Objective: In this study, we constructed a model for evaluating the binding of hepatitis E virus (HEV) mutated capsid proteins to cells in a way that allowed us to evaluate the role of various amino acid sites in the cell binding process. This method helps us understand the invasion mechanism of HEV and lays a foundation for studying the receptor of HEV. Methods: We used a genotype IV recombinant virus-like particle-like antigen called D66, which contains the key domain of a.a.459-606.We performed site-specific mutations of amino acids at key D66 domain sites, and then confirmed the normal conformation of all mutated proteins by SDS-PAGE and ELISA methods. After confirming the conformation of the mutated proteins, we analyzed the effect of these mutated proteins on adsorption by flow cytometry to find out the key sites that affect the absorption of virus into the cell. Results: We successfully prepared and ide.pngied 45 correctly folded and conformationally normal mutant proteins by polymerase chain reaction site-specific mutagenesis. ELISA results showed that the single point mutation of recombinant viral particle-like antigen D66 did not affect the conformation of the protein. In the application of the model to simulate the binding process of mutant proteins to cells, we found that the mutant proteins of T484A, S488A, T489A, P491A, R512A, Y561A, N562A and T585A significantly affected their adsorption of C3A. Conclusion: In this study, a model was established to evaluate the cell binding of HEV capsid protein, and it was found that alanine mutations at T484A, S488A, T489A, P491A and R512A significantly weakened the cell binding of HEV capsid proteins. These sites may be the key sites that affect HEV binding to cells.
Objective: To study the changes of protein methylation in mouse pituitary tumor cell (AtT20) after exposure to cold, and to efficiently enrich and ide.pngy the methylated peptide of AtT20 cells. Methods: Trypsin and Peptide-N-Glycosidase F (PNGase F) were used to digest protein at the same time. Methylated peptides were enriched with the tip of hydrophilic interaction liquid chromatography (HILIC). Finally, the methylated peptides were analyzed by mass spectrometry. The PhosphoSitePlus® database was used to find new methylation sites and proteins, and the DAVID database was used to perform GO function enrichment and KEGG pathway enrichment analysis. Finally, the difference analysis was performed on the methylation peptides and methylation sites obtained from the two groups of cells. Results: 55 methylation proteins and 83 methylation sites were ide.pngied, of which 78.3% were unreported. The ide.pngied methylation proteins are mainly distributed in nuclear chromosomes, nucleosomes and nuclei, and they participate in the regulation of gene silencing, RNA splicing, mRNA processing and other biological processes. The enrichment of KEGG pathway indicates that these proteins are related to neutrophil exocytosis, alcoholism and systemic lupus erythematosus. The difference analysis of methylation peptides and methylation sites showed that there were 7 significantly changed methylation peptides and 2 significantly changed sites. Conclusion: The changes of protein methylation in AtT20 cells after short-term exposure to cold are not significant. For the first time, comprehensive ide.pngication of methylated proteins in AtT20 cells was carried out, and several unreported methylated proteins and methylation sites were found,which may provide new insights into the study of AtT20 cells.
