In Silico Characterization of African Swine Fever Virus Nucleoprotein p10 Interaction with DNA.

African swine fever virus (ASFV) is the etiological agent of a highly contagious, hemorrhagic infectious swine disease, with a tremendous sanitary and economic impact on a global scale. Currently, there are no globally available vaccines or treatments. The p10 protein, a structural nucleoprotein encoded by ASFV, has been previously described as capable of binding double-stranded DNA (dsDNA), which may have implications for viral replication. However, the molecular mechanism that governs this interaction is still unknown, mostly due to the lack of a structural model for this protein. In this work, we have generated an ab initio model of the p10 protein and performed extensive structural characterization, using molecular dynamics simulations to identify the motifs and residues regulating DNA recognition. The helix-turn-helix motif identified at the C-terminal region of the protein was shown to be crucial to the dsDNA-binding efficiency. As with other DNA-binding proteins, two distinct serine and lysine-rich regions found in the two helices were identified as key players in the binding to DNA, whose importance was later validated using experimental binding assays. Altogether, these findings may contribute to a better understanding of the p10 function in ASFV replication.

Thermal destabilization mechanism of cytochrome c' from psychrophilic Shewanella violacea.

Cytochrome c' is a nitric oxide (NO)-binding heme protein found in Gram negative bacteria. The thermal stability of psychrophilic Shewanella violacea cytochrome c' (SVCP) is lower than those of its homologues from other 2 psychrophilic Shewanella species, indicating that thermal destabilization mechanism for low-temperature adaptation accumulates in SVCP. In order to understand this mechanism at the amino acid level, here the stability and function of SVCP variants, modeled using the 2 homologues, were examined. The variants exhibited increased stability, and they bound NO similar to the wild type. The vulnerability as to the SVCP stability could be attributed to less hydrogen bond at the subunit interface, more flexible loop structure, and less salt bridge on the protein surface, which appear to be its destabilization mechanism. This study provides an example for controlling stability without spoiling function in psychrophilic proteins.

Albumin binding function is a novel biomarker for early liver damage and disease progression in non-alcoholic fatty liver disease.

PURPOSE: Indicators to assess early liver damage and disease progression in nonalcoholic fatty liver disease (NAFLD) remain unsatisfactory. Albumin binding function has been reported to be an early indicator of liver damage in hepatitis and liver cirrhosis. However, its role in NAFLD patients is unknown. METHODS: An age/sex-matched, case-control study was performed. Albumin-binding capacity (ABiC) and albumin metal ion binding ability, assessed by ischemia modified albumin (IMA), were measured. Correlation analysis was performed to assess the association of albumin binding function with liver function enzymes and noninvasive liver fibrosis markers. RESULTS: A total of 80 NAFLD patients and 41 healthy controls were included. Albumin binding function was significantly lower in NAFLD (ABiC: 196.00%, p < 0.001; IMA transformed (IMAT): 0.461, p < 0.001; and IMAT/albumin: 0.947 × 10-2, p < 0.001) than controls (ABiC: 211.00%; IMAT: 0.575; and IMAT/albumin: 1.206 × 10-2). Albumin binding function was also found to be significantly different among healthy participants and different severity groups of NAFLD (p < 0.001). Besides, albumin binding function showed positive correlation with BMI (ABiC: r = -0.247, p = 0.011; IMAT: r = -0.243, p = 0.013; IMAT/albumin: r = -0.254, p = 0.009) and FIB-4 index (ABiC: r = 0.230, p = 0.029). The ROC curve suggested that albumin binding function combined with BMI and triglyceride may predict the presence of NAFLD (area under ROC (AUROC) = 0.935, p < 0.001). CONCLUSION: Our findings suggest albumin binding function is a novel biomarker for early liver damage and disease progression in NAFLD.

Recognizing Proteins with Binding Function in Elymus nutans Based on Machine Learning Methods.

BACKGROUND: We research the binding function proteins in Elymus nutans. Recognition for proteins is essential for study of biology. Machine learning methods have been widely used for the prediction of proteins. METHODS: We used BLAST software for the function annotations of Elymus nutans. Besides, we used machine learning methods to recognize proteins which are not annotated by the software. In the process, we focused on identifying the proteins with binding functions. In our research, features are extracted by four algorithms, and then selected by mutual information estimator. Here three classifiers are constructed based on K-nearest neighbour algorithm and gradient boosting algorithm. RESULTS AND CONCLUSION: Experimental results show that there are 848 proteins with ATP binding function, 113 proteins with heme binding function, 315 proteins with zinc-ion binding function, 135 proteins with GTP binding function and 21 proteins with ADP binding function. Furthermore, we have successfully predicted the functions of 10 special protein sequences whose function annotations cannot be obtained by making sequence alignment with seven famous protein databases. Among them, seven sequences have ATP binding functions, one sequence has heme binding function, one sequence has zinc-ion binding function and the other one has GTP binding function.

