8 research outputs found

    MOESM1 of Regioselectivity of oxidation by a polysaccharide monooxygenase from Chaetomium thermophilum

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    Additional file 1: Figure S1. SDS-PAGE of the purified Cu2+-CtPMO1 produced in Pichia pastoris. Figure S2. The N-terminal amino acid sequence analysis of CtPMO1 using LC-MS/MS. Figure S3. MALDI-TOF-MS/MS analysis of m/z 525 from MALDI-TOF-MS analysis. Figure S4. Types of fragmentation of CtPMO1 C4- and C6-oxidized products (m/z 525). Figure S5. 1H NMR spetra of CtPMO1 soluble reaction products with PASC as substrate in DMSO-d 6 . Figure S6. Sequence alignment of CtPMO1 and NCLPMO9C using ClastalW2. Figure S7. Homology model of the catalytic domain of CtPMO1 using SWISS-MODEL. Figure S8. Homology model of CtPMO1 binding with cellopentaose. Figure S9. Identification of the mutated CtPMO1 soluble reaction products oxidized by Br2 using with PASC as substrate MALDI-TOF-MS. Table S1. List of primers used for PCR of the CtPMO1 protein. Table S2. Fragmentation analysis of the peak of DP3-2 (m/z 525) according to Additional file 1: Figure S3, S4

    Exploring the Molecular Mechanism of Cross-Resistance to HIV‑1 Integrase Strand Transfer Inhibitors by Molecular Dynamics Simulation and Residue Interaction Network Analysis

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    The rapid emergence of cross-resistance to the integrase strand transfer inhibitors (INSTIs) has become a serious problem in the therapy of human immunodeficiency virus type 1 (HIV-1) infection. Understanding the detailed molecular mechanism of INSTIs cross-resistance is therefore critical for the development of new effective therapy against cross-resistance. On the basis of the homology modeling constructed structure of tetrameric HIV-1 intasome, the detailed molecular mechanism of the cross-resistance mutation E138K/Q148K to three important INSTIs (Raltegravir (RAL, FDA approved in 2007), Elvitegravir (EVG, FDA approved in 2012), and Dolutegravir (DTG, phase III clinical trials)) was investigated by using molecular dynamics (MD) simulation and residue interaction network (RIN) analysis. The results from conformation analysis and binding free energy calculation can provide some useful information about the detailed binding mode and cross-resistance mechanism for the three INSTIs to HIV-1 intasome. Binding free energy decomposition analysis revealed that Pro145 residue in the 140s 1oop (Gly140 to Gly149) of the HIV-1 intasome had strong hydrophobic interactions with INSTIs and played an important role in the binding of INSTIs to HIV-1 intasome active site. A systematic comparison and analysis of the RIN proves that the communications between the residues in the resistance mutant is increased when compared with that of the wild-type HIV-1 intasome. Further analysis indicates that residue Pro145 may play an important role and is relevant to the structure rearrangement in HIV-1 intasome active site. In addition, the chelating ability of the oxygen atoms in INSTIs (e.g., RAL and EVG) to Mg<sup>2+</sup> in the active site of the mutated intasome was reduced due to this conformational change and is also responsible for the cross-resistance mechanism. Notably, the cross-resistance mechanism we proposed could give some important information for the future rational design of novel INSTIs overcoming cross-resistance. Furthermore, the combination use of molecular dynamics simulation and residue interaction network analysis can be generally applicable to investigate drug resistance mechanism for other biomolecular systems

    Mass spectrometry-based quantification of the cellular response to ultraviolet radiation in HeLa cells

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    <div><p>Ultraviolet (UV) irradiation is a common form of DNA damage that can cause pyrimidine dimers between DNA, which can cause gene mutations, even double-strand breaks and threaten genome stability. If DNA repair systems default their roles at this stage, the organism can be damaged and result in disease, especially cancer. To better understand the cellular response to this form of damage, we applied highly sensitive mass spectrometry to perform comparative proteomics of phosphorylation in HeLa cells. A total of 4367 phosphorylation sites in 2100 proteins were identified, many of which had not been reported previously. Comprehensive bioinformatics analysis revealed that these proteins were involved in many important biological processes, including signaling, localization and cell cycle regulation. The nuclear pore complex, which is very important for RNA transport, was changed significantly at phosphorylation level, indicating its important role in response to UV-induced cellular stress. Protein–protein interaction network analysis and DNA repair pathways crosstalk were also examined in this study. Proteins involved in base excision repair, nucleotide repair and mismatch repair changed their phosphorylation pattern in response to UV treatment, indicating the complexity of cellular events and the coordination of these pathways. These systematic analyses provided new clues of protein phosphorylation in response to specific DNA damage, which is very important for further investigation. And give macroscopic view on an overall phosphorylation situation under UV radiation.</p></div

    Network of phosphoproteins derived from data and the expanding view of phosphorylation level changes for parts of representative proteins.

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    <p>The different colors represent different ratios from -2 to +2. The highlight part are mismatch repair related protein—MSH6 network picture, DNA replication related protein—POLA1 and POLE network and nuclear pore complex protein—Nup153, Nup50, Nup188 and Nup214 network part.</p

    Influenced phosphoproteins inrelated to DNA repair pathway under UV treatment.

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    <p>Diamond represents mismatch repair proteins, ellipse represents base excision repair proteins and rectangle stands for nucleotide excision repair proteins. The different colors represent different ratios of phosphorylation level from -2 to +2.</p

    Quantitative overview of phosphorylated peptides and proteins in <i>HeLa</i> cells following UV treatment.

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    <p>(A) Number of phosphorylated proteins and proteins’sites quantified in HeLa cells in response to UV treatment. (B) Distribution of identified phosphorylated peptides at Serine, Threonine and Tyrosine sites. (C) Distribution of phosphorylated and non-phosphorylated amino acids in secondary structure. (D) Comparison of phosphorylated peptides identified in this study (I) and Phospho.ELM database (II) (<a href="http://phospho.elm.eu.org/" target="_blank">http://phospho.elm.eu.org/</a>).Validation of phosphorylation sites in RPA1 (S38) and RFC3 (T76) by western blot. Plasmids with 3xFLAG-S38A or 3xFLAG-T76A mutation were transfected to HeLa cells and precipitated by M2 beads. The phosphorylation level between wildtype and mutant proteins were evaluated by western blotting using specific antibody.</p

    Phosphorylation-specific motifs using the Motif-X algorithm.

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    <p>(A) Pro-directed motif centered on Thr and Ser with a strong preference for additional Pro residues C-terminal to the phosphate. (B) Double-phosphorylation motifs found in our study.</p
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