) domain with the phosphorylated tyrosine residue in the receptor tyrosine kinases (RTKs). Here, we present the solution NMR structure of the SH2 domain of Drk (Drk-SH2), which was determined in the presence of a phosphotyrosine (pY)-containing peptide derived from a receptor tyrosine kinase, Sevenless (Sev). The solution structure of Drk-SH2 possess a common SH2 domain architecture, consisting of three β strands imposed between two α helices. Additionally, we interpret the site-specific interactions of the Drk-SH2 domain with the pY-containing peptide through NMR titration experiments. The dynamics of Drk-SH2 were also analysed through NMR-relaxation experiments as well as the molecular dynamic simulation. The docking simulations of the pY-containing peptide onto the protein surface of Drk-SH2 provided the orientation of the peptide, which showed a good agreement with the analysis of the SH2 domain of GRB2. Full article

Capitellacin is the β-hairpin membrane-active cationic antimicrobial peptide from the marine polychaeta Capitella teleta. Capitellacin exhibits antibacterial activity, including against drug-resistant strains. To gain insight into the mechanism of capitellacin action, we investigated the structure of the peptide in the membrane-mimicking environment of dodecylphosphocholine (DPC) micelles using high-resolution NMR spectroscopy. In DPC solution, two structural forms of capitellacin were observed: a monomeric β-hairpin was in equilibrium with a dimer formed by the antiparallel association of the N-terminal β-strands and stabilized by intermonomer hydrogen bonds and Van der Waals interactions. The thermodynamics of the enthalpy-driven dimerization process was studied by varying the temperature and molar ratios of the peptide to detergent. Cooling the peptide/detergent system promoted capitellacin dimerization. Paramagnetic relaxation enhancement induced by lipid-soluble 12-doxylstearate showed that monomeric and dimeric capitellacin interacted with the surface of the micelle and did not penetrate into the micelle interior, which is consistent with the “carpet” mode of membrane activity. An analysis of the known structures of β-hairpin AMP dimers showed that their dimerization in a membrane-like environment occurs through the association of polar or weakly hydrophobic surfaces. A comparative analysis of the physicochemical properties of β-hairpin AMPs revealed that dimer stability and hemolytic activity are positively correlated with surface hydrophobicity. An additional positive correlation was observed between hemolytic activity and AMP charge. The data obtained allowed for the provision of a more accurate description of the mechanism of the oligomerization of β-structural peptides in biological membranes. Full article

Gram-negative bacteria are intrinsically more resistant to many frontline antibiotics, which is attributed to the permeability barrier of the outer membrane, drug efflux pumps and porins. Consequently, discovery of new small molecules antibiotics to kill drug-resistant Gram-negative bacteria presents a significant challenge. Thanatin, a 21-residue insect-derived antimicrobial peptide, is known for its potent activity against Enterobacter Gram-negative bacteria, including drug-resistant strains. Here, we investigated a 15-residue N-terminal truncated analog PM15 (P¹IIYCNRRTGKCQRM¹⁵) of thanatin to determine modes of action and antibacterial activity. PM15 and the P¹ to Y and A substituted variants PM15Y and PM15A delineated interactions and permeabilization of the LPS–outer membrane. In antibacterial assays, PM15 and the analogs showed growth inhibition of strains of Gram-negative bacteria that is largely dependent on the composition of the culture media. Atomic-resolution structures of PM15 and PM15Y in free solution and in complex with LPS micelle exhibited persistent β-hairpin structures similar to native thanatin. However, in complex with LPS, the structures of peptides are more compact, with extensive packing interactions among residues across the two anti-parallel strands of the β-hairpin. The docked complex of PM15/LPS revealed a parallel orientation of the peptide that may be sustained by potential ionic and van der Waals interactions with the lipid A moiety of LPS. Further, PM15 and PM15Y bind to LptA_m, a monomeric functional variant of LptA, the periplasmic component of the seven-protein (A-G) complex involved in LPS transport. Taken together, the structures, target interactions and antibacterial effect of PM15 presented in the current study could be useful in designing thanatin-based peptide analogs. Full article

Heterologous interactions between different amyloid-forming proteins, also called cross-interactions, may have a critical impact on disease-related amyloid formation. β-hairpin conformers of amyloid-forming proteins have been shown to affect homologous interactions in the amyloid self-assembly process. Here, we applied two β-hairpin-forming peptides derived from immunoglobulin light chains as models to test how heterologous β-hairpins modulate the fibril formation of Parkinson’s disease-associated protein α-synuclein (αSyn). The peptides SMAhp and LENhp comprise β-strands C and C′ of the κ4 antibodies SMA and LEN, which are associated with light chain amyloidosis and multiple myeloma, respectively. SMAhp and LENhp bind with high affinity to the β-hairpin-binding protein β-wrapin AS10 according to isothermal titration calorimetry and NMR spectroscopy. The addition of SMAhp and LENhp affects the kinetics of αSyn aggregation monitored by Thioflavin T (ThT) fluorescence, with the effect depending on assay conditions, salt concentration, and the applied β-hairpin peptide. In the absence of agitation, substoichiometric concentrations of the hairpin peptides strongly reduce the lag time of αSyn aggregation, suggesting that they support the nucleation of αSyn amyloid fibrils. The effect is also observed for the aggregation of αSyn fragments lacking the N-terminus or the C-terminus, indicating that the promotion of nucleation involves the interaction of hairpin peptides with the hydrophobic non-amyloid-β component (NAC) region. Full article

