Publications by authors named "Yue-He Ding"

22 Publications

  • Page 1 of 1

CRL4Cdt2 ubiquitin ligase regulates Dna2 and Rad16 (XPF) nucleases by targeting Pxd1 for degradation.

PLoS Genet 2020 07 21;16(7):e1008933. Epub 2020 Jul 21.

National Institute of Biological Sciences, Beijing, China.

Structure-specific endonucleases (SSEs) play key roles in DNA replication, recombination, and repair. SSEs must be tightly regulated to ensure genome stability but their regulatory mechanisms remain incompletely understood. Here, we show that in the fission yeast Schizosaccharomyces pombe, the activities of two SSEs, Dna2 and Rad16 (ortholog of human XPF), are temporally controlled during the cell cycle by the CRL4Cdt2 ubiquitin ligase. CRL4Cdt2 targets Pxd1, an inhibitor of Dna2 and an activator of Rad16, for degradation in S phase. The ubiquitination and degradation of Pxd1 is dependent on CRL4Cdt2, PCNA, and a PCNA-binding degron motif on Pxd1. CRL4Cdt2-mediated Pxd1 degradation prevents Pxd1 from interfering with the normal S-phase functions of Dna2. Moreover, Pxd1 degradation leads to a reduction of Rad16 nuclease activity in S phase, and restrains Rad16-mediated single-strand annealing, a hazardous pathway of repairing double-strand breaks. These results demonstrate a new role of the CRL4Cdt2 ubiquitin ligase in genome stability maintenance and shed new light on how SSE activities are regulated during the cell cycle.
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http://dx.doi.org/10.1371/journal.pgen.1008933DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7394458PMC
July 2020

Improving mass spectrometry analysis of protein structures with arginine-selective chemical cross-linkers.

Nat Commun 2019 09 2;10(1):3911. Epub 2019 Sep 2.

National Institute of Biological Sciences (NIBS), 102206, Beijing, China.

Chemical cross-linking of proteins coupled with mass spectrometry analysis (CXMS) is widely used to study protein-protein interactions (PPI), protein structures, and even protein dynamics. However, structural information provided by CXMS is still limited, partly because most CXMS experiments use lysine-lysine (K-K) cross-linkers. Although superb in selectivity and reactivity, they are ineffective for lysine deficient regions. Herein, we develop aromatic glyoxal cross-linkers (ArGOs) for arginine-arginine (R-R) cross-linking and the lysine-arginine (K-R) cross-linker KArGO. The R-R or K-R cross-links generated by ArGO or KArGO fit well with protein crystal structures and provide information not attainable by K-K cross-links. KArGO, in particular, is highly valuable for CXMS, with robust performance on a variety of samples including a kinase and two multi-protein complexes. In the case of the CNGP complex, KArGO cross-links covered as much of the PPI interface as R-R and K-K cross-links combined and improved the accuracy of Rosetta docking substantially.
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http://dx.doi.org/10.1038/s41467-019-11917-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6718413PMC
September 2019

A high-speed search engine pLink 2 with systematic evaluation for proteome-scale identification of cross-linked peptides.

Nat Commun 2019 07 30;10(1):3404. Epub 2019 Jul 30.

Key Laboratory of Intelligent Information Processing of Chinese Academy of Sciences (CAS), Institute of Computing Technology, CAS, Beijing, 100190, China.

We describe pLink 2, a search engine with higher speed and reliability for proteome-scale identification of cross-linked peptides. With a two-stage open search strategy facilitated by fragment indexing, pLink 2 is ~40 times faster than pLink 1 and 3~10 times faster than Kojak. Furthermore, using simulated datasets, synthetic datasets, N metabolically labeled datasets, and entrapment databases, four analysis methods were designed to evaluate the credibility of ten state-of-the-art search engines. This systematic evaluation shows that pLink 2 outperforms these methods in precision and sensitivity, especially at proteome scales. Lastly, re-analysis of four published proteome-scale cross-linking datasets with pLink 2 required only a fraction of the time used by pLink 1, with up to 27% more cross-linked residue pairs identified. pLink 2 is therefore an efficient and reliable tool for cross-linking mass spectrometry analysis, and the systematic evaluation methods described here will be useful for future software development.
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http://dx.doi.org/10.1038/s41467-019-11337-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6667459PMC
July 2019

Comprehensive identification of peptides in tandem mass spectra using an efficient open search engine.

