Protein and Peptide Letters最新文献

Background: The six-helix bundle (6-HB) is a core structure formed during the membrane fusion process of viruses with the Class I envelope proteins. Peptide inhibitors, including the marketed Enfuvirtide, blocking the membrane fusion to exert inhibitory activity were designed based on the heptads repeat interactions in 6-HB. However, the drawbacks of Enfuvirtide, such as drug resistance and short half-life in vivo, have been confirmed in clinical applications. Therefore, novel design strategies are pivotal in the development of next-generation peptide-based fusion inhibitors.

Objective: The de novo design of α-helical peptides against MERS-CoV and IAVs has successfully expedited the development of fusion inhibitors. The reported sequences were completely nonhomologous with natural peptides, which can provide some inspirations for the antiviral design against other pathogenic viruses with class I fusion proteins. Here, we design a series of artificial C-peptides based on the similar mechanism of 6-HB formation and general rules of heptads repeat interaction.

Methods: The inhibitory activity of peptides against HIV-1 was assessed by HIV-1 Env-mediated cell-cell fusion assays. Interaction between artificial C-peptides and target peptides was evaluated by circular dichroism, polyacrylamide gel electrophoresis, size-exclusion chromatography, and sedimentation velocity analysis. Molecular docking studies were performed by using Schrödinger molecular modelling software.

Results: The best-performing artificial C-peptide, 1SR, was highly active against HIV-1 env-mediated cell-cell fusion. 1SR binds to the gp41 NHR region, assembling polymer to prevent endogenous 6-HB formation.

Conclusion: We have found an artificial C-lipopeptide lead compound with inhibitory activity against HIV-1. Also, this paper enriched both N- and C-teminal heptads repeat interaction rules in 6-HB and provided an effective idea for next-generation peptide-based fusion inhibitors against HIV-1.

Introduction: Insulin-like growth factor-1 (IGF-1) is a single-chain polypeptide with various physiological functions. Escherichia coli is one of the most desirable hosts for recombinant protein production, especially for human proteins whose post-translation modifications are not essential for their bioactivity, such as hIGF-1.

Objectives: In this study, bacterial thioredoxin (Trx) was studied as a fused and non-fused protein to convert the insoluble form of recombinant human IGF-1 (rhIGF-1) to its soluble form in E. coli.

Methods: The rhIGF-1 was expressed in the E. coli Origami strain in the form of fused-Trx. It was co-expressed with Trx and then purified and quantified. In the next step, the biological activity of rhIGF-1 was evaluated by alkaline phosphatase (ALP) activity assay in human adipose- derived stem cells (hASCs) regarding the differentiation enhancement effect of IGF-1 through the osteogenic process.

Results: Results showed that Trx in both the fused and non-fused forms had a positive effect on the production of the soluble form of rhIGF-1. A significant increase in ALP activity in hASCs after rhIGF-1 treatment was observed, confirming protein bioactivity.

Conclusion: It was strongly suggested that the overproduction of Trx could increase the solubility of co-expressed recombinant proteins by changing the redox state in E. coli cells.

Background: The spread of the COVID-19 disease is the result of an infection caused by the SARS-CoV2 virus. Four crucial proteins, spike (S), membrane (M), nucleocapsid (N), and envelope (E) in coronaviruses have been considered to a large extent.

Objective: This research aimed to express the recombinant protein of a multiepitope immunogen construct and evaluate the immunogenicity of the multiepitope vaccine that was previously designed as a candidate immunogenic against SARS-Cov-2.

Materials and methods: Plasmid pET26b was transferred to the expression host E. coli BL21 (DE3) and the recombinant protein was expressed with IPTG induction. The recombinant protein was purified by Ni-NTA column affinity chromatography, and western blotting was used to confirm it. Finally, mice were immunized with recombinant protein in three doses. Then, the interaction of the 3D structure of the vaccine with the human neutralizing antibodies3D structures (7BWJ and 7K8N) antibody was evaluated by docking and molecular dynamics simulation.

