Search Results (112)

Proteins play an important role in living organisms, and, for most of them, the function depends on their structure. There are some proteins that have similar properties but different structures. An example of this is ice-binding proteins (IBPs), which have different structures but share the ability to bind to ice. Many organisms have evolved such proteins to help them survive in cold environments. Therefore, it is important to study the oligomeric state of the active form in solutions. The activity of IBP is related to the area of their ice-binding site. We have demonstrated the presence of oligomeric forms of protein in solution using multiple techniques, such as mass spectrometry, native gel electrophoresis, atomic force microscopy (AFM), isothermal titration calorimetry (ITC) and small-angle X-ray scattering (SAXS). It is noteworthy that, to date, there have been no reports of the oligomerization of ice-binding protein from Longhorn sculpin. Additionally, our findings suggest that larger molecules may influence the ability of proteins to bind to ice. In our study, the ice-binding protein forms elongated assemblies with limited intermonomer interfaces. The combination of SAXS and AFM data indicates a structure that combines compactness and flexibility and probably consists of four monomeric units. The employment of molecular modelling methodologies resulted in the attainment of a tetrameric complex that is in alignment with AFM data. Details of oligomers observed using the methods in our study emphasize the importance of different techniques that complement each other in resolving structural features. Additionally, we suggest that the protein particles, which were dispersed on the surface, exhibit softness or the form planar complexes with loose quaternary structures. It is conceivable that, depending on ionic strength and/or temperature, the various oligomeric forms of the ice-binding protein form thermodynamically more favorable complexes than their monomeric forms. Full article

Protein–protein interactions (PPIs) take place in many cellular processes, including the activation of cellular cascades, such as the MAPK/ERK (Mitogen-Activated Protein Kinase/Extracellular-Regulated Kinase) pathway. Deregulation of these pathways leads to the development of diseases, such as cancer. DEL-22379 is an ERK2 dimerization inhibitor, which presents anti-tumoral effects, without affecting ERK2 phosphorylation. Our aim was to identify new therapeutic molecules targeting ERK2 dimerization, based on DEL-22379 structure. In this study, we implemented a combination of computational and experimental workflow, which includes in silico techniques, such as scaffold hopping and virtual screening to generate a dataset of candidate compounds, a native PAGE (PolyAcrylamide Gel Electrophoresis) electrophoresis to experimentally screen the potential inhibitors, and a detailed molecular docking and chemical profile prediction to understand the potential mechanism of action of the selected compounds. From an initial dataset of 536 compounds, we obtained two hit molecules that exhibited inhibitory effects on ERK2 dimerization: Drug73 and Drug120. A computational analysis of the mechanism of action, unveiled that Drug73 and Drug120 presented an improved docking score, and better drug-like properties when compared to DEL-22379. This study shows that computational studies, in combination with experimental evaluation, can be useful and efficient to find new therapeutic compounds. Full article

Marinesco–Sjögren syndrome (MSS) is a rare autosomal recessive neuromuscular disorder marked by ataxia, muscle weakness, cataracts, and often intellectual and skeletal abnormalities. It is commonly caused by loss-of-function variants in the SIL1 gene, which impair binding immunoglobulin protein (BiP) function, leading to protein misfolding and activation of the unfolded protein response. In a 2-year-old patient with typical MSS symptoms, we identified a previously unreported c.1024G>A (p.E342K) variant in SIL1 via whole-exome sequencing. The pathogenicity of this Sil1 variant was supported by evidence of structural changes revealed through in silico predictions, circular dichroism, and native gel electrophoresis. Patient-derived fibroblasts exhibited reduced Sil1 protein levels, likely due to misfolding and degradation, which was partially rescued by proteasome inhibition. Proteomics revealed a profile similar to known MSS cases and a distinctive MSS transcriptional signature. Ultrastructural analysis confirmed typical MSS features, such as autophagic vacuoles and lipid droplets. Although the p.E342K phenotype appears milder than the reference pathogenic variant R111X, our findings support the reclassification of this novel variant as pathogenic, in accordance with the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) 2015 guidelines and the refinements proposed by the Clinical Genome Resource Sequence Variant Interpretation (ClinGen SVI) recommendations. Furthermore, the overall evidence also provides important insights into the genotype–phenotype correlation and the underlying pathogenic mechanism of the p.E342K variant. Full article