Nat Biotechnol 2018 Oct 8. Epub 2018 Oct 8.

Key Laboratory of Intelligent Information Processing of Chinese Academy of Sciences (CAS), Institute of Computing Technology, CAS, Beijing, China.

We present a sequence-tag-based search engine, Open-pFind, to identify peptides in an ultra-large search space that includes coeluting peptides, unexpected modifications and digestions. Our method detects peptides with higher precision and speed than seven other search engines. Open-pFind identified 70-85% of the tandem mass spectra in four large-scale datasets and 14,064 proteins, each supported by at least two protein-unique peptides, in a human proteome dataset.
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http://dx.doi.org/10.1038/nbt.4236DOI Listing
October 2018

Identification of piRNA Binding Sites Reveals the Argonaute Regulatory Landscape of the C. elegans Germline.

Cell 2018 02 15;172(5):937-951.e18. Epub 2018 Feb 15.

RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA; Howard Hughes Medical Institute. Electronic address:

piRNAs (Piwi-interacting small RNAs) engage Piwi Argonautes to silence transposons and promote fertility in animal germlines. Genetic and computational studies have suggested that C. elegans piRNAs tolerate mismatched pairing and in principle could target every transcript. Here we employ in vivo cross-linking to identify transcriptome-wide interactions between piRNAs and target RNAs. We show that piRNAs engage all germline mRNAs and that piRNA binding follows microRNA-like pairing rules. Targeting correlates better with binding energy than with piRNA abundance, suggesting that piRNA concentration does not limit targeting. In mRNAs silenced by piRNAs, secondary small RNAs accumulate at the center and ends of piRNA binding sites. In germline-expressed mRNAs, however, targeting by the CSR-1 Argonaute correlates with reduced piRNA binding density and suppression of piRNA-associated secondary small RNAs. Our findings reveal physiologically important and nuanced regulation of individual piRNA targets and provide evidence for a comprehensive post-transcriptional regulatory step in germline gene expression.
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http://dx.doi.org/10.1016/j.cell.2018.02.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5905434PMC
February 2018

Protocol for analyzing protein ensemble structures from chemical cross-links using DynaXL.

Biophys Rep 2017 20;3(4):100-108. Epub 2017 Nov 20.

CAS Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, and National Center for Magnetic Resonance at Wuhan, Wuhan Institute of Physics and Mathematics of the Chinese Academy of Sciences, Wuhan, 430071 China.

Chemical cross-linking coupled with mass spectroscopy (CXMS) is a powerful technique for investigating protein structures. CXMS has been mostly used to characterize the predominant structure for a protein, whereas cross-links incompatible with a unique structure of a protein or a protein complex are often discarded. We have recently shown that the so-called over-length cross-links actually contain protein dynamics information. We have thus established a method called DynaXL, which allow us to extract the information from the over-length cross-links and to visualize protein ensemble structures. In this protocol, we present the detailed procedure for using DynaXL, which comprises five steps. They are identification of highly confident cross-links, delineation of protein domains/subunits, ensemble rigid-body refinement, and final validation/assessment. The DynaXL method is generally applicable for analyzing the ensemble structures of multi-domain proteins and protein-protein complexes, and is freely available at www.tanglab.org/resources.
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http://dx.doi.org/10.1007/s41048-017-0044-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5719800PMC
November 2017

Polη O-GlcNAcylation governs genome integrity during translesion DNA synthesis.

Nat Commun 2017 12 5;8(1):1941. Epub 2017 Dec 5.

CAS Key Laboratory of Genomics and Precision Medicine, Beijing Institute of Genomics, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Beijing, 100101, China.

