The protein journal最新文献

An essential modulator of cell growth and division is the evolutionarily conserved kinase (S6K1). It is triggered by certain stimulants, including amino acids, insulin, and other growth hormones. The Akt/phosphatidylinositol 3-kinase pathway's downstream effector, the serine/threonine kinase S6K1, is consistently activated in a variety of cancer types. Rho family guanosine triphosphate (GTPase) activation and actin filament cross-linking are two of S6K1's roles. The p70 (∆2-146/∆CT104) S6K is a truncated variant of p70S6 kinase, created by removing 146 amino acids from the N-terminal and 104 amino acids from the C-terminal end of the original protein, resulting in a total of 275 amino acids. The p70 (∆2-146/∆CT104) S6K was effectively expressed in the E. coli BL21 (DE3)pLysS strain after being cloned in E. coli DH5α. A rabbit polyclonal anti-GST antibody had been employed during Western blot analysis throughout the protein's production and purification process. Protein purification was achieved by affinity chromatography using glutathione resin-agarose beads, and chromatography onto a spin concentration column was performed. Rabbit polyclonal anti-(p70S6Kinase and GST) antibodies confirmed the presence of the purified protein.

Over the past three years, AlphaFold-a deep learning-based protein structure prediction system-has transformed structural biology by providing near-experimental accuracy models directly from amino acid sequences. This narrative review synthesizes applications reported in the 2022-2025 literature across human, microbial, and viral systems, drawing on peer-reviewed studies as our data source. Representative examples include modeling of SARS-CoV-2 spike and nucleocapsid proteins in virology, assisting cryo-EM interpretation of bacterial ribosomal and membrane-protein complexes in microbiology, and refining conformational hypotheses for human GPCRs in biomedicine. Across these cases, AlphaFold predictions have complemented experimental workflows by accelerating hypothesis generation, improving model fitting within ambiguous density regions (poorly resolved areas of cryo-EM maps), and guiding mutagenesis strategies to probe dynamic conformational states. We also summarize recent method extensions: AlphaFold-Multimer improves multi-chain complex assembly prediction, while molecular dynamics (MD) simulations augment AlphaFold's static models by sampling conformational flexibility and testing stability. Despite these advances, important limitations remain-particularly for intrinsically disordered regions, protein-ligand and protein-cofactor interactions, and very large or transient assemblies-and current community benchmarks indicate that approximately one-third of residues may lack atomistic precision, underscoring uncertainty in flexible or modified segments. Framed within a clear chronological window and evidence base, our analysis highlights both the practical impact and the remaining challenges of integrating AlphaFold with experiment, outlining priorities where further methodological innovation and orthogonal validation are needed.

Although biological drugs have been considered as one of the effective and growing therapeutic approaches in the pharmaceutical industry in recent decades, the largest concern about them is the insufficient stability and rapid degradation in the bloodstream due to their structural nature. One of the effective methods for increasing the circulating half-life of peptide and protein drugs is the addition of half-life-extending tags, which prevent both the degradation of the biological drug and its glomerular filtration by various mechanisms, thereby increasing its half-life. This review focuses on peptide and protein tags used to enhance the pharmacokinetic profiles of biological drugs by increasing their half-life. It discusses various tags, including HSA (Human Serum Albumin), ABD (Albumin Binding Domain), DARPINS (Designed Ankyrin Repeat proteins), XTEN, CTP (Carboxy Terminal Peptide), ELP (Elastin Like Peptide), and others, and highlights both FDA-approved products and candidates currently in different stages of clinical development. In the meantime, special attention has been paid to albumin-binding domains and albumin-binding domain antibody (AlbudAb), which increases the half-life of biological drugs by binding to albumin, as the most abundant and stable protein in the body.