We report an efficient, high-yield method for synthesizing dumbbell-shaped conjugates composed of gold nanoparticles (AuNPs) connected by double-stranded (ds) DNA. The dsDNA, bearing terminal thiol groups, was covalently attached to two AuNPs to form uniform constructs comprising either 15 nm or 25 nm particles bridged by 38 base pairs (bp) or 100 bp dsDNA. The dumbbells were purified by gel electrophoresis and exhibited high stability, remaining intact for several days in pure water or buffers at ambient temperature. Deposition onto solid substrates followed by drying, however, led to their partial structural collapse. TEM imaging showed that deposition on carbon grids typically yielded dumbbell structures with interparticle gaps of only 1–2 nm, suggesting that the dsDNA bridge contracts during deposition and drying. However, deposition on polylysine-coated mica for AFM imaging preserved the native geometry, with the gaps consistent with the expected DNA length. Our results reveal that deposition significantly affects the structure and integrity of dsDNA bridges in dumbbell constructs, highlighting the importance of appropriate substrate and surface coating selection for reliable characterization of DNA properties in dried dumbbells. Full article

Mitochondrial fission and fusion appear to be relatively infrequent in cardiac cells compared to other cell types; however, the proteins involved in these events are highly expressed in adult cardiomyocytes (ACM). Therefore, these proteins likely have additional non-canonical roles. We have previously shown that DRP1 not only participates in mitochondrial fission processes but also regulates mitochondrial bioenergetics in cardiac tissue. However, it is still unknown where the DRP1 that does not participate in mitochondrial fission is located and what its role is at those non-fission spots. Therefore, this manuscript will clarify whether oligomeric DRP1 is located at the SR–mitochondria interface, a specific region that harbors the Ca²⁺ microdomains created by Ca²⁺ release from the SR through the RyR2. The high Ca²⁺ microdomains and the subsequent Ca²⁺ uptake by mitochondria through the mitochondrial Ca²⁺ uniporter complex (MCUC) are essential to regulate mitochondrial bioenergetics during excitation–contraction (EC) coupling. Herein, we aimed to test the hypothesis that mitochondria-bound DRP1 preferentially accumulates at the mitochondria–SR contacts to deploy its function on regulating mitochondrial bioenergetics and that this strategic position is modulated by calcium in a beat-to-beat manner. In addition, the mechanism responsible for such a biased distribution and its functional implications was investigated. High-resolution imaging approaches, cell fractionation, Western blot, 2D blue native gel electrophoresis, and immunoprecipitations were applied to both electrically paced ACM and Langendorff-perfused beating hearts to elucidate the mechanisms of the strategic DRP1 localization. Our data show that in ACM, mitochondria-bound DRP1 clusters in high molecular weight protein complexes at mitochondria-associated membrane (MAM). This clustering requires DRP1 interaction with β-ACTIN and is fortified by EC coupling-mediated Ca²⁺ transients. In ACM, DRP1 is anchored at the mitochondria–SR contacts through interactions with β-ACTIN and Ca²⁺ transients, playing a fundamental role in regulating mitochondrial physiology. Full article

Co-infections pose significant challenges not only clinically, but also in terms of simultaneous diagnoses. The development of sensitive, multiplexed analytical platforms is critical for accurately detecting viral co-infections, particularly in complex biological environments. In this study, we present a mass spectrometry (MS)-based detection strategy employing a target-triggered hybridization chain reaction (HCR) to amplify signals and in situ photocleavable mass tags (PMTs) for the simultaneous detection of multiple targets. Hairpin DNAs modified with PMTs and immobilized loop structures on magnetic particles (Loop@MPs) were engineered for each target, and their hybridization and amplification efficiency was validated using native polyacrylamide gel electrophoresis (PAGE) and laser desorption/ionization MS (LDI-MS), with silica@gold core–shell hybrid (SiAu) nanoparticles being employed as an internal standard to ensure quantitative reliability. The system exhibited excellent sensitivity, with a detection limit of 415.12 amol for the hepatitis B virus (HBV) target and a dynamic range spanning from 1 fmol to 100 pmol. Quantitative analysis in fetal bovine serum confirmed high accuracy and precision, even under low-abundance conditions. Moreover, the system successfully and simultaneously detected multiple targets, i.e., HBV, human immunodeficiency virus (HIV), and hepatitis C virus (HCV), mixed in various ratios, demonstrating clear PMT signals for each. These findings establish our approach as a robust and reliable platform for ultrasensitive multiplexed detection, with strong potential for clinical and biomedical research. Full article