DNA polymerase η (Polη) facilitates translesion DNA synthesis (TLS) across ultraviolet (UV) irradiation- and cisplatin-induced DNA lesions implicated in skin carcinogenesis and chemoresistant phenotype formation, respectively. However, whether post-translational modifications of Polη are involved in these processes remains largely unknown. Here, we reported that human Polη undergoes O-GlcNAcylation at threonine 457 by O-GlcNAc transferase upon DNA damage. Abrogation of this modification results in a reduced level of CRL4-dependent Polη polyubiquitination at lysine 462, a delayed p97-dependent removal of Polη from replication forks, and significantly enhanced UV-induced mutagenesis even though Polη focus formation and its efficacy to bypass across cyclobutane pyrimidine dimers after UV irradiation are not affected. Furthermore, the O-GlcNAc-deficient T457A mutation impairs TLS to bypass across cisplatin-induced lesions, causing increased cellular sensitivity to cisplatin. Our findings demonstrate a novel role of Polη O-GlcNAcylation in TLS regulation and genome stability maintenance and establish a new rationale to improve chemotherapeutic treatment.
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http://dx.doi.org/10.1038/s41467-017-02164-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5717138PMC
December 2017

Characterizing Protein Dynamics with Integrative Use of Bulk and Single-Molecule Techniques.

Biochemistry 2018 01 2;57(3):305-313. Epub 2017 Oct 2.

CAS Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, and National Center for Magnetic Resonance at Wuhan, Wuhan Institute of Physics and Mathematics of the Chinese Academy of Sciences , Wuhan, Hubei 430071, China.

A protein dynamically samples multiple conformations, and the conformational dynamics enables protein function. Most biophysical measurements are ensemble-based, with the observables averaged over all members of the ensemble. Though attainable, the decomposition of the observables to the constituent conformational states can be computationally expensive and ambiguous. Here we show that the incorporation of single-molecule fluorescence resonance energy transfer (smFRET) data resolves the ambiguity and affords protein ensemble structures that are more precise and accurate. Using K63-linked diubiquitin, we characterize the dynamic domain arrangements of the model system, with the use of chemical cross-linking coupled with mass spectrometry (CXMS), small-angle X-ray scattering (SAXS), and smFRET techniques. CXMS allows the modeling of protein conformational states that are alternatives to the crystal structure. SAXS provides ensemble-averaged low-resolution shape information. Importantly, smFRET affords state-specific populations, and the FRET distances validate the ensemble structures obtained by refining against CXMS and SAXS restraints. Together, the integrative use of bulk and single-molecule techniques affords better insight into protein dynamics and shall be widely implemented in structural biology.
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http://dx.doi.org/10.1021/acs.biochem.7b00817DOI Listing
January 2018

Architecture of the ATG2B-WDR45 complex and an aromatic Y/HF motif crucial for complex formation.

Autophagy 2017 13;13(11):1870-1883. Epub 2017 Sep 13.

a State Key Laboratory of Membrane Biology , Tsinghua University-Peking University Joint Center for Life Sciences, School of Life Sciences, Tsinghua University , Beijing , China.

PtdIns3P signaling is critical for dynamic membrane remodeling during autophagosome formation. Proteins in the Atg18/WIPI family are PtdIns3P-binding effectors which can form complexes with proteins in the Atg2 family, and both families are essential for macroautophagy/autophagy. However, little is known about the biophysical properties and biological functions of the Atg2-Atg18/WIPI complex as a whole. Here, we demonstrate that an ortholog of yeast Atg18, mammalian WDR45/WIPI4 has a stronger binding capacity for mammalian ATG2A or ATG2B than the other 3 WIPIs. We purified the full-length Rattus norvegicus ATG2B and found that it could bind to liposomes independently of PtdIns3P or WDR45. We also purified the ATG2B-WDR45 complex and then performed 3-dimensional reconstruction of the complex by single-particle electron microscopy, which revealed a club-shaped heterodimer with an approximate length of 22 nm. Furthermore, we performed cross-linking mass spectrometry and identified a set of highly cross-linked intermolecular and intramolecular lysine pairs. Finally, based on the cross-linking data followed by bioinformatics and mutagenesis analysis, we determined the conserved aromatic H/YF motif in the C terminus of ATG2A and ATG2B that is crucial for complex formation.
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http://dx.doi.org/10.1080/15548627.2017.1359381DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5788475PMC
June 2019

Phosphorylation of LSD1 by PLK1 promotes its chromatin release during mitosis.

Cell Biosci 2017 23;7:15. Epub 2017 Mar 23.

Beijing Key Laboratory of DNA Damage Response and College of Life Sciences, Capital Normal University, Beijing, 100048 China.

Background: Lysine-specific histone demethylase 1 (LSD1) modulates chromatin status through demethylation of H3K4 and H3K9. It has been demonstrated that LSD1 is hyperphosphorylated and dissociates from chromatin during mitosis. However, the molecular mechanism of LSD1 detachment is unknown.

