The Protein Journal最新文献

Molecular chaperones known as heat shock proteins (HSPs) are evolutionarily conserved and are critical in regulating immune system functions. To assess the immunostimulatory potential of HSPs, we employed advanced computational and experimental techniques. Bioinformatics tools were employed to analyze and confirm the physicochemical characteristics of the full-length HSPA1A, the fragments of HSPA1A known as minichaperones (C-terminal region of HSPA1A: CHSPA1A and N-terminal region of HSPA1A: NHSPA1A), and the full length HSP27, confirming their advantageous properties and structural integrity through 3D refinement and validation. Docking studies revealed significant interactions of HSPs with CD91 and TLR4 receptors respectively, notably a strong and stable binding of the full length HSPA1A and CHSPA1A with CD91. Molecular dynamics simulations were conducted to confirm the structural stability of the complexes formed between HSPs and CD91. Immunological simulations demonstrated robust and stable responses from both innate and adaptive immune cells against HSPs. All HSPs were produced in E. coli, isolated via Ni-NTA affinity chromatography, and subsequently utilized to stimulate immune cells from mice in vitro. The results showed significant secretion of TNF-α and IFN-γ in comparison to IL-10, indicating a directed T-helper 1 (cellular) immunity. The in silico and in vitro findings represented that HSPA1A and then CHSPA1A resulted in significantly greater TNF-α secretion in splenocytes co-cultured with the pulsed DCs compared to NHSPA1A and HSP27, suggesting their significant role in activating adaptive immunity. In contrast, macrophages exhibited an increased TNF-α secretion in response to HSP27, suggesting its involvement in modulating innate inflammatory processes. Understanding these differences can help in designing more effective immunotherapies.

Homocysteine thiolactone is a reactive thiol known for its interaction with various proteins. Nevertheless, there exists a paucity of information concerning the interaction between homocysteine thiolactone and human insulin, particularly regarding the mechanism by which homocysteine facilitates the reduction of disulfide bonds within insulin. In the present study, we have elucidated the binding sites of homocysteine to the cysteine residues (A6-B7 and A20-B19) that are implicated in the formation of intermolecular disulfide bonds in insulin through an in vitro reaction analyzed via LC-ESI MS/MS. This results in a reduction of disulfide bonds linking the A and B chains, which was corroborated by MALDI-TOF-MS and ESI-MS analysis. The secondary structure of insulin is affected by this modification, as evidenced by circular dichroism spectroscopy. In-silico studies also show that homocysteine affects the insulin structure. A glucose uptake assay conducted in Chinese hamster ovary (CHO) cells that stably express the insulin receptor revealed that HC-modified insulin is less effective in inducing glucose uptake compared to native insulin, suggesting that HC-induced structural modifications in insulin influence functional activity. This study provides insight into the HC-induced structural and functional changes in insulin and discusses the consequent implications for diabetes.

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Superoxide dismutase (SOD) is found in a variety of organisms, including animals, plants, and microorganisms, and is widely used in medicine, food, and cosmetics. In this study, a novel heat-resistant SOD from Rhodothermus sp. XMH10 (RhSOD) has been found to have no loss of activity at 80 °C and exhibit high thermal stability across a temperature range from 20 °C to 80 °C. Unlike other reported SODs, RhSOD was found to have a unique small α-helix tail at the C-terminus, consisting of 11 amino acid residues. The absence of the C-terminal α-helix tail of RhSOD was shown to reduce its activity and thermal stability at 80 °C, suggesting that the C-terminal α-helix tail is crucial for the high thermal stability of RhSOD. Furthermore, the fusion of the C-terminal α-helix tail to the C-terminus of a thermophilic SOD from Anoxybacillus caldiproteolyticus (AcSOD) enhances its thermal stability at 70 °C and 80 °C. Circular dichroism (CD) spectral analysis further indicated that the C-terminal α-helix tail could improve the α-helix content, thus enhancing the structural stability of AcSOD. Thus, a novel C-terminal α-helix tail was firstly discovered, which could confer significant thermal stability to host proteins. This finding provides a new theoretical basis for the study of protein thermostability mechanism.

Plasmodium falciparum, the parasite responsible for the most severe form of malaria, encodes three small heat shock proteins (sHsps), PfHsp20a, PfHsp20b, and PfHsp20c, each containing a conserved α-crystallin domain (ACD). These class I sHsps are hypothesized to play critical roles in proteostasis under stress, yet their specific functions have remained poorly defined. In this study, all three sHsps were recombinantly expressed and purified for structural and functional characterization. Circular dichroism and thermal shift assays revealed distinct conformational properties, with PfHsp20a exhibiting the highest thermal and chemical stability. Functional assays using malate dehydrogenase and citrate synthase confirmed that all three isoforms possess autonomous chaperone activity, although with varying efficiency. Notably, the plant-derived flavonoid quercetin disrupted both the structure and function of the sHsps in a concentration-dependent manner, with PfHsp20c being the most sensitive. Quercetin also inhibited the growth of P. falciparum Nf54 and Dd2 strains in vitro with IC₅₀ values of 5.4 μM and 7.8 μM, respectively. These results provide the first direct evidence of independent chaperone activity in P. falciparum sHsps and highlight their vulnerability to small molecule inhibition. This establishes their potential as novel drug targets for antimalarial intervention.