A periplasmic D-malate:cytochrome c oxidoreductase (DMCO) was identified in Ectopseudomonas oleovorans CECT5344, utilizing 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride (INT) as an artificial electron acceptor. The assay was adapted for a spectrophotometric or native polyacrylamide gel electrophoresis (PAGE) analysis. The DMCO-encoding gene (BN5_4044) was cloned and expressed in Escherichia coli, enabling a partial purification and biochemical characterization. In addition to D-malate, the enzyme oxidizes D-2-hydroxyglutarate and, to a lesser extent, D-lactate, with cytochrome c also serving as an electron acceptor. DMCO requires Zn²⁺ for activity and exists as a dimer, as determined by gel filtration. The in vitro reconstitution of the electron transfer from D-malate to oxygen was achieved using spheroplasts, enriched periplasmic fractions, and cytochrome c. This extracytoplasmic respiration, unique among homologs of this protein, may eliminate the need for a dedicated inner membrane transporter, thereby avoiding potential upstream respiratory bottlenecks. In the context of bioremediation, and particularly regarding the cyanide metabolism, this D-malate oxidation to oxaloacetate facilitates detoxification by forming the corresponding cyanohydrin, which can be subsequently assimilated for growth. Full article

The enzyme transglutaminase 2 (TG2) has an open conformation with transamidase activity which crosslinks matrix proteins contributing to fibrosis development. LDN-27219 promotes the closed conformation of TG2, which can enhance vasodilation, but its effects in renal tissue are unknown. We investigated whether LDN-27219 treatment affects albuminuria and markers of renal fibrosis as well as ex vivo vasodilatation. Male C57BL/6 mice (n = 48) underwent unilateral nephrectomy plus insertion of a deoxycorticosterone acetate pellet (DOCA group) or nephrectomy only (sham group). Both groups were randomized to intraperitoneal treatment with either LDN-27219 (8 mg/kg twice daily) or vehicle for 2 weeks. Urine albumin excretion was evaluated by metabolic cages. Kidney tissue fibrosis markers were assessed by qPCR and Western blotting, while the TG2 conformational state was evaluated using native gel electrophoresis. Collagen staining was performed using Picrosirius red and quantified under circularly polarized light. Mesenteric arteries were mounted in wire myographs for evaluation of vasorelaxation. DOCA mouse developed significant albuminuria (p < 0.001 vs. sham), but neither TG2 mRNA nor protein expression was upregulated in the kidney. However, the relative amount of TG2 in the closed conformation was higher in DOCA mice. LDN-27219 did not affect albuminuria, but LDN-27219-treated DOCA mice showed less urine production and less collagen staining than vehicle-treated DOCA mice. LDN-27219 did not affect TG2 mRNA or TG2 protein expression or mRNA of fibrosis markers. LDN-27219-treated mice had enhanced vasorelaxation to the nitric oxide donor sodium nitroprusside. In conclusion, LDN-27219 treatment in the one-kidney DOCA–salt model did not affect renal TG2 mRNA and protein expression or albuminuria but still exerted beneficial effects in terms of reduced kidney fibrosis and urine production in addition to enhanced vasodilatation. Full article

We investigated the interaction of HspB7 and its α-crystallin domain with the wild-type (WT) C-terminal fragment of human filamin C (FLNC), containing immunoglobulin-like domains 22–24 and its three mutants associated with cardio- and myopathies. The physicochemical properties of the WT FLNC fragment and its three mutants, p.Glu2472_Asn2473delinsAsp (EN/D) located in the 22nd domain, p.P2643_L2645del (ΔPGL), and p.W2710X (Wmut) both located in the 24th immunoglobulin-like domain were analyzed. Although all FLNC fragments had similar secondary structures, WT FLNC and its EN/D and ΔPGL mutants formed dimers, whereas Wmut formed either monomers or aggregates. The surface hydrophobicity of EN/D, ΔPGL, and especially Wmut mutants was larger than that of the WT fragment. Size exclusion chromatography, native gel electrophoresis, and chemical crosslinking indicated that the efficiency of interaction with HspB7 or its α-crystallin domain decreased in the order WT~EN/D > ΔPGL. Wmut was unable to interact with either HspB7 or its α-crystallin domain. Modeling via Alphafold 3 indicated that EN/D mutation affected the orientation of two loops connecting β-strands in the 22nd domain, while the ΔPGL and Wmut mutations exposed a hydrophobic groove in the 24th domain thereby reducing their interaction with HspB7. These findings reveal the molecular mechanisms underlying filaminopathies associated with three mutations in the C-terminal region of filamin C. Full article