Results: In this report, we found that polo-like kinase 1 (PLK1) directly interacted with LSD1 and phosphorylated LSD1 at Ser-126 . Nocodazole-induced metaphase arrest promoted release of LSD1 from chromatin, and the phosphorylation-defective mutant LSD1 (S126A) failed to dissociate from chromatin upon nocodazole treatment.

Conclusions: Taken together, our findings demonstrate that phosphorylation of LSD1 at Ser-126 by PLK1 promotes its release from chromatin during mitosis.
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http://dx.doi.org/10.1186/s13578-017-0142-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5364692PMC
March 2017

Modeling Protein Excited-state Structures from "Over-length" Chemical Cross-links.

J Biol Chem 2017 01 19;292(4):1187-1196. Epub 2016 Dec 19.

From the CAS Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, National Center for Magnetic Resonance at Wuhan, Wuhan Institute of Physics and Mathematics of the Chinese Academy of Sciences, Wuhan, Hubei Province 430071,

Chemical cross-linking coupled with mass spectroscopy (CXMS) provides proximity information for the cross-linked residues and is used increasingly for modeling protein structures. However, experimentally identified cross-links are sometimes incompatible with the known structure of a protein, as the distance calculated between the cross-linked residues far exceeds the maximum length of the cross-linker. The discrepancies may persist even after eliminating potentially false cross-links and excluding intermolecular ones. Thus the "over-length" cross-links may arise from alternative excited-state conformation of the protein. Here we present a method and associated software DynaXL for visualizing the ensemble structures of multidomain proteins based on intramolecular cross-links identified by mass spectrometry with high confidence. Representing the cross-linkers and cross-linking reactions explicitly, we show that the protein excited-state structure can be modeled with as few as two over-length cross-links. We demonstrate the generality of our method with three systems: calmodulin, enzyme I, and glutamine-binding protein, and we show that these proteins alternate between different conformations for interacting with other proteins and ligands. Taken together, the over-length chemical cross-links contain valuable information about protein dynamics, and our findings here illustrate the relationship between dynamic domain movement and protein function.
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http://dx.doi.org/10.1074/jbc.M116.761841DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5270465PMC
January 2017

Structural characterization of coatomer in its cytosolic state.

Protein Cell 2016 08 29;7(8):586-600. Epub 2016 Jul 29.

National Key Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.

Studies on coat protein I (COPI) have contributed to a basic understanding of how coat proteins generate vesicles to initiate intracellular transport. The core component of the COPI complex is coatomer, which is a multimeric complex that needs to be recruited from the cytosol to membrane in order to function in membrane bending and cargo sorting. Previous structural studies on the clathrin adaptors have found that membrane recruitment induces a large conformational change in promoting their role in cargo sorting. Here, pursuing negative-stain electron microscopy coupled with single-particle analyses, and also performing CXMS (chemical cross-linking coupled with mass spectrometry) for validation, we have reconstructed the structure of coatomer in its soluble form. When compared to the previously elucidated structure of coatomer in its membrane-bound form we do not observe a large conformational change. Thus, the result uncovers a key difference between how COPI versus clathrin coats are regulated by membrane recruitment.
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http://dx.doi.org/10.1007/s13238-016-0296-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4980336PMC
August 2016

Increasing the Depth of Mass-Spectrometry-Based Structural Analysis of Protein Complexes through the Use of Multiple Cross-Linkers.

Anal Chem 2016 Apr 4;88(8):4461-9. Epub 2016 Apr 4.

National Institute of Biological Sciences, Beijing , Beijing 102206, China.

Chemical cross-linking of proteins coupled with mass spectrometry (CXMS) is a powerful tool to study protein folding and to map the interfaces between interacting proteins. The most commonly used cross-linkers in CXMS are BS(3) and DSS, which have similar structures and generate the same linkages between pairs of lysine residues in spatial proximity. However, there are cases where no cross-linkable lysine pairs are present at certain regions of a protein or at the interface of two interacting proteins. In order to find the cross-linkers that can best complement the performance of BS(3) and DSS, we tested seven additional cross-linkers that either have different spacer arm structures or that target different amino acids (BS(2)G, EGS, AMAS, GMBS, Sulfo-GMBS, EDC, and TFCS). Using BSA, aldolase, the yeast H/ACA protein complex, and E. coli 70S ribosomes, we showed that, in terms of providing structural information not obtained through the use of BS(3) and DSS, EGS and Sulfo-GMBS worked better than the other cross-linkers that we tested. EGS generated a large number of cross-links not seen with the other amine-specific cross-linkers, possibly due to its hydrophilic spacer arm. We demonstrate that incorporating the cross-links contributed by the EGS and amine-sulfhydryl cross-linkers greatly increased the accuracy of Rosetta in docking the structure of the yeast H/ACA protein complex. Given the improved depth of useful information it can provide, we suggest that the multilinker CXMS approach should be used routinely when the amount of a sample permits.
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http://dx.doi.org/10.1021/acs.analchem.6b00281DOI Listing
April 2016

