Publications by authors named "Meng-Qiu Dong"

120 Publications

Toward Increased Reliability, Transparency, and Accessibility in Cross-linking Mass Spectrometry.

Structure 2020 11 15;28(11):1259-1268. Epub 2020 Oct 15.

University of Konstanz, Department of Biology, Universitätsstrasse 10, 78457 Konstanz, Germany; Konstanz Research School Chemical Biology, University of Konstanz, Universitätsstrasse 10, 78457 Konstanz, Germany.

Cross-linking mass spectrometry (MS) has substantially matured as a method over the past 2 decades through parallel development in multiple labs, demonstrating its applicability to protein structure determination, conformation analysis, and mapping protein interactions in complex mixtures. Cross-linking MS has become a much-appreciated and routinely applied tool, especially in structural biology. Therefore, it is timely that the community commits to the development of methodological and reporting standards. This white paper builds on an open process comprising a number of events at community conferences since 2015 and identifies aspects of Cross-linking MS for which guidelines should be developed as part of a Cross-linking MS standards initiative.
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http://dx.doi.org/10.1016/j.str.2020.09.011DOI Listing
November 2020

Atg1 kinase in fission yeast is activated by Atg11-mediated dimerization and cis-autophosphorylation.

Elife 2020 09 10;9. Epub 2020 Sep 10.

National Institute of Biological Sciences, Beijing, China.

Autophagy is a proteolytic pathway that is conserved from yeasts to mammals. Atg1 kinase is essential for autophagy, but how its activity is controlled remains insufficiently understood. Here, we show that, in the fission yeast Atg1 kinase activity requires Atg11, the ortholog of mammalian FIP200/RB1CC1, but does not require Atg13, Atg17, or Atg101. Remarkably, a 62 amino acid region of Atg11 is sufficient for the autophagy function of Atg11 and for supporting the Atg1 kinase activity. This region harbors an Atg1-binding domain and a homodimerization domain. Dimerizing Atg1 is the main role of Atg11, as it can be bypassed by artificially dimerizing Atg1. In an Atg1 dimer, only one Atg1 molecule needs to be catalytically active, suggesting that Atg1 activation can be achieved through cis-autophosphorylation. We propose that mediating Atg1 oligomerization and activation may be a conserved function of Atg11/FIP200 family proteins and cis-autophosphorylation may be a general mechanism of Atg1 activation.
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http://dx.doi.org/10.7554/eLife.58073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7511232PMC
September 2020

Discovery of a molecular glue promoting CDK12-DDB1 interaction to trigger cyclin K degradation.

Elife 2020 08 17;9. Epub 2020 Aug 17.

National Institute of Biological Sciences, Beijing, China.

Molecular-glue degraders mediate interactions between target proteins and components of the ubiquitin-proteasome system to cause selective protein degradation. Here, we report a new molecular glue HQ461 discovered by high-throughput screening. Using loss-of-function and gain-of-function genetic screening in human cancer cells followed by biochemical reconstitution, we show that HQ461 acts by promoting an interaction between CDK12 and DDB1-CUL4-RBX1 E3 ubiquitin ligase, leading to polyubiquitination and degradation of CDK12-interacting protein Cyclin K (CCNK). Degradation of CCNK mediated by HQ461 compromised CDK12 function, leading to reduced phosphorylation of a CDK12 substrate, downregulation of DNA damage response genes, and cell death. Structure-activity relationship analysis of HQ461 revealed the importance of a 5-methylthiazol-2-amine pharmacophore and resulted in an HQ461 derivate with improved potency. Our studies reveal a new molecular glue that recruits its target protein directly to DDB1 to bypass the requirement of a substrate-specific receptor, presenting a new strategy for targeted protein degradation.
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http://dx.doi.org/10.7554/eLife.59994DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7462607PMC
August 2020

A UPR-Induced Soluble ER-Phagy Receptor Acts with VAPs to Confer ER Stress Resistance.

Mol Cell 2020 09 30;79(6):963-977.e3. Epub 2020 Jul 30.

