Tianjin Joint Academy of Biotechnology and Medicine

Tianjin, China

Tianjin Joint Academy of Biotechnology and Medicine

Tianjin, China
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PubMed | CAS Tianjin Institute of Industrial Biotechnology, Nankai University and Tianjin Joint Academy of Biotechnology and Medicine
Type: | Journal: Analytica chimica acta | Year: 2015

Binding affinity of a small molecule drug candidate to a therapeutically relevant biomolecular target is regarded the first determinant of the candidates efficacy. Although the ultrafiltration-LC/MS (UF-LC/MS) assay enables efficient ligand discovery for a specific target from a mixed pool of compounds, most previous analysis allowed for relative affinity ranking of different ligands. Moreover, the reliability of affinity measurement for multiple ligands with UF-LC/MS has hardly been strictly evaluated. In this study, we examined the accuracy of K(d) determination through UF-LC/MS by comparison with classical ITC measurement. A single-point K(d) calculation method was found to be suitable for affinity measurement of multiple ligands bound to the same target when binding competition is minimized. A second workflow based on analysis of the unbound fraction of compounds was then developed, which simplified sample preparation as well as warranted reliable ligand discovery. The new workflow implemented in a fragment mixture screen afforded rapid and sensitive detection of low-affinity ligands selectively bound to the RNA polymerase NS5B of hepatitis C virus. More importantly, ligand identification and affinity measurement for mixture-based fragment screens by UF-LC/MS were in good accordance with single ligand evaluation by conventional SPR analysis. This new approach is expected to become a valuable addition to the arsenal of high-throughput screening techniques for fragment-based drug discovery.

PubMed | CAS Tianjin Institute of Industrial Biotechnology, Tianjin University of Traditional Chinese Medicine, Tianjin Joint Academy of Biotechnology and Medicine, Tianjin University and Shanghai University
Type: | Journal: Scientific reports | Year: 2016

The nucleoprotein (NP) of Ebola virus (EBOV) and Marburg virus (MARV) is an essential component of the viral ribonucleoprotein complex and significantly impacts replication and transcription of the viral RNA genome. Although NP is regarded as a promising antiviral druggable target, no chemical ligands have been reported to interact with EBOV NP or MARV NP. We identified two compounds from a traditional Chinese medicine Gancao (licorice root) that can bind both NPs by combining affinity mass spectrometry and metabolomics approaches. These two ligands, 18-glycyrrhetinic acid and licochalcone A, were verified by defined compound mixture screens and further characterized with individual ligand binding assays. Accompanying biophysical analyses demonstrate that binding of 18-glycyrrhetinic acid to EBOV NP significantly reduces protein thermal stability, induces formation of large NP oligomers, and disrupts the critical association of viral ssRNA with NP complexes whereas the compound showed no such activity on MARV NP. Our study has revealed the substantial potential of new analytical techniques in ligand discovery from natural herb resources. In addition, identification of a chemical ligand that influences the oligomeric state and RNA-binding function of EBOV NP sheds new light on antiviral drug development.

PubMed | CAS Tianjin Institute of Industrial Biotechnology, Nankai University and Tianjin Joint Academy of Biotechnology and Medicine
Type: | Journal: Scientific reports | Year: 2015

In fragment-based lead discovery (FBLD), a cascade combining multiple orthogonal technologies is required for reliable detection and characterization of fragment binding to the target. Given the limitations of the mainstream screening techniques, we presented a ligand-observed mass spectrometry approach to expand the toolkits and increase the flexibility of building a FBLD pipeline especially for tough targets. In this study, this approach was integrated into a FBLD program targeting the HCV RNA polymerase NS5B. Our ligand-observed mass spectrometry analysis resulted in the discovery of 10 hits from a 384-member fragment library through two independent screens of complex cocktails and a follow-up validation assay. Moreover, this MS-based approach enabled quantitative measurement of weak binding affinities of fragments which was in general consistent with SPR analysis. Five out of the ten hits were then successfully translated to X-ray structures of fragment-bound complexes to lay a foundation for structure-based inhibitor design. With distinctive strengths in terms of high capacity and speed, minimal method development, easy sample preparation, low material consumption and quantitative capability, this MS-based assay is anticipated to be a valuable addition to the repertoire of current fragment screening techniques.