Trifunctional cross-linker for mapping protein-protein interaction networks and comparing protein conformational states.

Elife 2016 Mar 8;5. Epub 2016 Mar 8.

National Institute of Biological Sciences, Beijing, China.

To improve chemical cross-linking of proteins coupled with mass spectrometry (CXMS), we developed a lysine-targeted enrichable cross-linker containing a biotin tag for affinity purification, a chemical cleavage site to separate cross-linked peptides away from biotin after enrichment, and a spacer arm that can be labeled with stable isotopes for quantitation. By locating the flexible proteins on the surface of 70S ribosome, we show that this trifunctional cross-linker is effective at attaining structural information not easily attainable by crystallography and electron microscopy. From a crude Rrp46 immunoprecipitate, it helped identify two direct binding partners of Rrp46 and 15 protein-protein interactions (PPIs) among the co-immunoprecipitated exosome subunits. Applying it to E. coli and C. elegans lysates, we identified 3130 and 893 inter-linked lysine pairs, representing 677 and 121 PPIs. Using a quantitative CXMS workflow we demonstrate that it can reveal changes in the reactivity of lysine residues due to protein-nucleic acid interaction.
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http://dx.doi.org/10.7554/eLife.12509DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4811778PMC
March 2016

Structural basis for receptor recognition and pore formation of a zebrafish aerolysin-like protein.

EMBO Rep 2016 Feb 28;17(2):235-48. Epub 2015 Dec 28.

Hefei National Laboratory for Physical Sciences at the Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, China

Various aerolysin-like pore-forming proteins have been identified from bacteria to vertebrates. However, the mechanism of receptor recognition and/or pore formation of the eukaryotic members remains unknown. Here, we present the first crystal and electron microscopy structures of a vertebrate aerolysin-like protein from Danio rerio, termed Dln1, before and after pore formation. Each subunit of Dln1 dimer comprises a β-prism lectin module followed by an aerolysin module. Specific binding of the lectin module toward high-mannose glycans triggers drastic conformational changes of the aerolysin module in a pH-dependent manner, ultimately resulting in the formation of a membrane-bound octameric pore. Structural analyses combined with computational simulations and biochemical assays suggest a pore-forming process with an activation mechanism distinct from the previously characterized bacterial members. Moreover, Dln1 and its homologs are ubiquitously distributed in bony fishes and lamprey, suggesting a novel fish-specific defense molecule.
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http://dx.doi.org/10.15252/embr.201540851DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5290818PMC
February 2016

ESCRTs Cooperate with a Selective Autophagy Receptor to Mediate Vacuolar Targeting of Soluble Cargos.

Mol Cell 2015 Sep 10;59(6):1035-42. Epub 2015 Sep 10.

National Institute of Biological Sciences, Collaborative Innovation Center for Cancer Medicine, Beijing, 102206, China. Electronic address:

Autophagy transports cytosolic materials into lysosomes/vacuoles either in bulk or selectively. Selective autophagy requires cargo receptor proteins, which usually link cargos to the macroautophagy machinery composed of core autophagy-related (Atg) proteins. Here, we show that fission yeast Nbr1, a homolog of mammalian autophagy receptor NBR1, interacts with and facilitates the transport of two cytosolic hydrolases into vacuoles, in a way reminiscent of the budding yeast cytoplasm-to-vacuole targeting (Cvt) pathway, a prototype of selective autophagy. We term this pathway Nbr1-mediated vacuolar targeting (NVT). Surprisingly, unlike the Cvt pathway, the NVT pathway does not require core Atg proteins. Instead, it depends on the endosomal sorting complexes required for transport (ESCRTs). NVT components colocalize with ESCRTs at multivesicular bodies (MVBs) and rely on ubiquitination for their transport. Our findings demonstrate the ability of ESCRTs to mediate highly selective autophagy of soluble cargos, and suggest an unexpected mechanistic versatility of autophagy receptors.
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http://dx.doi.org/10.1016/j.molcel.2015.07.034DOI Listing
September 2015

Visualizing the Ensemble Structures of Protein Complexes Using Chemical Cross-Linking Coupled with Mass Spectrometry.

