A protocol for high-throughput screening of histone lysine demethylase 4 inhibitors using TR-FRET assay.

Identification of diverse chemotypes of selective KDM4 inhibitors is important for exploring and validating the roles of KDM4s in the pathogenesis of human disease and for developing therapies. Here, we report a protocol for high-throughput screening of KDM4 inhibitors using TR-FRET demethylation functional assay. We describe this protocol for screen of KDM4B inhibitors, which can be modified to screen inhibitors of other JmjC-domain-containing KDMs. For complete details on the use and execution of this protocol, please refer to Singh et al. (2021).

Roles of conserved residues in the receptor binding sites of human parainfluenza virus type 3 HN protein.

Human parainfluenza virus type 3 (hPIV-3) entry and intrahost spread through membrane fusion are initiated by two envelope glycoproteins, hemagglutinin-neuraminidase (HN) and fusion (F) protein. Binding of HN protein to the cellular receptor via its receptor-binding sites triggers conformational changes in the F protein leading to virus-cell fusion. However, little is known about the roles of individual amino acids that comprise the receptor-binding sites in the fusion process. Here, residues R192, D216, E409, R424, R502, Y530 and E549 located within the receptor-binding site Ⅰ, and residues N551 and H552 at the putative site Ⅱ were replaced by alanine with site-directed mutagenesis. All mutants except N551A displayed statistically lower hemadsorption activities ranging from 16.4% to 80.2% of the wild-type (wt) level. With standardization of the number of bound erythrocytes, similarly, other than N551A, all mutants showed reduced fusogenic activity at three successive stages: lipid mixing (hemifusion), content mixing (full fusion) and syncytium development. Kinetic measurements of the hemifusion process showed that the initial hemifusion extent for R192A, D216A, E409A, R424A, R502A, Y530A, E549A and H552A was decreased to 69.9%, 80.6%, 71.3%, 67.3%, 50.6%, 87.4%, 84.9% and 25.1%, respectively, relative to the wt, while the initial rate of hemifusion for the E409A, R424A, R502A and H552A mutants was reduced to 69.0%, 35.4%, 62.3%, 37.0%, respectively. In addition, four mutants with reduced initial hemifusion rates also showed decreased percentages of F protein cleavage from 43.4% to 56.3% of the wt. Taken together, Mutants R192A, D216A, E409A, R424A, R502A, Y530A, E549A and H552A may lead to damage on the fusion activity at initial stage of hemifusion, of which decreased extent and rate may be associated with impaired receptor binding activity resulting in the increased activation barrier of F protein and the cleavage of it, respectively.

Angiogenesis in tissue-engineered nerves evaluated objectively using MICROFIL perfusion and micro-CT scanning.

Angiogenesis is a key process in regenerative medicine generally, as well as in the specific field of nerve regeneration. However, no convenient and objective method for evaluating the angiogenesis of tissue-engineered nerves has been reported. In this study, tissue-engineered nerves were constructed in vitro using Schwann cells differentiated from rat skin-derived precursors as supporting cells and chitosan nerve conduits combined with silk fibroin fibers as scaffolds to bridge 10-mm sciatic nerve defects in rats. Four weeks after surgery, three-dimensional blood vessel reconstructions were made through MICROFIL perfusion and micro-CT scanning, and parameter analysis of the tissue-engineered nerves was performed. New blood vessels grew into the tissue-engineered nerves from three main directions: the proximal end, the distal end, and the middle. The parameter analysis of the three-dimensional blood vessel images yielded several parameters, including the number, diameter, connection, and spatial distribution of blood vessels. The new blood vessels were mainly capillaries and microvessels, with diameters ranging from 9 to 301 µm. The blood vessels with diameters from 27 to 155 µm accounted for 82.84% of the new vessels. The microvessels in the tissue-engineered nerves implanted in vivo were relatively well-identified using the MICROFIL perfusion and micro-CT scanning method, which allows the evaluation and comparison of differences and changes of angiogenesis in tissue-engineered nerves implanted in vivo.

R237 and h238 are key amino acids in the rubella virus E1 neutralization epitope.

Residues 221-239 of rubella virus E1 glycoprotein contain antibody neutralization domains, and the solvent-exposed charged amino acids at the binding interface may be crucial for binding ability. However, the role of charged amino acid residues on the E1 epitope in peptide-antibody binding is unknown. To investigate the role of single amino acid substitutions on the important neutralizing epitope, biolayer interferometry and serological tests were performed. There are three charged residues in the neutralizing epitope: D229, R237, and H238. Substitution of D229 for amino acid A had no influence on the binding activity of the antibody to the peptide. However, substitutions of R237 or H238 for charged amino acid H or R were found to abolish the binding activity. Furthermore, substitution of an uncharged amino acid Q236 for a charged amino acid D was found to reduce the binding activity significantly. Thus, R237 and H238 are key amino acids in the rubella virus E1 neutralization epitope.

Role of N-linked glycosylation of the human parainfluenza virus type 3 hemagglutinin-neuraminidase protein.