National Institute of Biological Sciences, 102206 Beijing, China; Tsinghua Institute of Multidisciplinary Biomedical Research, Tsinghua University, 102206 Beijing, China. Electronic address:

Autophagic degradation of the endoplasmic reticulum (ER-phagy) is triggered by ER stress in diverse organisms. However, molecular mechanisms governing ER stress-induced ER-phagy remain insufficiently understood. Here we report that ER stress-induced ER-phagy in the fission yeast Schizosaccharomyces pombe requires Epr1, a soluble Atg8-interacting ER-phagy receptor. Epr1 localizes to the ER through interacting with integral ER membrane proteins VAPs. Bridging an Atg8-VAP association is the main ER-phagy role of Epr1, as it can be bypassed by an artificial Atg8-VAP tether. VAPs contribute to ER-phagy not only by tethering Atg8 to the ER membrane, but also by maintaining the ER-plasma membrane contact. Epr1 is upregulated during ER stress by the unfolded protein response (UPR) regulator Ire1. Loss of Epr1 reduces survival against ER stress. Conversely, increasing Epr1 expression suppresses the ER-phagy defect and ER stress sensitivity of cells lacking Ire1. Our findings expand and deepen the molecular understanding of ER-phagy.
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http://dx.doi.org/10.1016/j.molcel.2020.07.019DOI Listing
September 2020

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

Structural snapshots of human pre-60S ribosomal particles before and after nuclear export.

Nat Commun 2020 07 15;11(1):3542. Epub 2020 Jul 15.

State Key Laboratory of Membrane Biology, Peking-Tsinghua Joint Centre for Life Sciences, School of Life Sciences, Peking University, 100871, Beijing, China.

Ribosome biogenesis is an elaborate and energetically expensive program that involve two hundred protein factors in eukaryotes. Nuclear export of pre-ribosomal particles is one central step which also serves as an internal structural checkpoint to ensure the proper completion of nuclear assembly events. Here we present four structures of human pre-60S particles isolated through a nuclear export factor NMD3, representing assembly stages immediately before and after nuclear export. These structures reveal locations of a dozen of human factors, including an uncharacterized factor TMA16 localized between the 5S RNA and the P0 stalk. Comparison of these structures shows a progressive maturation for the functional regions, such as peptidyl transferase centre and peptide exit tunnel, and illustrate a sequence of factor-assisted rRNA maturation events. These data facilitate our understanding of the global conservation of ribosome assembly in eukaryotes and species-specific features of human assembly factors.
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http://dx.doi.org/10.1038/s41467-020-17237-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7363849PMC
July 2020

New and Improved Tools for the Omics Crew.

J Proteome Res 2020 07;19(7):2525-2528

Biodesign Institute, Arizona State University.

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http://dx.doi.org/10.1021/acs.jproteome.0c00328DOI Listing
July 2020

Spatial confinement of receptor activity by tyrosine phosphatase during directional cell migration.

Proc Natl Acad Sci U S A 2020 06 8;117(25):14270-14279. Epub 2020 Jun 8.

Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing 100084, China;

Directional cell migration involves signaling cascades that stimulate actin assembly at the leading edge, and additional pathways must inhibit actin polymerization at the rear. During neuroblast migration in , the transmembrane protein MIG-13/Lrp12 acts through the Arp2/3 nucleation-promoting factors WAVE and WASP to guide the anterior migration. Here we show that a tyrosine kinase, SRC-1, directly phosphorylates MIG-13 and promotes its activity on actin assembly at the leading edge. In GFP knockin animals, SRC-1 and MIG-13 distribute along the entire plasma membrane of migrating cells. We reveal that a receptor-like tyrosine phosphatase, PTP-3, maintains the F-actin polarity during neuroblast migration. Recombinant PTP-3 dephosphorylates SRC-1-dependent MIG-13 phosphorylation in vitro. Importantly, the endogenous PTP-3 accumulates at the rear of the migrating neuroblast, and its extracellular domain is essential for directional cell migration. We provide evidence that the asymmetrically localized tyrosine phosphatase PTP-3 spatially restricts MIG-13/Lrp12 receptor activity in migrating cells.
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http://dx.doi.org/10.1073/pnas.2003019117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7321996PMC
June 2020

The acetyltransferase Eco1 elicits cohesin dimerization during S phase.

J Biol Chem 2020 05 20;295(22):7554-7565. Epub 2020 Apr 20.