Biophys Rep 2015 28;1:127-138. Epub 2015 Dec 28.

CAS Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan, 430071 China.

Graphical Abstract:

Abstract: Chemical cross-linking coupled with mass spectrometry (CXMS) identifies protein residues that are close in space, and has been increasingly used for modeling the structures of protein complexes. Here we show that a single structure is usually sufficient to account for the intermolecular cross-links identified for a stable complex with sub-µmol/L binding affinity. In contrast, we show that the distance between two cross-linked residues in the different subunits of a transient or fleeting complex may exceed the maximum length of the cross-linker used, and the cross-links cannot be fully accounted for with a unique complex structure. We further show that the seemingly incompatible cross-links identified with high confidence arise from alternative modes of protein-protein interactions. By converting the intermolecular cross-links to ambiguous distance restraints, we established a rigid-body simulated annealing refinement protocol to seek the minimum set of conformers collectively satisfying the CXMS data. Hence we demonstrate that CXMS allows the depiction of the ensemble structures of protein complexes and elucidates the interaction dynamics for transient and fleeting complexes.
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http://dx.doi.org/10.1007/s41048-015-0015-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4871902PMC
December 2015

Fission yeast Pxd1 promotes proper DNA repair by activating Rad16XPF and inhibiting Dna2.

PLoS Biol 2014 Sep 9;12(9):e1001946. Epub 2014 Sep 9.

National Institute of Biological Sciences, Beijing, China; Graduate School of Peking Union Medical College, Beijing, China.

Structure-specific nucleases play crucial roles in many DNA repair pathways. They must be precisely controlled to ensure optimal repair outcomes; however, mechanisms of their regulation are not fully understood. Here, we report a fission yeast protein, Pxd1, that binds to and regulates two structure-specific nucleases: Rad16XPF-Swi10ERCC1 and Dna2-Cdc24. Strikingly, Pxd1 influences the activities of these two nucleases in opposite ways: It activates the 3' endonuclease activity of Rad16-Swi10 but inhibits the RPA-mediated activation of the 5' endonuclease activity of Dna2. Pxd1 is required for Rad16-Swi10 to function in single-strand annealing, mating-type switching, and the removal of Top1-DNA adducts. Meanwhile, Pxd1 attenuates DNA end resection mediated by the Rqh1-Dna2 pathway. Disabling the Dna2-inhibitory activity of Pxd1 results in enhanced use of a break-distal repeat sequence in single-strand annealing and a greater loss of genetic information. We propose that Pxd1 promotes proper DNA repair by differentially regulating two structure-specific nucleases.
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http://dx.doi.org/10.1371/journal.pbio.1001946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4159138PMC
September 2014

CAMKII and calcineurin regulate the lifespan of Caenorhabditis elegans through the FOXO transcription factor DAF-16.

Elife 2013 Jun 25;2:e00518. Epub 2013 Jun 25.

Graduate Program in Chinese Academy of Medical Sciences and Peking Union Medical College , Beijing , China ; National Institute of Biological Sciences, Beijing , Beijing , China.

The insulin-like signaling pathway maintains a relatively short wild-type lifespan in Caenorhabditis elegans by phosphorylating and inactivating DAF-16, the ortholog of the FOXO transcription factors of mammalian cells. DAF-16 is phosphorylated by the AKT kinases, preventing its nuclear translocation. Calcineurin (PP2B phosphatase) also limits the lifespan of C. elegans, but the mechanism through which it does so is unknown. Herein, we show that TAX-6•CNB-1 and UNC-43, the C. elegans Calcineurin and Ca(2+)/calmodulin-dependent kinase type II (CAMKII) orthologs, respectively, also regulate lifespan through DAF-16. Moreover, UNC-43 regulates DAF-16 in response to various stress conditions, including starvation, heat or oxidative stress, and cooperatively contributes to lifespan regulation by insulin signaling. However, unlike insulin signaling, UNC-43 phosphorylates and activates DAF-16, thus promoting its nuclear localization. The phosphorylation of DAF-16 at S286 by UNC-43 is removed by TAX-6•CNB-1, leading to DAF-16 inactivation. Mammalian FOXO3 is also regulated by CAMKIIA and Calcineurin. DOI:http://dx.doi.org/10.7554/eLife.00518.001.
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http://dx.doi.org/10.7554/eLife.00518DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3691573PMC
June 2013