Objective: To investigate the mechanism of steroid receptor coactivator 3 (SRC3) on renal dysfunction and podocyte injury in the diabetic kidney disease (DKD). Methods: Streptozotocin (STZ) was injected into C57BL/6 mice to induce DKD disease model. Fasting blood glucose (FBG), serum creatinine (Scr), blood urea nitrogen (BUN), 24-hour urine and urine albumin creatinine ratio (UACR) were detected to assess renal function. The glomerular pathology change and glomerular ultrastructure were observed by HE, PAS staining and transmission electron microscopy (TEM). Immunofluorescence was used to observe the relationship between SRC3 and glomerular injury in DKD patients and mice, and Western blot was used to detect the expression levels of SRC3, Podocin and WT1 in DKD glomeruli to verify the relationship between SRC3 and DKD mouse. SRC3-/- DKD mouse model was induced by STZ injection, and the effect of SRC3 on kidney injury was investigated by renal function, pathological structure, and ultrastructural changes compared with WT DKD mice. Western blot and immunofluorescence were used to detect the levels of SRC3 and podocyte markers WT1, Podocin and Nephrin in SRC3-/- DKD and WT DKD mice. Next, SRC3-silenced RNA and SRC3-overexpressed podocytes were constructed, and the negative control (NC), high glucose group (HG), high glucose+SRC3 silenced group (HG+si-SRC3), and overexpression of SRC3 group (SRC3OE) were set. Western blot was used to detect the levels of SRC3, WT1, Podocin and apoptosis-related protein Caspase3, and immunofluorescence was used to detect the levels of SRC3 in podocytes under HG environment. To verify the effect of SRC3 on DKD glomerular injury under high glucose environment, all the groups including Normal, HG, HG+si-SRC3 and SRC3OE were set to determine the relationship between SRC3 and the expression of inflammatory cytokines by detecting the levels of TNF-α and IL-1β of supernatant in both podocyte and mesangial cells. Results: Compared with WT group, FBG, BUN and Scr in WT+STZ group were significantly increased(P<0.001, P<0.001, P<0.001). The 24 h urine volume and UACR of WT DKD mice were the highest at 12 weeks(P<0.001, P<0.001). Glomerular injury shows glomerular sclerosis, mesangial hyperplasia and glomerular glycogen accumulation (P<0.01, P<0.01). The ultrastructure of the glomeruli showed that the podocyte process effaced obviously and the basement membrane thickness increased(P<0.001, P<0.001). The level of SRC3 in local glomeruli was significantly increased in DKD patients(P<0.001). Compared with WT mice, the levels of WT1 and Podocin in WT DKD mice were decreased (P<0.05, P<0.05), while the level of SRC3 was increased(P<0.05). Immunofluorescence also showed that SRC3 level was increased (P<0.001) while WT1 level was decreased (P<0.05). Compared with WT DKD mice, SRC3-/- DKD mice had no significant changes in FBG, but Scr and BUN were decreased (P<0.01, P<0.05). The 24 h urine volume of SRC3-/- DKD mice decreased significantly at 12 weeks (P<0.05). UACR was at a lower level in SRC3-/- DKD mice at 12 weeks (P<0.001). Furthermore, the glomerular injury was alleviated in SRC3-/- DKD mice. Western blot showed that SRC3-/- DKD mice had almost no expression of SRC3(P<0.001), while levels of WT1 and Podocin were significantly increased (P<0.05, P<0.01). Immunofluorescence also showed a negative correlation between SRC3 and Nephrin (P<0.001, P<0.05). In vitro, within high glucose (HG), the levels of SRC3 and Caspase3 were increased (P<0.05, P<0.01), while the levels of WT1 and Podocin were decreased (P<0.01, P<0.05). The levels of SRC3 and Caspase3 were significantly decreased in group HG+si-SRC3 (P<0.01, P<0.05), while WT1 and Podocin were increased (P<0.05, P<0.05). The level of SRC3 in SRC3OE was significantly increased (P<0.001), but there were no significant changes in podocyte markers and Caspase3 compared with NC group. Immunofluorescence also showed that SRC3 was significantly increased in podocytes in the high glucose environment(P<0.01). In podocytes and mesangial cells, the levels of TNF-α and IL-1β in HG group were significantly higher than those in Normal group (P<0.001, P<0.001). But all of them are decreased in HG+si-SRC3 (P<0.01, P<0.01). There was no significant difference between Normal and SRC3OE. Conclusion: High expression levels of SRC3 were found in glomeruli of DKD patients and mice, and podocytes in the high glucose environment, confirming that SRC3 is positively correlated with glomerular injury in DKD. In addition in vitro experiments show that SRC3 could promote the production of inflammatory cytokines in podocytes and mesangial cells only under a high glucose environment.