Human parainfluenza virus type 3 (hPIV-3) is a major respiratory tract pathogen that affects infants and young children. The hPIV-3 hemagglutinin-neuraminidase (HN) protein is a multifunctional protein mediating hemadsorption (HAD), neuraminidase (NA), and fusion promotion activities, each of which affects the ability of HN to promote viral fusion and entry. The hPIV-3 HN protein contains four potential sites (N308, N351, N485 and N523) for N-linked glycosylation. Electrophoretic mobility analysis of mutated HN proteins indicated that N308, N351 and N523 sites, but not the N485 site in HN protein, were targeted for the addition of glycans in BHK-21 cells. These functional glycosylation sites were systematically eliminated in various combinations from HN to form a panel of mutants in which the roles of individual carbohydrate chains and groups of carbohydrate chains could be analyzed. Removal of individual or multiple N-glycans on the hPIV-3 HN protein had no effects on transport to the cell surface, expression and NA activity. Single glycosylation site mutants (G1, G2 and G4) not only impaired fusion promotion activity but also reduced HAD activity of HN protein, which was even more obvious for all three double mutants (G12, G14 and G24) and the triple mutant (G124). In addition, every mutant protein retained F-interactive capability that was equal to the wild-type protein capability. Interestingly, the F protein that could be co-immunoprecipitated with the G12 mutated protein or immunoprecipitated with anti-F antibody was not efficiently cleaved. For G14, G24 and G124, little cleaved F protein was detected in co-immuoprecipitation F protein assay and its total amounts where in the cell lysates. The mechanism underlying hPIV-3 HN and F protein remained associated before and after receptor engagement and the strength of the HN-receptor interaction modulated the activation of F the protein which could determine the extent of fusion. Finally, we demonstrated that single or multiple N-glycosylation site mutations inhibited fusion at the earliest stages. Taken together, these results indicated that N-glycosylation of hPIV-3 HN is critical to its receptor recognition activity, cleavage of the F protein, and fusion promotion activity, but had no influence on its interaction with the homologous F protein and NA activity.

[Study on functions of N-carbohydrate chains in human parainfluenza virus type 3 hemagglutinin-neuraminidase protein].

To determine the functions of N-carbohydrate chains in human parainfluenza virus type 3 hemagglutinin-neuraminidase(HN) protein, a PCR-based site-directed mutagenesis method was used to obtain N-glycan mutants. Protein electrophoresis rate, cell surface expression,receptor binding activity, neuraminidase activity and cell fusion promotion activity were determined. The HN proteins of single mutants (G1, G2, and G4) and multiple mutants (G12, G14, G24 and G124) migrated faster than the wild-type (wt) HN protein on polyacrylamide gels, while G3-mutated protein and wt HN protein migrated at the same position. There was no statistic difference in cell surface expression and neuraminidase activity between wt and each mutant HN protein (P>0.05), but receptor binding activity and cell fusion promotion activity of each mutant protein was reduced to significant extent (P<0.05). G1, G2 and G4 mutants exhibited re duced receptor binding activity, which was 83.94%, 76.45% and 55.32% of the wt level, respectively. G1, G2 and G4-mutated proteins also showed reductions in fusion promotion activity, which was 80.84%, 77.83% and 64.16%, respectively. Multiple mutants with G12-, G14-, G24- and G124- substitutions could further reduce receptor binding activities, 33.07%, 20.67%, 19.96% and 15.11% of the wt HN level, respectively. G12, G14, G24 and G124 mutants exhibited levels of fusion promotion activity that were only 46.360, 12.04%, 13.43% and 4.05% of the wt amount, respectively. As N-glycans of hPIV3 HN protein play an important role in receptor binding activity and cell fusion promotion activity of HN protein. We propose that the loss of N-glycans change the conformation or orientation of globular domain that is responsible for receptor binding and lower receptor binding activity and cell fusion promotion activi ty.

[Progress on mechanism of cell apoptosis induced by rubella virus].

Rubella virus (RV), a member of the family Togaviridae, can induce apoptosis of host cells in vitro. Protein kinases of the Ras-Raf-MEK-ERK pathway and PI3K-Akt pathway play essential roles in virus multiplication, cell survival and apoptosis. Proteins p53 and TAp63 that bind to specific DNA sequences stimulate Bax in a manner to produce functional pores that facilitate release of mitochondrial cytochrome c and downstream caspase activation. In this review, the molecular mechanisms of RV-induced cell apoptosis, including RV-infected cell lines, pathological changes in cell components and apoptosis signaling pathways are summarized.

[Expression and bioassay of rubella virus E1-374 glycoprotein in yeast cells].

OBJECTIVE: To express the rubella virus E1-374 glycoprotein in Pichia pastoris and study the immunogenecity of the recombinant protein. METHODS: The cDNA of protein E1-374 was cloned into the expression vector pGAPZalphaA and transformed into Pichia pastoris GS115 cells by electrotransfection. The expressed protein was confirmed by indirect immunofluorescence and demonstrated immunoreactivity by Western Blot. Rubella virus IgG antibody was assayed with ELISA after mice were inmmunized by E1-374 glycoprotein. RESULTS: SDS-PAGE analysis and Western Blot analysis of E1-374 protein revealed this protein to be 46.89 x 10(3). Antiserum (1:100) and E1-374 (5.5 microg/ml) was chosen for ELISA optimization. The intra-assay coefficient of variation for the ELISA was 0.36%-12.45%. CONCLUSION: Protein E1-374 was highly expressed in Pichia pastoris cells, and it was a good choice to prepare rubella virus recombinant protein vaccines.