Xu J.,Nankai University | Xu J.,Tianjin Joint Academy of Biotechnology and Medicine | Wei X.,Nankai University | Wei X.,Tianjin Joint Academy of Biotechnology and Medicine | And 15 more authors.
Protein and Cell | Year: 2013

Arabidopsis BOTRYTIS-INDUCED KINASE1 (BIK1) is a receptor-like cytoplasmic kinase acting early in multiple signaling pathways important for plant growth and innate immunity. It is known to form a signaling complex with a cell-surface receptor FLS2 and a co-receptor kinase BAK1 to transduce signals upon perception of pathogen-associated molecular patterns (PAMPs). Although site-specific phosphorylation is speculated to mediate the activation and function of BIK1, few studies have been devoted to complete profiling of BIK1 phosphorylation residues. Here, we identified nineteen in vitro autophosphorylation sites of BIK1 including three phosphotyrosine sites, thereby proving BIK1 is a dual-specificity kinase for the first time. The kinase activity of BIK1 substitution mutants were explicitly assessed using quantitative mass spectrometry (MS). Thr-237, Thr-242 and Tyr-250 were found to most significantly affect BIK1 activity in autophosphorylation and phosphorylation of BAK1 in vitro. A structural model of BIK1 was built to further illustrate the molecular functions of specific phosphorylation residues. We also mapped new sites of FLS2 phosphorylation by BIK1, which are different from those by BAK1. These in vitro results could provide new hypotheses for more in-depth in vivo studies leading to deeper understanding of how phosphorylation contributes to BIK1 activation and mediates downstream signaling specificity. © 2013 Higher Education Press and Springer-Verlag Berlin Heidelberg.

Zhou H.,Nankai University | Zhou H.,Tsinghua University | Sun Y.,CAS Institute of Biophysics | Wang Y.,Tianjin Joint Academy of Biotechnology and Medicine | And 13 more authors.
Protein and Cell | Year: 2013

Severe fever with thrombocytopenia syndrome virus (SFTSV), a member of the Phlebovirus genus from the Bunyaviridae family endemic to China, is the causative agent of life-threatening severe fever with thrombocytopenia syndrome (SFTS), which features high fever and hemorrhage. Similar to other negative-sense RNA viruses, SFTSV encodes a nucleocapsid protein (NP) that is essential for viral replication. NP facilitates viral RNA encapsidation and is responsible for the formation of ribonucleoprotein complex. However, recent studies have indicated that NP from Phlebovirus members behaves in inhomogeneous oligomerization states. In the present study, we report the crystal structure of SFTSV NP at 2.8 Å resolution and demonstrate the mechanism by which it processes a ringshaped hexameric form to accomplish RNA encapsidation. Key residues essential for oligomerization are identified through mutational analysis and identified to have a significant impact on RNA binding, which suggests that correct formation of highly ordered oligomers is a critical step in RNA encapsidation. The findings of this work provide new insights into the discovery of new antiviral reagents for Phlebovirus infection. © 2013 Higher Education Press and Springer-Verlag Berlin Heidelberg.

Shan S.,Tsinghua University | Chen X.,CAS Institute of Biophysics | Liu T.,Tianjin Joint Academy of Biotechnology and Medicine | Zhao H.,Tsinghua University | And 5 more authors.
FASEB Journal | Year: 2011

Isoprenoid precursors, which are a large group of natural products and play key roles in many biological pathways, can only be biosynthesized by the 2-C-methyl-D-erythritol 4-phosphate pathway in Mycobacterium tuberculosis. The 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE), which is an essential enzyme in the isoprenoid precursor biosynthesis pathway, catalyzes ATP-dependent phosphorylation of 4-diphosphocytidyl- 2-C-methyl-D-erythritol (CDP-ME) to 4-diphosphocytidyl-2C-methyl-D-erythritol-2-phosphate and plays a crucial role in M. tuberculosis survival. Therefore, IspE is characterized as an attractive and potential target for antimicrobial drug discovery. However, no experimental structure of M. tuberculosis IspE has been reported, which has hindered our understanding of its structural details and mechanism of action. Here, we report the expression and purification of fully active full-length M. tuberculosis IspE and solve the high-resolution crystal structures of IspE alone and in complex with either the substrate CDP-ME or nonhydrolyzable ATP analog or ADP. The structures present a characteristic galactose/homoserine/mevalonate/ phosphomevalonate kinase superfamily α/β-fold with a catalytic center located in a cleft between 2 domains and display clear substrate and ATP binding pockets. Our results also indicate distinct differences in ligand binding of M. tuberculosis IspE with other reported IspEs. Combined with the results of mutagenesis and enzymatic studies, our results provide useful information on the structural basis of IspE for future anti-M. tuberculosis drug discovery targeting this kinase. © FASEB.

Hao J.,Nankai University | Hao J.,Tianjin Joint Academy of Biotechnology and Medicine | Li W.,Tianjin Joint Academy of Biotechnology and Medicine | Dan J.,Nankai University | And 9 more authors.
Journal of Proteomics | Year: 2013

Induced pluripotent stem cells (iPSCs), derived from somatic cells and functionally very similar to embryonic stem cells (ESCs), are at the center stage of intense research in regenerative medicine. We carried out the first membrane proteomic profiling of mouse iPSCs, in comparison with ESCs and adult mouse tail tip fibroblasts (TTFs) from which iPSCs were generated. Using a proteomic workflow combining membrane fractionation, SDS-PAGE separation and nanoUPLC-MSE technology, we identified 673, 679 and 682 non-redundant proteins from mouse iPSC, ESC and TTF membrane fractions, respectively. Label-free quantitation revealed 155 reprogramming-associated and 128 pluripotency-associated transmembrane proteins. Furthermore, a small group of 23 membrane proteins mainly involved in amino acid/glucose/ion transport, membrane fusion and vesicular trafficking were found potentially regulated between miPSCs and mESCs. Expression changes of selected proteins were verified by qPCR, western blot and/or immunofluorescence analyses in a wider array of cell types. Notably, epithelial cell adhesion molecules, glucose transporters 1 and 3, transferrin receptor and several nuclear membrane-associated components were highly expressed in both iPSCs and ESCs, relative to TTFs. Moreover, knock-down of glucose transporter 3 in ESCs impaired the beating function of ESC-derived cardiomyocytes, suggesting its potential role in mediating stem cell differentiation. © 2013.