State Key Laboratory of Agro-Biotechnology and Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Biological Sciences, China Agricultural University, No. 2 Yuan-Ming-Yuan West Road, Beijing 100193, China

Cohesin is a DNA-associated protein complex that forms a tripartite ring controlling sister chromatid cohesion, chromosome segregation and organization, DNA replication, and gene expression. Sister chromatid cohesion is established by the protein acetyltransferase Eco1, which acetylates two conserved lysine residues on the cohesin subunit Smc3 and thereby ensures correct chromatid separation in yeast () and other eukaryotes. However, the consequence of Eco1-catalyzed cohesin acetylation is unknown, and the exact nature of the cohesive state of chromatids remains controversial. Here, we show that self-interactions of the cohesin subunits Scc1/Rad21 and Scc3 occur in a DNA replication-coupled manner in both yeast and human cells. Using cross-linking MS-based and disulfide cross-linking analyses of purified cohesin, we show that a subpopulation of cohesin may exist as dimers. Importantly, upon temperature-sensitive and auxin-induced degron-mediated Eco1 depletion, the cohesin-cohesin interactions became significantly compromised, whereas deleting either the deacetylase Hos1 or the Eco1 antagonist Wpl1/Rad61 increased cohesin dimer levels by ∼20%. These results indicate that cohesin dimerizes in the S phase and monomerizes in mitosis, processes that are controlled by Eco1, Wpl1, and Hos1 in the sister chromatid cohesion-dissolution cycle. These findings suggest that cohesin dimerization is controlled by the cohesion cycle and support the notion that a double-ring cohesin model operates in sister chromatid cohesion.
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http://dx.doi.org/10.1074/jbc.RA120.013102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7261783PMC
May 2020

Author Correction: Cryo-EM structure and biochemical analysis reveal the basis of the functional difference between human PI3KC3-C1 and -C2.

Cell Res 2020 Jun;30(6):551-552

Ministry of Education Key Laboratory of Protein Sciences, Tsinghua-Peking Joint Center for Life Sciences, Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, 100084, China.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41422-020-0311-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7264212PMC
June 2020

Rett syndrome-causing mutations compromise MeCP2-mediated liquid-liquid phase separation of chromatin.

Cell Res 2020 05 28;30(5):393-407. Epub 2020 Feb 28.

Beijing Advanced Innovation Center for Structural Biology, Beijing Frontier Research Center for Biological Structure, Tsinghua University-Peking University Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, China.

Rett syndrome (RTT), a severe postnatal neurodevelopmental disorder, is caused by mutations in the X-linked gene encoding methyl-CpG-binding protein 2 (MeCP2). MeCP2 is a chromatin organizer regulating gene expression. RTT-causing mutations have been shown to affect this function. However, the mechanism by which MeCP2 organizes chromatin is unclear. In this study, we found that MeCP2 can induce compaction and liquid-liquid phase separation of nucleosomal arrays in vitro, and DNA methylation further enhances formation of chromatin condensates by MeCP2. Interestingly, RTT-causing mutations compromise MeCP2-mediated chromatin phase separation, while benign variants have little effect on this process. Moreover, MeCP2 competes with linker histone H1 to form mutually exclusive chromatin condensates in vitro and distinct heterochromatin foci in vivo. RTT-causing mutations reduce or even abolish the ability of MeCP2 to compete with histone H1 and to form chromatin condensates. Together, our results identify a novel mechanism by which phase separation underlies MeCP2-mediated heterochromatin formation and reveal the potential link between this process and the pathology of RTT.
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http://dx.doi.org/10.1038/s41422-020-0288-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7196128PMC
May 2020

Atg38-Atg8 interaction in fission yeast establishes a positive feedback loop to promote autophagy.

Autophagy 2020 11 19;16(11):2036-2051. Epub 2020 Jan 19.

National Institute of Biological Sciences , Beijing, China.