Rotaviruses (RV) are a major cause of severe childhood diarrhoea, particularly in developing nations, necessitating stable vaccines. Therefore, the presented preliminary study aimed to assess the impact of altered physicochemical properties on the structural stability of recombinant rotavirus capsid protein VP6 (RV-VP6). The expression system used in this study was designed by genetically engineering the RV-VP6 into E. coli (NiCo21(DE3))-pET28a host-vector system and purified using liquid chromatography. The purified RV-VP6 homology detection and structure prediction were conducted using LC-MS and HHpred computational analysis, which indicated a 100% probability of 1QHD_A Viral Capsid VP6 (1.95 Å), representing the crystal structure of VP6. The secondary and tertiary structural stability of RV-VP6 was evaluated in altered pH and Ca²⁺ concentrations using far UV-CD and intrinsic tryptophan fluorescence spectroscopy, respectively. The computational analysis of the far-UV CD spectra revealed a significant increase in the composition of α-helices and β-sheets in altered pH and Ca²⁺ environments compared to the denatured protein (p < 0.0001). Intrinsic fluorescence analysis of RV-VP6 at pH 7 yielded an emission λmax of 339 nm, which shifted to 342 nm at pH 5. In 1 mM Ca²⁺, a λmax of 340 nm was observed, with an increase in intensity in 10 mM Ca²⁺, accompanied by a slight blue shift to 338 nm. Investigation of RV-VP6 under thermal stress yielded unfolding concomitant with aggregation, rendering the process irreversible and nullifying analysis using equilibrium thermodynamics. These findings form the preliminary basis for our future evaluation of manufacturing stable and enhanced RV-VP6 vaccines through the downstream process control of (1) pH, which alters the charge distribution on the surface of the protein, leading to conformational changes, and (2) Ca²⁺ ions, which interact with specific amino acid residues in the protein, thereby affecting its structure and function.

Refolding of protein from denatured structure caused by sodium dodecyl sulfate (SDS) was examined using agarose native gel electrophoresis and circular dichroism (CD). Refolding of protein from SDS complex was induced with the addition of non-ionic and zwitterionic detergents followed by agarose native gel electrophoresis. The native gel electrophoresis was done without both SDS and non-ionic detergents in the agarose gel and running buffer. The electrophoretic mobility of bovine serum albumin (BSA) drastically increased with the addition of 1% SDS to the samples indicative of SDS-BSA complex formation. The SDS-denatured BSA returned to the native mobility by the addition of non-ionic Tween 20 and Triton X-100 and zwitterionic CHAPS as a function of detergent concentration. Refolding, at least partially, was confirmed by CD, which was done in the presence of both SDS and non-ionic detergents, a condition different from the native gel electrophoresis done in their absence. When BSA was denatured by both 1% SDS and a disulfide-reducing dithiothreitol, even 10% Tween 20 was insufficient to restore the native BSA mobility on agarose native gel electrophoresis. When BSA was denatured by 1% Sarkosyl and sodium lauroyl-glutamate, Tween 20 restored the native structure at Tween 20 concentration lower than the Tween 20 concentration used for SDS denaturation. A similar refolding by non-ionic detergents was also observed for a rabbit monoclonal IgG, but not for lysozyme. The results with lysozyme suggest strong SDS binding and difficulty in dissociating the bound SDS by non-ionic detergents due to high isoelectric point of the protein and thereby more SDS binding.

Serum albumin (SA) is a key biomarker routinely used in clinical practice. Multiple analytical methods exist for its measurement, including bromocresol green (BCG) colorimetry and capillary zone electrophoresis (CZE). However, discrepancies between methods raise concerns regarding result consistency and clinical interpretation. This study aimed to compare the analytical concordance between BCG and CZE methods for SA measurement, and to assess their clinical interchangeability. A cross-sectional study conducted on 109 serum samples collected from patients undergoing serum protein electrophoresis at Bechir Hamza Children's Hospital. SA was measured using BCG and CZE. Descriptive statistics were calculated for each method. Paired t-test and Wilcoxon signed-rank test were used to assess differences in means. Method agreement was evaluated using Passing-Bablok regression, Intraclass Correlation Coefficient (ICC), Bland-Altman plot, and a confusion matrix. BCG significantly overestimated SA compared to CZE (41.49 ± 7.30 g/L vs. 37.48 ± 6.85 g/L; p < 0.001). Passing-Bablok regression revealed a regression line of y = 1.02x + 3.21, indicating a consistent positive bias. The R² value was 0.891, suggesting strong correlation. The ICC was 0.81 (95% CI 0.081-0.932), reflecting good agreement. The Bland-Altman analysis confirmed a mean difference of 4.01 g/L. Confusion matrix analysis showed perfect concordance in low albumin values but significant misclassification at higher levels, with BCG shifting many values into higher categories. While BCG and CZE methods show strong correlation, BCG consistently overestimates albumin concentrations. These findings underscore the need for method standardization and careful interpretation of results in clinical settings.