Chen X.,Nankai University | Li L.,Nankai University | Chen S.,Nankai University | Xu Y.,Nankai University | And 11 more authors.
Analytical Chemistry | Year: 2013

Mass spectrometry-based platforms have gained increasing success in discovery of ligands bound to therapeutic targets as drug candidates. We established both a nanoelectrospray ionization mass spectrometry (nanoESI-MS) assay and an ultrafiltration liquid chromatography/mass spectrometry (LC/MS) assay to identify new ligands for New Delhi metallo-β-lactamase 1 (NDM-1), responsible for worldwide antibiotic resistance. To alleviate nonspecific binding of hydrophobic compounds and eliminate false positives typically encountered in the indirect LC/MS-based assay, we introduced a blocking protein in the control, which remarkably enhances the selectivity and accuracy of the indirect approach. Side-by-side comparison of the two MS-based approaches for the first time further reveals unique advantages of the indirect approach, including better reproducibility and tolerance of interference. Moreover, the success of fishing out a potent ligand from a mixture of small-molecule fragments demonstrates great potential of the indirect LC/MS-based approach for constructing a robust screening platform against combinatorial libraries or natural product extracts. More importantly, by combining the results of MS-based analyses, enzymatic activity assay, competition experiments, and structural simulation, we discovered a new compound as a promising drug candidate targeting NDM-1. © 2013 American Chemical Society.

Zhang L.,Nankai University | Liu S.,Nankai University | Liu S.,Tianjin Joint Academy of Biotechnology and Medicine | Liu N.,Nankai University | And 9 more authors.
Protein and Cell | Year: 2014

ABSTRACT: Histone deacetylase 6 (HDAC6), a predominantly cytoplasmic protein deacetylase, participates in a wide range of cellular processes through its deacetylase activity. However, the diverse functions of HDAC6 cannot be fully elucidated with its known substrates. In an attempt to explore the substrate diversity of HDAC6, we performed quantitative proteomic analyses to monitor changes in the abundance of protein lysine acetylation in response to HDAC6 deficiency. We identified 107 proteins with elevated acetylation in the liver of HDAC6 knockout mice. Three cytoplasmic proteins, including myosin heavy chain 9 (MYH9), heat shock cognate protein 70 (Hsc70), and dnaJ homolog subfamily A member 1 (DNAJA1), were verified to interact with HDAC6. The acetylation levels of these proteins were negatively regulated by HDAC6 both in the mouse liver and in cultured cells. Functional studies reveal that HDAC6-mediated deacetylation modulates the actin-binding ability of MYH9 and the interaction between Hsc70 and DNAJA1. These findings consolidate the notion that HDAC6 serves as a critical regulator of protein acetylation with the capability of coordinating various cellular functions. © 2014, The Author(s).

PubMed | Tianjin Joint Academy of Biotechnology and Medicine, Tianjin Medical University and University of Manitoba
Type: Journal Article | Journal: Biochimica et biophysica acta | Year: 2016

Posttranslational modifications of certain stress granule (SG) proteins are closely related to the assembly of SGs, a type of cytoplasmic foci structure. Our previous studies revealed that the Tudor staphylococcal nuclease (Tudor-SN) protein participates in the formation of SGs. However, the functional significance of potential Tudor-SN modifications during stress has not been reported. In this study, we demonstrated that the Tudor-SN protein was phosphorylated at threonine 103 (T103) upon stimulation with arsenite. In addition, c-Jun N-terminal kinase (JNK) was found to be responsible for Tudor-SN phosphorylation at the T103 site. We further illustrated that either a T103A mutation or the suppression of phosphorylation of T103 by the JNK inhibitor SP600125 inhibited the efficient recruitment of Tudor-SN into SGs. In addition, the T103A mutation could affect the physical binding of Tudor-SN with the G3BP (Ras-GAP SH3 domain-binding protein) protein but not with the HuR (Hu antigen R) protein and AGTR1-3UTR (3-untranslated region of angiotensin II receptor, type 1) mRNA cargo. These data suggested that JNK-enhanced Tudor-SN phosphorylation promotes the interaction between Tudor-SN and G3BP and facilitates the efficient recruitment of Tudor-SN into SGs under conditions of sodium arsenite-induced oxidative stress. This finding provides novel insights into the physiological function of Tudor-SN modification.

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