Macroautophagy (autophagy) is driven by the coordinated actions of core autophagy-related (Atg) proteins. Atg8, the core Atg protein generally considered acting most downstream, has recently been shown to interact with other core Atg proteins via their Atg8-family-interacting motifs (AIMs). However, the extent, functional consequence, and evolutionary conservation of such interactions remain inadequately understood. Here, we show that, in the fission yeast , Atg38, a subunit of the phosphatidylinositol 3-kinase (PtdIns3K) complex I, interacts with Atg8 via an AIM, which is highly conserved in Atg38 proteins of fission yeast species, but not conserved in Atg38 proteins of other species. This interaction recruits Atg38 to Atg8 on the phagophore assembly site (PAS) and consequently enhances PAS accumulation of the PtdIns3K complex I and Atg proteins acting downstream of the PtdIns3K complex I, including Atg8. The disruption of the Atg38-Atg8 interaction leads to the reduction of autophagosome size and autophagic flux. Remarkably, the loss of this interaction can be compensated by an artificial Atg14-Atg8 interaction. Our findings demonstrate that the Atg38-Atg8 interaction in fission yeast establishes a positive feedback loop between Atg8 and the PtdIns3K complex I to promote efficient autophagosome formation, underscore the prevalence and diversity of AIM-mediated connections within the autophagic machinery, and reveal unforeseen flexibility of such connections. : AIM: Atg8-family-interacting motif; AP-MS: affinity purification coupled with mass spectrometry; Atg: autophagy-related; FLIP: fluorescence loss in photobleaching; PAS: phagophore assembly site; ; PE: phosphatidylethanolamine; PtdIns3K: phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol 3-phosphate.
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http://dx.doi.org/10.1080/15548627.2020.1713644DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7595586PMC
November 2020

Characterization of the dimeric CMG/pre-initiation complex and its transition into DNA replication forks.

Cell Mol Life Sci 2020 Aug 15;77(15):3041-3058. Epub 2019 Nov 15.

State Key Laboratory of Agro-Biotechnology and Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Biological Sciences China Agricultural University, Beijing, China.

The pre-initiation complex (pre-IC) has been proposed for two decades as an intermediate right before the maturation of the eukaryotic DNA replication fork. However, its existence and biochemical nature remain enigmatic. Here, through combining several enrichment strategies, we are able to isolate an endogenous dimeric CMG-containing complex (designated as d-CMG) distinct from traditional single CMG (s-CMG) and in vitro reconstituted dimeric CMG. D-CMG is assembled upon entry into the S phase and shortly matures into s-CMG/replisome, leading to the fact that only ~ 5% of the total CMG-containing complexes can be detected as d-CMG in vivo. Mass spectra reveal that RPA and DNA Pol α/primase co-purify with s-CMG, but not with d-CMG. Consistently, the former fraction is able to catalyze DNA unwinding and de novo synthesis, while the latter catalyzes neither. The two CMGs in d-CMG display flexibly orientated conformations under an electronic microscope. When DNA Pol α-primase is inactivated, d-CMG % rose up to 29%, indicating an incomplete pre-IC/fork transition. These findings reveal biochemical properties of the d-CMG/pre-IC and provide in vivo evidence to support the pre-IC/fork transition as a bona fide step in replication initiation.
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http://dx.doi.org/10.1007/s00018-019-03333-9DOI Listing
August 2020

A Pandas complex adapted for piRNA-guided transcriptional silencing and heterochromatin formation.

Nat Cell Biol 2019 10 30;21(10):1261-1272. Epub 2019 Sep 30.

Key Laboratory of RNA Biology, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.

The repression of transposons by the Piwi-interacting RNA (piRNA) pathway is essential to protect animal germ cells. In Drosophila, Panoramix enforces transcriptional silencing by binding to the target-engaged Piwi-piRNA complex, although the precise mechanisms by which this occurs remain elusive. Here, we show that Panoramix functions together with a germline-specific paralogue of a nuclear export factor, dNxf2, and its cofactor dNxt1 (p15), to suppress transposon expression. The transposon RNA-binding protein dNxf2 is required for animal fertility and Panoramix-mediated silencing. Transient tethering of dNxf2 to nascent transcripts leads to their nuclear retention. The NTF2 domain of dNxf2 competes dNxf1 (TAP) off nucleoporins, a process required for proper RNA export. Thus, dNxf2 functions in a Panoramix-dNxf2-dependent TAP/p15 silencing (Pandas) complex that counteracts the canonical RNA exporting machinery and restricts transposons to the nuclear peripheries. Our findings may have broader implications for understanding how RNA metabolism modulates heterochromatin formation.
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http://dx.doi.org/10.1038/s41556-019-0396-0DOI Listing
October 2019

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

Chk2-dependent phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) regulates centrosome maturation.