Objective: A.pngicial compartmentalization is among the important approaches in metabolic engineering and synthetic biology. Due to the insufficient evaluation of compartmentalization in lipid droplets (LDs), the aim of this work is to ide.pngy efficient LD localization signals in the oleaginous yeast Xanthophyllomyces dendrorhous. Methods: Based on the three-dimensional structure and the predicted transmembrane domain (TMD) of sesame oleosin, LD localization signals were predicted and fused with eGFP to generate a series of expression vectors, which were used to transform X. dendrorhous. Green and orange fluorescence was observed and used to assess the efficiency of the predicted LD localization signals. Results: The intact oleosin protein Ols1(1-145) and truncated peptides Ols2(1-63), Ols4(32-63), and Ols5(44-63) successfully guided eGFP is localization in LDs, while truncated peptide Ols3(64-145) was ineffective and most eGFP proteins were expressed in cytoplasm. Conclusion: A short LD localization signal Ols5(44-63) consisting of 19 amino acids was ide.pngied, which can precisely lead the eGFP to localize inside the LDs.
Objective: To improve the soluble expression level of human interleukin-11 in prokaryotic expression system and enhance its circulating half-life in vivo. Methods: The recombinant protein expression vector (PBV220-ABD-rhIL11) was constructed, and it was successfully expressed in prokaryotic expression system in soluble form. Based on hydrophobic chromatography, ion exchange chromatography and gel chromatography, a method for purification of ABD-rhIL-11 from the supernatant of bacteria was successfully established. The circular dichroism spectrum and fluorescence emission spectrum analysis confirmed that the spatial structure of the purified ABD-rhIL-11 was folded correctly. In vivo and in vitro activity and pharmacodynamic evaluation was carried out. Results: The purity of recombinant fusion protein was higher than 95%. The results of in vitro binding experiments showed that ABD-rhIL-11 could rapidly bind to human serum albumin. The results of cell proliferation experiment showed that the activity of ABD-rhIL-11 was equivalent to that of the control group. Pharmacokinetic results showed that the fusion protein could significantly prolong the circulating half-life of interleukin-11 in rats. The results of platelet count test showed that ABD-rhIL-11 could effectively stimulate platelet proliferation in rats. Conclusion: The results of this study would provide a new idea for the long-term design of interleukin-11 drugs based on albumin binding peptide fusion strategy.
Objective: Mercury is a highly toxic and widespread pollutant in the environment, which can cause harm to human body even in low concentration. Based on the T-Hg2+-T base mismatch principle, a new biosensor was constructed to detect the concentration of Hg2+ in water with high sensitivity and selectivity. Methods: A new Gelred/TRO biosensor was constructed by using thymine rich oligonucleotide (TRO) as the specific Hg2+ recognition probe, and Gelred as the fluorescence indicator probe. The sensor is used to detect Hg2+ in water. The influence of many factors on the detection system was studied. Results: Under the optimized detection conditions, the linear range of Hg2+ detection was 10 ~ 800 nmol/L, which was fitted as follows: y=0.005 96x+0.881 44 (r2=0.991 47). The detection limit was 0.14 nmol/L. The presence of ten interfering ions such as Fe2+, Co2+, Cu2+, K+, Mg2+, Ca2+, Ag+, Cl-,
Protein assembly technology has developed rapidly and is widely used in biocatalysis, biosensors, and drug release. It has become an important part of bioengineering. Due to the complexity of protein-protein interactions and insufficient understanding of protein folding and molecular recognition, designing complex protein assemblies is very challenging. With the continuous development of computer technology and molecular simulation, researchers have gradually realized the precise design of protein assemblies with atomic-and molecular-level accuracy, predicted the structure of protein assemblies, and further designed catalytic sites on protein assemblies to obtain a.pngicial biocatalysis of assembled enzymes. In recent years, a.pngicial intelligence technologies such as machine learning have also been applied to protein assembly design, contributing to the development of protein assembly research. Here, the research progress of computational simulation technology in design of protein assembly, prediction of assembly structure, and catalytic site design and its application in new enzyme design, drug release, biosensor and other areas are reviewed, in order to guide the design and optimization of protein assembly for more different application fields through basic theoretical research.