The cohesin protein complex plays a very important role in chromosome segregation, transcription, DNA replication and chromosome condensation. Mutations in cohesin proteins give rise to a disease collectively referred to as Cohesinopathies. The major cause of cohesinopathies arise due to defects associated with gene expression, that give rise to developmental disorders. We have used Saccharomyces cerevisiae to mimic the cohesinopathy disorder Roberts syndrome with mutations (eco1W216G) homologous to that of humans (esco2). Our data suggests that polyol sugars like sorbitol, can repair misfolded proteins and reduce ER and proteostatic stress. We have used sorbitol as a chemical chaperone, to check how it can restore chromosome segregation, gene expression, misregulation, protein misfolding, autophagy and translational defects in the cohesin mutant of the Roberts’ phenotype. Molecular docking has helped us identify the possible sites on Eco1, which could possibly alter the phenotypic traits.

A phytochemical investigation of Glycyrrhiza echinata led to the isolation and structural characterization of twelve phenolic compounds. An in silico target fishing analysis identified protein tyrosine phosphatase 1B (PTP1B) as a potential biological target for these phytochemicals, prompting an in vitro evaluation of their PTP1B inhibitory activities. Gancaonin Q and licoflavone C exhibited notably low IC₅₀ values (1.61 ± 0.32 µM and 1.39 ± 0.33 µM, respectively), outperforming the reference inhibitor ursolic acid (IC₅₀ = 7.17 ± 0.69 µM), while norartocarpetin showed moderate activity (IC₅₀ = 42.41 ± 2.12 µM). Enzyme kinetic studies revealed that gancaonin Q and licoflavone C act as noncompetitive inhibitors of PTP1B. Subsequent in silico analyses supported these findings and provided mechanistic insights. Molecular docking confirmed robust binding interactions for gancaonin Q and licoflavone C at the PTP1B allosteric site. Free energy landscape (FEL) calculations indicated that both compounds stabilized the enzyme within low-energy conformations, and MM/PBSA estimations corroborated their favorable binding free energies. Molecular dynamics simulations further demonstrated the stability of the ligand-enzyme complexes, characterized by reduced structural fluctuations in comparison with the free enzyme and norartocarpetin-bound states. Finally, ADMET assessments indicated promising pharmacokinetic and toxicity profiles, with some scope for structural refinement. Overall, these results highlight gancaonin Q and licoflavone C as promising lead compounds for the development of PTP1B inhibitors with therapeutic potential.

Phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) of cestodes is considered a possible anthelmintic target because of its differential role in their hosts. In an earlier study, the recombinant PEPCK from Raillietina echinobothrida (rePEPCK) was overexpressed as inclusion bodies and was solubilized following renaturation with chemical additives, specifically L-arginine. Molecular chaperones are alternatives to chemical additives and detergents because they preserve the stability and conformation of the proteins. Hence, in this study, the recombinant rePEPCK was subcloned into the pE-SUMO vector and co-expressed along with the molecular chaperones (e.g. pG-KJE8, pG-Tf2) in Escherichia coli BL21 (DE3) cells. The protein was purified using affinity chromatography and subsequently characterized. The overexpressed rePEPCK was found to be a monomer of ~ 75 kDa. The optimum activity of the enzyme was observed in 50 mM Tris–HCl buffer at pH 7.0. In comparison, Mn²⁺ at 4.0 mM and GDP at 0.6 mM were observed to be the ideal cofactor and nucleotide, respectively. The V_max of the purified rePEPCK was found to be ~ 0.279 U/mg protein and K_m value of ~ 35.87 μM for its substrate. The turnover number (k_cat) of rePEPCK was found to be 4.7 s⁻¹ with catalytic efficiency (k_cat/K_m) 1.31 × 10⁵ M⁻¹ s⁻¹. The chaperones interacted with the key amino acids of PEPCK. This investigation explored the role of the chaperones in producing biologically active rePEPCK for its characterisation and may improve the understanding of the biochemical and biophysical properties of the enzyme as an anthelmintic target.

Resveratrol, a natural phytoalexin synthesized by certain plants in response to injury, exhibits antimicrobial properties and various medically significant effects, including anticancer and antiaging activities. Although its exact mechanism of action is still under investigation, it is believed to involve its interaction with a group of protein deacetylases known as sirtuins. Sirtuins are crucial in regulating metabolism, stress responses, and processes such as lifespan extension through caloric restriction. In this study, we report the inhibitory effect of resveratrol on the growth of Leishmania amazonensis promastigotes, highlighting its potential as a microbicide. Through fluorescence spectroscopy assays and in silico analysis, we identified and characterized the interaction between resveratrol and Sir2-related protein 1 from L. amazonensis (rLaSir2RP1). Our results demonstrate a direct interaction between resveratrol and rLaSir2RP1, characterized by a binding constant of 10⁵ M⁻¹. This interaction involves a single binding site located near a hydrophobic pocket, which includes its solely tryptophan residue.

Insulin lispro, a fast-acting insulin analogue, is widely used for the treatment of diabetes due to its rapid onset and high flexibility. This study established a three-step chromatography purification process, L fermentation broth can obtain 475 mg/L insulin lispro. Through a multi-step process involving fermentation, recovery of inclusion bodies, renaturation, enzymatic digestion, and chromatographic purification, a high-purity insulin lispro product was obtained. The final product achieved a purity of 99.7%. The high molecular weight protein was ≤ 0.1%, zinc content was 0.41%, host cell protein contamination was 2.31 ng/mg, and residual DNA was 1.57 ng/mg. Analytical results, including retention time, molecular weight, and peptide mapping, were consistent with theoretical expectations and matched the control. This purification process provides a streamlined and cost-effective method for large-scale production of insulin lispro, supporting further development of recombinant insulin analogs and offering high-quality active pharmaceutical ingredients for pharmaceutical formulations.