Alternative binding sites at the vitamin D receptor and their ligands.

In recent decades, the majority of ligands developed for the vitamin D receptor (VDR) bind at its deeply buried genomic ligand binding pocket. Theses ligands can be categorized into agonists and partial agonists/antagonists. A limited number of ligands, most of them peptides, bind the VDR‒coactivator binding site that is formed in the presence of an agonist and inhibit coactivator recruitment, and therefore transcription. Another solvent exposed VDR‒ligand binding pocket was identified for lithocholic acid, improving the overall stability of the VDR complex. Additional proposed interactions with VDR are discussed herein that include the alternative VDR‒ligand binding pocket that may mediate both non-genomic cellular responses and binding function 3 that was identified for the androgen receptor. Many VDR ligands increase blood calcium levels at therapeutic concentrations in vivo, thus the identification of alternative VDR‒ligand binding pockets might be crucial to develop non-calcemic and potent ligands for VDR to treat cancer and inflammatory disease.

Recognition mechanism of Wilms' tumour suppressor protein and DNA triplets: insights from molecular dynamics simulation and free energy analysis.

The Wilms' tumour suppressor protein (WT1) plays a multifaceted role in human cancer processes. Mutations on its DNA recognition domain could lead to Denys-Drash syndrome, and alternate splicing results in insertion of the tripeptide Lys-Thr-Ser (KTS) between the third and fourth zinc fingers (ZFs), leading to changes in the DNA-binding function. However, detailed recognition mechanisms of the WT1-DNA complex have not been explored. To clarify the mutational effects upon WT1 towards DNA binding at the atomic level, molecular dynamics simulations and the molecular mechanics/Poisson Boltzmann surface area (MM/PBSA) method were employed. The simulation results indicate that mutations in ZF domains (E427Q and Q369H) may weaken the binding affinity, and the statistical analyses of the hydrogen bonds and hydrophobic interactions show that eight residues (Lys351, Arg366, Arg375, Arg376, Lys399, Arg403, Arg424 and Arg430) have a significant influence on recognition and binding to DNA. Insertion of the tripeptide KTS could form an immobilized hydrogen-bonding network with Arg403, affecting the flexibility and angle of the linker between ZF3 and ZF4, thus influencing the recognition between the protein and the DNA triplet at its 5' terminus. These results represent the first step towards a thorough characterization of the WT1 recognition mechanisms, providing a better understanding of the structure-function relationship of WT1 and its mutants.

UCH-L1 Inhibition Decreases the Microtubule-Binding Function of Tau Protein.

Ubiquitin C-terminal hydrolase L1 (UCH-L1) is critical for protein degradation and free ubiquitin recycling. In Alzheimer's disease brains, UCH-L1 is negatively related to neurofibrillary tangles whose major component is hyperphosphorylated tau protein, but the direct action of UCH-L1 on tau has not been reported. In the current study, mouse neuroblastoma Neuro2a (N2a) cells were treated by the different concentrations of UCH-L1 inhibitor LDN (2.5, 5 and 10 µM) to inhibit the hydrolase activity of UCH-L1. In addition, we also used UCH-L1 siRNA to treat the HEK293/tau441 cells to decrease the expression of UCH-L1. After LDN and UCH-L1 siRNA treatment, we used immunofluorescence, immunoprecipitation, and tau-microtubule binding assay to measure the microtubule-binding ability and post-translational modifications of tau protein. All the results presented that both inhibition of the activity and expression of UCH-L1 induced the decreased microtubule-binding ability and increased phosphorylation of tau protein. Abnormal aggregation and ubiquitination of tau protein was also observed after UCH-L1 inhibition. The above results suggested that aggregation of tau protein might be devoted to the abnormal post-translational modifications of tau protein. Our study first indicates that dysfunction of UCH-L1 most likely affected normal biological function of tau protein through decreasing degradation of ubiquitinated and hyperphosphorylated tau.

Recent Developments in Androgen Receptor Antagonists.

The androgen receptor (AR), a ligand-dependent transcription factor that regulates the expression of a series of downstream target genes after the binding of androgens, has been a target for the discovery of drugs used to treat prostate cancer. Prostate cancer always progresses to castration-resistant prostate cancer after a period of androgen deprivation therapy. Thus, developing potent androgen receptor antagonists for the therapy of castration-resistant prostate cancer possesses great significance. This review summarizes the preclinical development of androgen receptor antagonists, conventional androgen receptor antagonists that competitively bind to the ligand binding domain of the androgen receptor and coactivator antagonists of the androgen receptor, including both activation function-2 antagonists and binding function-3 antagonists. We hope that this review can help other researchers find new scaffolds and sites for the treatment of prostate cancer.

Carbonic Anhydrase Glycoinhibitors belonging to the Aminoxysulfonamide Series.