RNA-binding proteins (RBPs) can bind RNAs through RNA-binding domains and regulate the fate or functions of their bound RNAs. RBPs are responsible for all the steps of RNA metabolism throughout their life cycle and are involved in many other cellular processes related to cell’s survival, replication, and adaption to environmental changes. RNA-protein interactions are essential to cell homeostasis, and their defects are associated with various diseases. Therefore, the characterization of RBPs is crucial to depict their functions and underlying molecular mechanisms. Recently, several methods have been developed and extensively applied to ide.pngy RBPs, uncovering many previously unannotated RBPs. However, the majority of the newly discovered RBPs lack classical RNA-binding domains, thus highlighting the complexity and diversity of RNA-protein interactions. Here, the progress in the ide.pngication of RBPs based on mass spectrometry was reviewed, from two aspects: high-throughput ide.pngication and targeted ide.pngication. Moreover, their technical principles, application areas, and strengths and limitations were discussed, providing new perspectives to better understand the biological significance and clinical implications of RNA-protein interactions.
Presently, to design epitope vaccines, the research and development process is generally to use computer-aided immunoinformatics tools and related technical methods to analyze acquired or known nucleotide and amino acid sequences to determine and pre-screen out possible dominant epitopes, and then prepare polypeptide vaccines with dominant epitopes through synthetic or genetic engineering techniques. The rapid development of immunoinformatics has been successfully applied to the field of vaccinology, and the immunoinformatics method is the most effective method to develop vaccines based on epitope polypeptide. Immunoinformatics, the general process of immunoinformatics in epitope vaccine design and validation, the immunoinformatics tools involved in the design of epitope vaccines, and the specific application of immunoinformatics in the design of epitope vaccines are reviewed, which will provide reference for reasonable design and development of effective epitope vaccines.
Early screening and diagnosis of cancer is an effective means to reduce its incidence rate and mortality. Breast cancer is the most common malignant tumor among women, and has surpassed lung cancer to become the tumor with the highest incidence rate among women. With the innovation of electrochemical technology and the advantages of biosensors such as high sensitivity, rapid detection, simple operation and low cost, electrochemical biosensors are widely used in the detection of breast cancer markers. This review first summarizes the classification of common breast cancer biomarkers, and then summarizes and discusses the application of electrochemical biosensors in tumor markers of breast cancer at home and abroad, with electrochemical related immunosensors and aptamer sensors as the classification, and discusses their development prospects.
Nanodrug delivery systems are widely used in disease treatment due to their special physical and chemical properties. However, the traditional synthetic nanoparticles cannot cross the natural barrier, and it is difficult to escape immune surveillance, so they are not effective in clinical applications. Biomimetic nanoparticles are obtained by modification of natural materials or by chemical methods imitating the key characteristics of biological structures. They are similar to biologically related structures in chemistry, physics or morphology. Therefore, they exhibit excellent intelligent delivery performance in inert rejection, barrier overcoming and active effects, and can be used for more efficient and safe drug delivery. Biomimetic nano-drug delivery systems are divided into four categories: nano-drug delivery systems designed based on natural biological macromolecules, drug delivery systems related to cells and cell membranes, drug delivery systems of extracellular vesicles such as extracellular secretions, and nano drug delivery systems mimicking biological structures. The design principle of biomimetic nanoparticles used for drug delivery is summarized, the preparation methods of four kinds of biomimetic nanoparticle drug delivery systems are elaborated, the research progress of their application in rare diseases, neurodegenerative diseases, tumors, antiviral vaccine research and development and other targeted therapies is summarized, and the clinical application challenges, potential solutions and future research directions in this field are also discussed.
Butenyl-spinosyn, a macrolide antibiotic produced by Saccharopolyspora pogona, is a green, environmentally-friendly novel bio-pesticide with high application value in pest control and grain storage. However, the low yield of the wild-type strain has limited the application of this compound; therefore, high-yield strain breeding becomes a question that needs to be solved urgently. In this paper, new ideas of genetic breeding for increasing butenyl-spinosyn in recent years were summarized. The studies on increasing butenyl-spinosyn yield were reviewed, based on the modification of carbon, nitrogen and phosphate metabolic pathways as well as transcriptional regulation of S.pogona, and the prospect of strain breeding through heterologous expression was also discussed, in order to provide effective reference for the construction and optimization of butenyl-spinosyn high yielding strains.