Results: The optimized gene had a codon compatibility index of 0.96. The expression of the recombinant protein of the SARS-Cov-2 vaccine in an E. coli host led to the production of the recombinant protein with a weight of about 70 kDa with a concentration of 0.7 mg/ml. Immunization of mice with recombinant protein of SARS-Cov-2 vaccine-induced IgG serum antibody response. Statistical analysis showed that the antibody titer in comparison with the control sample has a significant difference, and the antibody titer was acceptable up to 1/256000 dilution. The simulation of vaccine binding with human antibodies by molecular dynamics showed that Root Mean Square Deviation (RMSD), Root Mean Square Fluctuation (RMSF), Radius of Gyration, and H-bond as well as van der Waals energies and electrostatic of Molecular mechanics Poisson- Boltzmann surface area (MM/PBSA) analysis have stable interaction.

Conclusion: This recombinant protein can probably be used as an immunogen candidate for the development of vaccines against SARS-CoV2 in future research.

Background: The transformation of proteins from their native conformation into highly ordered fibrillar structures due to their misfolding and aggregation under particular conditions are described as beta-sheet enriched amyloid fibrils. The accumulation of these fibrils in different body parts is the major cause of several neurological and non-neurological conditions (proteinopathies).

Objectives: To prevent these proteinopathies, inhibition of protein aggregation is considered a promising strategy. Therefore, in this study, we synthesized montmorillonite (MMT) based poly- orthophenylenediamine (PoPD) nanocomposites (NCs) and characterized their size and morphology due to their remarkable biological properties. Further, the effect of these nanocomposites on inhibition of fibril formation was assessed.

Methods: These nanocomposites were evaluated for their anti-amyloidogenic potential on two model proteins of amyloidopathies, i.e., human lysozyme and human serum albumin (HL & HSA), by using several biophysical methods, such as Thioflavin T (ThT) and 1-anilino-8-naphthalene sulfonate (ANS) fluorescence, congo red dye binding assay (CR). Secondary structural content was evaluated by Circular dichroism (CD) spectroscopy.

Results: Results demonstrated that synthesized nanocomposites significantly inhibited fibril formation in dose-dependent manner that corresponds to their ability to arrest fibrillation. It is suggested that they may adsorb proteins to protect them against aggregation when they are subjected to aggregating conditions.

Conclusion: This study offers an opportunity to understand the mechanism of inhibition of fibril formation by nanocomposites, showing that they inhibit amyloid formation and amyloid diseases. Thus, the study concludes that these nanocomposites are promising candidates as therapeutic molecules for proteinopathies and are envisaged to enrich the area of personalized medicine, augmenting the human healthcare system.

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The high global burden of tuberculosis (TB) and the increasing emergence of the drugresistant (DR) strain of Mycobacterium tuberculosis (Mtb) emphasize the urgent need for novel antimycobacterial agents. Antimicrobial peptides (AMPs) are small peptides widely existing in a variety of organisms and usually have amphiphilic cationic structures, which have a selective affinity to the negatively charged bacterial cell wall. Besides direct bactericidal mechanisms, including interacting with the bacterial cell membrane and interfering with the biosynthesis of the cell wall, DNA, or protein, some AMPs are involved in the host's innate immunity. AMPs are promising alternative or complementary agents for the treatment of DR-TB, given their various antibacterial mechanisms and low cytotoxicity. A large number of AMPs, synthetic or natural, from human to bacteriophage sources, have displayed potent anti-mycobacterial activity in vitro and in vivo. In this review, we summarized the features, antimycobacterial activity, and mechanisms of action of the AMPs according to their sources. Although AMPs have not yet met the expectations for clinical application due to their low bioavailabilities, high cost, and difficulties in large-scale production, their potent antimycobacterial activity and action mechanisms, which are different from conventional antibiotics, make them promising antibacterial agents against DR-Mtb in the future.

Introduction: Sensitive methods are necessary to identify the residual structure in an unfolded protein, which may be similar to the functionally native structure. Signal intensity in NMR experiments is useful for analyzing the line width for a dynamic structure; however, another contribution is contained.

Methods: Here, the signal-intensity difference along the sequence was used for probability to calculate the standard deviation.

Results: The relative values of the standard deviations were 0.57, 0.57, and 0.66 for alpha-synuclein wild-type, A53T, and A30P, respectively. This revealed that the flexible region was mainly in the Cterminal region of alpha-synuclein at higher temperatures as observed by the amide-proton exchange studies.