Cell Cycle 2019 Oct 15;18(20):2651-2659. Epub 2019 Aug 15.

Beijing Key Laboratory of DNA damage Response, College of Life Sciences, Capital Normal University , Beijing , China.

Checkpoint kinase 2 (Chk2) is a pivotal effector kinase in the DNA damage response, with an emerging role in mitotic chromosome segregation. In this study, we show that Chk2 interacts with myosin phosphatase targeting subunit 1 (MYPT1), the targeting subunit of protein phosphatase 1cβ (PP1cβ). Previous studies have shown that MYPT1 is phosphorylated by CDK1 at S473 during mitosis, and subsequently docks to the polo-binding domain of PLK1 and dephosphorylates PLK1. Herein we present data that Chk2 phosphorylates MYPT1 at S507 and , which antagonizes pS473. Chk2 inhibition results in failure of γ-tubulin recruitment to the centrosomes, phenocopying Plk1 inhibition defects. These aberrancies were also observed in the MYPT1-S507A stable transfectants, suggesting that Chk2 exerts its effect on centrosomes via MYPT1. Collectively, we have identified a Chk2-MYPT1-PLK1 axis in regulating centrosome maturation. : Chk2: checkpoint kinase 2; MYPT1: myosin phosphatase targeting subunit 1; PP1cβ: protein phosphatase 1c β; Noc: nocodazole; IP: immunoprecipitation; IB: immunoblotting; LC-MS/MS: liquid chromatography-tandem mass spectrometry; Chk2: checkpoint kinase 2; KD: kinase domain; WT: wild type; Ub: ubiquitin; DAPI: 4',6-diamidino-2-phenylindole; IF: Immunofluorescence; IR: ionizing radiation; siCHK2: siRNA targeting CHK2.
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http://dx.doi.org/10.1080/15384101.2019.1654795DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6773232PMC
October 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

Functional Proteomics Identifies a PICS Complex Required for piRNA Maturation and Chromosome Segregation.

Cell Rep 2019 06;27(12):3561-3572.e3

Hefei National Laboratory for Physical Sciences at the Microscale, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, P.R. China; CAS Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Hefei, Anhui 230027, P.R. China. Electronic address:

piRNAs play significant roles in suppressing transposons and nonself nucleic acids, maintaining genome integrity, and defending against viral infections. In C. elegans, piRNA precursors are transcribed in the nucleus and are subjected to a number of processing and maturation steps. The biogenesis of piRNAs is not fully understood. We use functional proteomics in C. elegans and identify a piRNA biogenesis and chromosome segregation (PICS) complex. The PICS complex contains TOFU-6, PID-1, PICS-1, TOST-1, and ERH-2, which exhibit dynamic localization among different subcellular compartments. In the germlines, the PICS complex contains TOFU-6/PICS-1/ERH-2/PID-1, is largely concentrated at the perinuclear granule zone, and engages in piRNA processing. During embryogenesis, the TOFU-6/PICS-1/ERH-2/TOST-1 complex accumulates in the nucleus and plays essential roles in chromosome segregation. The functions of these factors in mediating chromosome segregation are independent of piRNA production. We speculate that differential compositions of PICS factors may help cells coordinate distinct cellular processes.
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http://dx.doi.org/10.1016/j.celrep.2019.05.076DOI Listing
June 2019

Architecture of SAGA complex.

Cell Discov 2019 7;5:25. Epub 2019 May 7.

1MOE Key Laboratory of Protein Sciences, Beijing Advanced Innovation Center for Structural Biology, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences and School of Medicine, Tsinghua University, Beijing, 100084 China.

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http://dx.doi.org/10.1038/s41421-019-0094-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6502868PMC
May 2019

First Community-Wide, Comparative Cross-Linking Mass Spectrometry Study.

Anal Chem 2019 06 22;91(11):6953-6961. Epub 2019 May 22.

Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, Safra Campus Givat Ram , The Hebrew University of Jerusalem , Jerusalem 91904 , Israel.