σ^B, a Staphylococcus aureus-encoded alternative sigma factor, is inhibited by RsbW, an anti-sigma factor. RsbW also dimerizes in solution and phosphorylates RsbV, an RsbW antagonist. Of the predicted RsbW residues involved in its dimerization and/or binding cognate factors, the roles of Arg 11, Arg 32, and Lys 44 have been demonstrated here using various tools. The structural models of three RsbW mutants, harboring Ala at positions 11, 32, and 44, were built up, and their analyses suggested that all of the above Lys/Arg residues may be involved in the dimerization of this protein. Similar analyses indicated that Lys 44 and Arg 32 may be needed for binding σ^B3, the domain 3 of σ^B. The docking studies have confirmed the roles of Arg 11 and Arg 32 in the dimerization of RsbW, and revealed that its association with σ^B3 requires Arg 32 and Lys 44. Additionally, the simulation studies proposed that all of the above Lys/Arg may be needed for its structure maintenance. To verify the computational data, three Ala-substituted RsbW mutants were also purified, and their characterization indicates that the change of Arg 11 or Lys 44 to Ala, compared to the change of Arg 32 to Ala, impacts the structure, RsbV/ σ^B3 binding affinity, and phosphorylation activity of RsbW more severely. Further, the dimerization ability of RsbW was partly affected by Ala substitution at positions 11 and 32. Jointly, Arg 11, Arg 32, and Lys 44 of RsbW are crucial residues for this σ^B inhibitor and kinase. Knowledge of crucial RsbW residues may be useful for designing antistaphylococcal drugs in the future.

Aldose reductase (AR) is the rate-limiting enzyme of the polyol pathway and a validated target for preventing micro- and neurovascular complications of diabetes. Here, we combined multi-scale in-silico analyses with biochemical testing to evaluate five commercially available bisbenzylisoquinoline alkaloids-cepharanthine, dauricine, isotetrandrine, fangchinoline and sinomenine-as potential AR inhibitors. Density-functional optimization, structure-based docking and 500 ns molecular-dynamics simulations revealed that the macrocyclic scaffolds of cepharanthine (ΔG_dock =  - 8.4 kcal mol^-1) and dauricine (- 9.7 kcal mol^-1) fully occupy the Phe122-Trp219-Trp111 aromatic cage and lock AR into a single, deep free-energy basin, whereas sinomenine explores a broad landscape. MM/PBSA calculations on the 150-200 ns of each trajectory ranked binding free energies as dauricine ≈ isotetrandrine ≈ cepharanthine < sinomenine < fangchinoline, with van-der-Waals forces dominating. ADMET profiling predicted high gastrointestinal absorption across the series but flagged a potential hERG potassium-channel liability for the four macrocycles. Enzyme-kinetic assays corroborated the computational hierarchy: cepharanthine, dauricine and isotetrandrine inhibited recombinant AR with IC₅₀ values of 4.25 ± 0.42, 5.38 ± 0.22 and 6.65 ± 0.40 µM, respectively, compared with 2.36 ± 0.32 µM for quercetin. Lineweaver-Burk and Michaelis-Menten analysis showed mixed inhibition for cepharanthine (K_i = 3.71 µM) and non-competitive inhibition for dauricine (K_i = 4.63 µM) and isotetrandrine (K_i = 6.99 µM). Fangchinoline and sinomenine were an order of magnitude weaker (IC₅₀ = 37-57 µM). Taken together, these data position cepharanthine and dauricine as mechanistically validated, hit-stage starting points for next-generation AR inhibitors, and identify isotetrandrine as an allosteric back-up scaffold. More broadly, the study illustrates a transparent, reproducible computational-experimental workflow for prioritizing structurally complex natural products against redox enzymes implicated in diabetic pathology.