Conclusion: In particular, the flexible structure was induced by the A30P mutation.

Necrotrophic phytopathogens pose a serious challenge to the productivity of several crops causing seedling damage, pre- and post-emergence damping-off and root rot thus reducing plant growth and yield. They are known to gain nutrition by secreting a diverse array of hydrolytic enzymes and thereby causing extensive host plant tissue maceration. Amongst the diverse hydrolases, proteases play a pivotal role in the necrotrophic mode of nutrition and thereby in determining pathogenic virulence. Host plants often counteract the necrotrophic proteolysis events by proteins (peptides), particularly through protease inhibitors (PIs). PIs play an important role in host innate immunity function by functioning as anti-metabolic proteins inhibiting the activity of phytopathogenic secretory proteases. Their abundance in plant storage organs explains their anti-nutritional interaction which stalls pathogenic invasion. PIs, therefore, constitute potential candidates that can be deployed as effective antimicrobials in agriculture, particularly against necrotrophic soil-borne pathogens. The present review traces the progress made in the identification of PIs from plants, and their inhibitory potential against necrotrophic phytopathogens and explores prospects of utilizing these molecules as effective anti-necrotrophic formulations for disease management.

Cold-induced RNA-binding protein (CIRP) and RNA-binding motif protein 3 (RBM3) have recently been reported to be involved in cold stress in mammals. These proteins are expressed at low levels in various normal cells, tissues, and organs but can be upregulated upon stimulation by multiple stressors. Studies have shown that CIRP and RBM3 are multifunctional RNA molecular chaperones with different biological functions in various physiological and pathophysiological processes, such as reproductive development, the inflammatory response, the immune response, nerve injury regulation, and tumorigenesis. This paper reviews recent studies on the structure, localization and correlation of CIRP and RBM3 with reproductive development and reproductive system diseases.

Background: The CRISPR-Cas system is an adaptive immune mechanism for bacteria and archaea to resist foreign invasion. Currently, Cas9 and Cpf1 have been widely studied and applied in gene editing. C2c1 is a newly discovered CRISPR-Cas system endonuclease. It has broad application prospects due to its small molecular weight and high substrate recognition specificity.

Objectives: Bacillus thermoamylovorans C2c1(BthC2c1) was expressed in E. coli C43 (DE3) competent cells, purified, and the BthC2c1-sgRNA-dsDNA complex was assembled. The effect of temperature on the cleavage ability of the BthC2c1 system was investigated.

Methods: The cDNA of BthC2c1 was cloned into the vector pGEX-6P-1. BthC2c1 was expressed in E. coli C43(DE3) cells and purified using a GST affinity column and FPLC. The sgRNAs were transcribed and purified in vitro, and the complexes were assembled by gel filtration chromatography. The enzyme cleavage activity of BthC2c1 at different temperatures was investigated using an in vitro cleavage assay. Microscale Thermophoresis detected the affinity of the BthC2c1-sgRNA complexes to substrate DNA.

Results: BthC2c1 proteins were prokaryotically expressed and purified. The complex of BthC2c1 with sgRNA and dsDNA was assembled. In vitro cleavage assay results showed that BthC2c1 cleaved the target DNA at temperatures ranging from 37°C to 67°C. The cleavage ability of BthC2c1 at 42^oC was stronger than that at 37^oC. The results of affinity detection showed that the affinity between the BthC2c1-sgRNA complex and ds36/36 at 42^oC was stronger than that at 37^oC.

Conclusion: In this study, BthC2c1 was expressed, purified, and assembled into a complex with sgRNA and dsDNA. BthC2c1 cleaved DNA within the temperature range of 37^oC to 67^oC. The affinity of BthC2c1-sgRNA to DNA at 42°C was significantly enhanced than that at 37°C. It may be related to its stringent substrate recognition pattern, which differs from Cas9 and Cpf1. The temperature-dependent affinity changes of substrate binding may be part of the reason for the stronger cleavage activity of BthC2c1 at 42^oC. This study may provide an experimental basis for optimizing and modifying the C2c1 gene editing system.