Characterization of PUD-1 and PUD-2, two proteins up-regulated in a long-lived daf-2 mutant.

PLoS One 2013 14;8(6):e67158. Epub 2013 Jun 14.

National Institute of Biological Sciences, Beijing, Beijing, China.

C. elegans PUD-1 and PUD-2, two proteins up-regulated in daf-2(loss-of-function) (PUD), are homologous 17-kD proteins with a large abundance increase in long-lived daf-2 mutant animals of reduced insulin signaling. In this study, we show that both PUD-1 and PUD-2 are abundantly expressed in the intestine and hypodermis, and form a heterodimer. We have solved their crystal structure to 1.9-Å resolution and found that both proteins adopt similar β-sandwich folds in the V-shaped dimer. In contrast, their homologs PUD-3, PUD-4, PUDL-1 and PUDL-2 are all monomeric proteins with distinct expression patterns in C. elegans. Thus, the PUD-1/PUD-2 heterodimer probably has a function distinct from their family members. Neither overexpression nor deletion of pud-1 and pud-2 affected the lifespan of WT or daf-2 mutant animals, suggesting that their induction in daf-2 worms does not contribute to longevity. Curiously, deletion of pud-1 and pud-2 was associated with a protective effect against paralysis induced by the amyloid β-peptide (1-42), which further enhanced the protection conferred by daf-2(RNAi) against Aβ.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0067158PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3683130PMC
January 2014

ALKBH4-dependent demethylation of actin regulates actomyosin dynamics.

Nat Commun 2013 ;4:1832

Genome Structure and Stability Group, BIG CAS-OSLO Genome Research Cooperation, Disease Genomics and Individualized Medicine Laboratory, Beijing Institute of Genomics, Chinese Academy of Sciences, No. 1-7 Beichen West Road, Chaoyang District, Beijing 100101, China.

Regulation of actomyosin dynamics by post-transcriptional modifications in cytoplasmic actin is still poorly understood. Here we demonstrate that dioxygenase ALKBH4-mediated demethylation of a monomethylated site in actin (K84me1) regulates actin-myosin interaction and actomyosin-dependent processes such as cytokinesis and cell migration. ALKBH4-deficient cells display elevated K84me1 levels. Non-muscle myosin II only interacts with unmethylated actin and its proper recruitment to and interaction with actin depend on ALKBH4. ALKBH4 co-localizes with the actomyosin-based contractile ring and midbody via association with methylated actin. ALKBH4-mediated regulation of actomyosin dynamics is completely dependent on its catalytic activity. Disorganization of cleavage furrow components and multinucleation associated with ALKBH4 deficiency can all be restored by reconstitution with wild-type but not catalytically inactive ALKBH4. Similar to actin and myosin knock-out mice, homozygous Alkbh4 mutant mice display early embryonic lethality. These findings imply that ALKBH4-dependent actin demethylation regulates actomyosin function by promoting actin-non-muscle myosin II interaction.
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http://dx.doi.org/10.1038/ncomms2863DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3674258PMC
December 2013

Identification of cross-linked peptides from complex samples.

Nat Methods 2012 Sep 8;9(9):904-6. Epub 2012 Jul 8.

College of Biological Sciences, China Agricultural University, Beijing, China.

We have developed pLink, software for data analysis of cross-linked proteins coupled with mass-spectrometry analysis. pLink reliably estimates false discovery rate in cross-link identification and is compatible with multiple homo- or hetero-bifunctional cross-linkers. We validated the program with proteins of known structures, and we further tested it on protein complexes, crude immunoprecipitates and whole-cell lysates. We show that it is a robust tool for protein-structure and protein-protein-interaction studies.
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http://dx.doi.org/10.1038/nmeth.2099DOI Listing
September 2012