Dendrimeric and branched peptides are polypeptides formed by diverse types of scaffolds to give them different forms. Previously, we reported a cascade-type, Lys-scaffolded antimicrobial peptide dendrimer D4R tethered with four RLYR tetrapeptides. Antimicrobial D4R is broad-spectrum, salt insensitive, and as potent as the natural-occurring tachyplesins, displaying minimum inhibitory concentrations (MIC) < 1 μM. However, the relationships between scaffolds and antimicrobial potency remain undefined. Here, we report the design of four novel types of peptide antimicrobials whose scaffolded backbones are lysine (Lys), iso-Lys, ornithine (Orn), or iso-Orn tethered with RLYR on their α- or sidechain-amines to give ε-, δ-, and their α-branched peptides. When assayed against ten microorganisms, the Lys-scaffolded α- and ε-branched peptides are broadly active, salt insensitive, and as potent as D4R and tachyplesins, whereas the corresponding Orn-scaffolded α- and δ-branched peptides are salt sensitive and much less potent, displaying MICs ranging from 1 to >500 μM. Structure-activity relationship studies suggested that Lys-scaffolds, but not Orn-scaffolds, can support a reverse turn to organize RLYR tetrapeptides as parallel β-strands to form an amphipathic structure with Leu-Tyr as a hydrophobic core. Together, these results provide a structural approach for designing potent and salt-insensitive dendrimeric or branched peptide antimicrobials. Full article

Thymalin is an immunomodulatory drug containing a polypeptide extract of thymus that has demonstrated efficacy in the therapy of acute respiratory distress syndrome and chronic obstructive pulmonary disease, as well as in complex therapy related to severe COVID-19 in middle-aged and elderly patients.. KE and EW dipeptides are active substances of Thymalin. There is evidence that KE stimulates cellular immunity and nonspecific resistance in organisms, exerting an activating effect on macrophages, blood lymphocytes, thymocytes, and neutrophils, while EW reduces angiotensin-induced vasoconstriction and preserves endothelium-dependent vascular relaxation by inhibiting ACE2, the target protein of SARS-CoV-2. However, the mechanism of the immunomodulatory action of Thymalin, KE, and EW during COVID-19 remains unclear. To identify the potential mechanism of action underlying the immunomodulatory activity of Thymalin and its active components, EW and KE dipeptides, we assessed inflammatory response in the context of COVID-19. Interactions between EW and KE dipeptides and double-stranded DNA (dsDNA) were investigated by molecular modeling and docking using ICM-Pro. Analysis of the possible effect of EW and KE dipeptides on gene expression and protein synthesis involved in the pathogenesis of COVID-19 was conducted through the use of bioinformatics methods, including a search for promoter sequences in the Eukaryotic Promoter Database, the determination of genes associated with the development of COVID-19 using the PathCards database of human biological pathways (pathway unification database), identification of the relationship between proteins through cluster analysis in the STRING database (‘Search Tool for Retrieval of Interacting Genes/Proteins’), and assessment of the functional enrichment of protein–protein interaction (PPI) using the terms of gene ontology (GO) and the Markov cluster algorithm (MCL). After that, in vitro studying of a lipopolysaccharide (LPS)-induced model of inflammation using human peripheral blood mononuclear cells was performed. ELISA was applied to assess the level of cytokines (IL-1β, IL-6, TNFα) in the supernatant of cells with or without the impact of EW and KE peptides. Blood samples were obtained from four donors; for each cytokine, ELISA was performed 2–4 times, with two parallel experimental or control samples for each experiment (experiments to assess the effects of peptides on LPS-stimulated cells were repeated four times, while additional experiments with unstimulated cells were performed two times). Using molecular docking, GGAG was found to be the best dsDNA sequence in the classical B-form for binding the EW dipeptide, while GCGC is the preferred dsDNA sequence in the curved nucleosomal form for the KE dipeptide. Cluster analysis revealed that potential target genes for the EW and KE peptides encode the AKT1 and AKT2 proteins involved in the development of the cytokine storm. The specific targets for the EW peptide are the ACE2 and CYSLTR1 genes, and specific target for the KE peptide is the CHUK gene. Protein products of the ACE2, CYSLTR1, and CHUK genes are functionally associated with IL-1β, IL-6, TNF-α, IL-4, and IL-10 cytokines. An in vitro model of an inflammatory reaction demonstrated that Thymalin and EW and KE dipeptides reduced the synthesis of IL-1β, IL-6, and TNF-α cytokines in human peripheral blood mononuclear cells by 1.4–6.0 times. The immunomodulatory effect of Thymalin under the inflammatory response conditions in COVID-19 is based on the potential ability of its active components, EW and KE dipeptides, to regulate protein synthesis involved in the development of the cytokine storm. Full article

Solid surfaces have been shown to affect the aggregation and assembly of many biomolecular systems. One important example is the formation of protein fibrils, which can occur on a range of biological and synthetic surfaces. The rate of fibrillation depends on both the protein structure and the surface chemistry, with the different molecular and oligomer structures adopted by proteins on surfaces likely to be crucial. In this paper, the aggregation of the model amyloidogenic peptide, A$\beta$(16–22), corresponding to a hydrophobic segment of the amyloid beta protein on a gold surface is studied using molecular dynamics simulation. Previous simulations of this peptide on gold surfaces have shown that it adopts conformations on surfaces that are quite different from those in bulk solution. These simulations show that this then leads to significant differences in the oligomer structures formed in solution and on gold surfaces. In particular, oligomers formed on the surface are low in beta-strands so are unlike the structures formed in bulk solution. When oligomers formed in solution adsorb onto gold surfaces they can then restructure themselves. This can then help explain the inhibition of A$\beta$(16–22) fibrillation by gold surfaces and nanoparticles seen experimentally. Full article