MicroRNA-155-3p (miR-155-3p) is an important biomarker in various pathological conditions, including cancer, making the development of sensitive and specific detection methods crucial. Here, we present a molecular beacon (MB-G4) that underwent a conformational switch upon hybridization with miR-155-3p, enabling the formation of a G-quadruplex (G4) structure. This G4 was recognized by the fluorogenic ligand N-methyl mesoporphyrin IX (NMM), producing a fluorescence signal proportional to the target concentration, making it a new detection method. The conformational dynamics of MB-G4 were characterized through circular dichroism (CD) spectroscopy and native polyacrylamide gel electrophoresis (PAGE), confirming the transition from a hairpin structure to an RNA–DNA hybrid duplex that facilitated G4 formation. The optimization of the experimental conditions, including the potassium chloride (KCl) and NMM concentrations, ensured selective detection with minimal background signal. The detection limit (LOD) was determined to be 10.85 nM, using a linear fluorescence response curve, and the specificity studies demonstrated a clear distinction between miR-155-3p and miR-155-5p. Furthermore, MB-G4 was studied with total RNA extracted from the lung cancer cell line A549 to evaluate its detection in a more complex environment and was able to detect its target, validating its potential for biological sample analysis. Full article

Thrombin binding aptamer (TBA) is one of the best-known G-quadruplex (G4)-forming aptamers that efficiently binds to thrombin, resulting in anticoagulant effects. TBA also possesses promising antiproliferative properties. As with most therapeutic oligonucleotides, chemical modifications are critical for therapeutic applications, particularly to improve thermodynamic stability, resistance in biological environment, and target affinity. To evaluate the effects of nucleobase and/or sugar moiety chemical modifications, five TBA analogues have been designed and synthesized considering that the chair-like G4 structure is crucial for biological activity. Their structural and biological properties have been investigated by Circular Dichroism (CD), Nuclear Magnetic Resonance (NMR), native polyacrylamide gel electrophoresis (PAGE) techniques, and PT and MTT assays. The analogue TBAB contains 8-bromo-2′-deoxyguanosine (B) in G-syn glycosidic positions, while TBAL and TBAM contain locked nucleic acid guanosine (L) or 2′-O-methylguanosine (M) in G-anti positions, respectively. Instead, both the two types of modifications have been introduced in TBABL and TBABM with the aim of obtaining synergistic effects. In fact, both derivatives include B in syn positions, exhibiting in turn L and M in the anti ones. The most appealing results have been obtained for TBABM, which revealed an interesting cytotoxic activity against breast and prostate cancer cell lines, while in the case of TBAB, extraordinary thermal stability (T_m approximately 30 °C higher than that of TBA) and an anticoagulant activity higher than original aptamer were observed, as expected. These data indicate TBAB as the best TBA anticoagulant analogue here investigated and TBABM as a promising antiproliferative derivative. Full article

Background/Objectives: This study aimed to design and evaluate Chol-PEG₂₀₀₀ micelles and Chol-PEG₅₀₀ vesicles as drug delivery system (DDS) carriers and inhibitors of amyloid-β (Aβ) aggregation, a key factor in Alzheimer’s disease (AD). Methods: The physical properties of Chol-PEG assemblies were characterized using dynamic light scattering (DLS), electrophoretic light scattering (ELS), and transmission electron microscopy (TEM). Inhibitory effects on Aβ aggregation were assessed via thioflavin T (ThT) assay, circular dichroism (CD) spectroscopy, and native polyacrylamide gel electrophoresis (native-PAGE). Results: Chol-PEG₂₀₀₀ micelles and Chol-PEG₅₀₀ vesicles were found to exhibit diameters of 20–30 nm and 70–80 nm, respectively, with neutral surface charges and those physical properties indicated the high affinity for Aβ. At a 10-fold molar ratio, thioflavin T (ThT) assay revealed that Chol-PEG₂₀₀₀ delayed Aβ fibril elongation by 20 hours, while Chol-PEG₅₀₀ delayed it by 40 hours against Aβ peptide. At a 50-fold molar ratio, both Chol-PEG₂₀₀₀ and Chol-PEG₅₀₀ significantly inhibited Aβ aggregation, as indicated by minimal fluorescence intensity increases over 48 hours. CD spectroscopy indicated that Aβ maintained its random coil structure in the presence of Chol-PEG assemblies at a 50-fold molar ratio. Native-PAGE analysis demonstrated a retardation in Aβ migration immediately after mixing with Chol-PEG assemblies, suggesting complex formation. However, this retardation disappeared within 5 min, implying rapid dissociation of the complexes. Conclusions: This study demonstrated that Chol-PEG₅₀₀ vesicles more effectively inhibit Aβ aggregation than Chol-PEG₂₀₀₀ micelles. Chol-PEG assemblies perform as DDS carriers to be capable of inhibiting Aβ aggregation. Chol-PEG assemblies can deliver additional therapeutics targeting other aspects of AD pathology. This dual-function platform shows promise as both a DDS carrier and a therapeutic agent, potentially contributing to a fundamental cure for AD. Full article