A general approach for the synthesis of carbonic anhydrases glycoinhibitors belonging to an aminoxysulfonamide series is presented using a Ferrier sulfonamidoglycosylation reaction on glycals. All the compounds showed good in vitro inhibitory activity against four human carbonic anhydrase isoforms, with selectivity against the cytosolic (hCA II) vs the tumor associated (hCA IX and XII) enzymes.

Coregulator control of androgen receptor action by a novel nuclear receptor-binding motif.

The androgen receptor (AR) is a ligand-activated transcription factor that is essential for prostate cancer development. It is activated by androgens through its ligand-binding domain (LBD), which consists predominantly of 11 α-helices. Upon ligand binding, the last helix is reorganized to an agonist conformation termed activator function-2 (AF-2) for coactivator binding. Several coactivators bind to the AF-2 pocket through conserved LXXLL or FXXLF sequences to enhance the activity of the receptor. Recently, a small compound-binding surface adjacent to AF-2 has been identified as an allosteric modulator of the AF-2 activity and is termed binding function-3 (BF-3). However, the role of BF-3 in vivo is currently unknown, and little is understood about what proteins can bind to it. Here we demonstrate that a duplicated GARRPR motif at the N terminus of the cochaperone Bag-1L functions through the BF-3 pocket. These findings are supported by the fact that a selective BF-3 inhibitor or mutations within the BF-3 pocket abolish the interaction between the GARRPR motif(s) and the BF-3. Conversely, amino acid exchanges in the two GARRPR motifs of Bag-1L can impair the interaction between Bag-1L and AR without altering the ability of Bag-1L to bind to chromatin. Furthermore, the mutant Bag-1L increases androgen-dependent activation of a subset of AR targets in a genome-wide transcriptome analysis, demonstrating a repressive function of the GARRPR/BF-3 interaction. We have therefore identified GARRPR as a novel BF-3 regulatory sequence important for fine-tuning the activity of the AR.

Thermodynamic effects of multiple protein conformations on stability and DNA binding.

The side-chain conformations of amino acids in the hydrophobic core are important for protein folding and function. A previous NMR study has shown that a mutant protein of transcriptional activator c-Myb, I155L/I181L R3, has multiple conformations and increased fluctuation in comparison with the wild type. To elucidate the quantitative correlation of structural fluctuation with stability and function, we analyzed the thermodynamic effects of I155L and I181L mutations, using R2R3 that encompasses the minimum specific DNA-binding region. Circular dichroism and differential scanning calorimetry measurements showed that the mutation of I155L had little effect on stability, while the I181L mutation significantly destabilized the protein. It is noteworthy that the decreased stability resulting from the I181L mutation was mainly due to decreased enthalpy change, which is partially compensated by decreased entropy change. Isothermal titration calorimetry measurements showed that the specific DNA-binding affinity was decreased owing to the I181L mutation, which was due to decreased binding entropy change. Entropy in the folded state, which corresponds to the DNA-free state, increases due to the I181L mutation because of the increased conformational fluctuation observed in I155L/I181L mutant of R2R3 by CLEANEX-PM NMR analysis, which in turn results in decreased folding entropy and DNA-binding entropy changes.

Enhancement effect of propofol on binding function of GABA_A receptor / 第二军医大学学报

Objective:To investigate the influence of propofol on the binding function of GABA A receptor. Methods: By using radioligand receptor binding assay, effects of propofol on the specific binding of 3H GABA and saturation curves of GABA A receptor were observed in cortical membrane preparations from mouse cerebral cortex. Results: Specific binding experiments showed that propofol at the concentrations of 10 300 ?mol/L markedly enhanced the specific binding of 3H GABA( P 0.05). Conclusion: Clinical concentrations of propofol can enhance the binding function of GABA A receptor through increasing the affinity of the low affinity binding site of GABA.

Effect of Chinese Herbs on ?-Globin Gene Cluster Locus Control Hepersensitive site 2 Site Binding with Nucleoprotein / 实用儿科临床杂志

Objective To explore molecular mechanism and the curative effect of Yisuishengxue powder and its function of the hepersensitive site 2 (HS2) in ?-globin gene cluster locus control region binding with nucleoprotein.Methods After 3 months treatment of Yisuishengxue powder, nucleoprotein was extracted from the morrow cell before and after treatment. The HS2 DNA probes was combined with nucleoproteins.Electrophoresis gel mobile lag was utilized for observing the mobile velocity of DNA segment.Observe the mobile velocity of DNA probes.Results The mobile velocity of probes combined with nucleoproteins before treatment was different form that of the controls, while it was very close to the controls after treatment.Conclusions It is suggested that this compounding medicine might affect the DNA segment of HS2 site in ?-LCR binding with nucleoprotein GATA-1, which may be one molecular mechanism of Chinese herb therapy.