The use of avenins as biochemical markers successfully complements the use of molecular markers in oat breeding. Currently, the genes controlling the synthesis of oat prolamins are insufficiently studied. The purpose of the work was to study the genetic variation of avenin components in populations of F₂ common oat hybrids and to describe new allelic variants of component blocks. The avenins component of F₂ grain in 19 hybrid oat populations was studied using the native electrophoresis method. Cultivars with new combinations of avenin components were used as parental genotypes to produce hybrids. The protein separation was conducted in vertical plates of 13.2% polyacrylamide gel. The number of avenin components in the spectra of cultivars varied from 8 to 12. The observed ratio of the grain number that compose the phenotypic classes for allele pairs at each of the loci corresponded to the theoretically expected one for codominant monohybrid inheritance. Our results confirm the assumption that avenin synthesis is controlled by three independent gene clusters located on three chromosomes. In the course of the studies, hybrid combinations were not identified in the spectra of which avenin components were manifested that were absent in both parents. The prolamin component blocks in oat are formed by 2–5 components, are characterized by high stability, and are inherited unchanged. Fifteen new allelic variants of blocks of components of the avenin electrophoretic spectrum have been identified: six for the Avn A locus, six for the Avn B locus, and three for the Avn C locus. This expands the possibilities of using prolamins as biochemical markers of economically valuable oat traits and certification of new cultivars and valuable breeding lines. Full article

The search for more sustainable reaction conditions has been necessary to obtain more selective processes. In this context, laccases have gained great notoriety in recent years. However, these enzymes are unstable in organic solvents and have low thermal stability. Alternatively, conjugation with PEG (PEGylation) can be essential to overcome these problems. In this work, the commercial laccase from Myceliophthora thermophila (LacMT) was subjected to PEGylation with PEG functionalized as succinimidyl carbonate (mPEG-SC), followed by assessing its thermal stability and catalytic activity. Mono-PEGylated LacMT derivatives were obtained, with less than 50% of the enzyme remaining in its native form. In addition, 10% of the bi-PEGylated species was successfully obtained according to gel electrophoresis analysis. The PEGylated derivatives showed a significantly reduced ABTS oxidation activity (98 ± 3 U/mg) compared to the native LacMT (407 ± 9 U/mg) but higher than the control enzyme without PEGylation (51 ± 2 U/mg), demonstrating that the addition of activated PEG to the protein resulted in better protection against the harmful action of the pH change required in the process. PEGylated LacMT retained more than twice the initial activity of the native protein at 40 °C during 24 h. In addition, PEGylated LacMT exhibited kinetic changes, whereas the catalytic turnover rate (k_cat) of the PEGylated enzyme was reduced by 27% compared to the control. These findings are being reported for the first time. This sets precedents for constructing efficient catalytic systems involving laccases since no immobilized biocatalyst or commercial conjugate contains these proteins. Full article

Concatemeric viral DNA is packaged into bacteriophage P22 procapsids via a headful packaging mechanism mediated by a molecular machine consisting of small (gp3) and large (gp2) terminase subunits. Although a negative stain reconstruction exists for the terminase holoenzyme, it is not clear how this complex binds the dodecameric portal protein located at a 5-fold mismatch vertex. Herein, we describe new assemblies for the holoenzyme. Both native mass spectrometry and transmission electron microscopy reveal that the P22 terminase complex adopts three main assemblies, which include a nonameric S-terminase bound to two L-terminase 1(gp3)₉:2(gp2), two nonameric S-terminase bound to five L-terminase 2(gp3)₉:5(gp2), and three nonameric S-terminase bound to seven L-terminase 3(gp3)₉:7(gp2). Native agarose gel electrophoresis shows that the terminase complex interacts with procapsids with mild crosslinking. These results herein illustrate the P22 terminase complex can adopt a variety of conformations and assembly states. Full article