The number of publications in the field of chemical cross-linking combined with mass spectrometry (XL-MS) to derive constraints for protein three-dimensional structure modeling and to probe protein-protein interactions has increased during the last years. As the technique is now becoming routine for in vitro and in vivo applications in proteomics and structural biology there is a pressing need to define protocols as well as data analysis and reporting formats. Such consensus formats should become accepted in the field and be shown to lead to reproducible results. This first, community-based harmonization study on XL-MS is based on the results of 32 groups participating worldwide. The aim of this paper is to summarize the status quo of XL-MS and to compare and evaluate existing cross-linking strategies. Our study therefore builds the framework for establishing best practice guidelines to conduct cross-linking experiments, perform data analysis, and define reporting formats with the ultimate goal of assisting scientists to generate accurate and reproducible XL-MS results.
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http://dx.doi.org/10.1021/acs.analchem.9b00658DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6625963PMC
June 2019

Mechanistic insights into the SNARE complex disassembly.

Sci Adv 2019 04 10;5(4):eaau8164. Epub 2019 Apr 10.

State Key Laboratory of Membrane Biology, Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China.

NSF (-ethylmaleimide-sensitive factor) and α-SNAP (α-soluble NSF attachment protein) bind to the SNARE (soluble NSF attachment protein receptor) complex, the minimum machinery to mediate membrane fusion, to form a 20S complex, which disassembles the SNARE complex for reuse. We report the cryo-EM structures of the α-SNAP-SNARE subcomplex and the NSF-D1D2 domain in the 20S complex at 3.9- and 3.7-Å resolutions, respectively. Combined with the biochemical and electrophysiological analyses, we find that α-SNAPs use R116 through electrostatic interactions and L197 through hydrophobic interactions to apply force mainly on two positions of the VAMP protein to execute disassembly process. Furthermore, we define the interaction between the amino terminus of the SNARE helical bundle and the pore loop of the NSF-D1 domain and demonstrate its essential role as a potential anchor for SNARE complex disassembly. Our studies provide a rotation model of α-SNAP-mediated disassembly of the SNARE complex.
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http://dx.doi.org/10.1126/sciadv.aau8164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6457932PMC
April 2019

Systematic analysis reveals the prevalence and principles of bypassable gene essentiality.

Nat Commun 2019 03 1;10(1):1002. Epub 2019 Mar 1.

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

Gene essentiality is a variable phenotypic trait, but to what extent and how essential genes can become dispensable for viability remain unclear. Here, we investigate 'bypass of essentiality (BOE)' - an underexplored type of digenic genetic interaction that renders essential genes dispensable. Through analyzing essential genes on one of the six chromosome arms of the fission yeast Schizosaccharomyces pombe, we find that, remarkably, as many as 27% of them can be converted to non-essential genes by BOE interactions. Using this dataset we identify three principles of essentiality bypass: bypassable essential genes tend to have lower importance, tend to exhibit differential essentiality between species, and tend to act with other bypassable genes. In addition, we delineate mechanisms underlying bypassable essentiality, including the previously unappreciated mechanism of dormant redundancy between paralogs. The new insights gained on bypassable essentiality deepen our understanding of genotype-phenotype relationships and will facilitate drug development related to essential genes.
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http://dx.doi.org/10.1038/s41467-019-08928-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6397241PMC
March 2019

DAF-16 stabilizes the aging transcriptome and is activated in mid-aged Caenorhabditis elegans to cope with internal stress.

Aging Cell 2019 06 17;18(3):e12896. Epub 2019 Feb 17.

National Institute of Biological Sciences, Beijing, China.

The roles and regulatory mechanisms of transcriptome changes during aging are unclear. It has been proposed that the transcriptome suffers decay during aging owing to age-associated down-regulation of transcription factors. In this study, we characterized the role of a transcription factor DAF-16, which is a highly conserved lifespan regulator, in the normal aging process of Caenorhabditis elegans. We found that DAF-16 translocates into the nucleus in aged wild-type worms and activates the expression of hundreds of genes in response to age-associated cellular stress. Most of the age-dependent DAF-16 targets are different from the canonical DAF-16 targets downstream of insulin signaling. This and other evidence suggest that activation of DAF-16 during aging is distinct from activation of DAF-16 due to reduced signaling from DAF-2. Further analysis showed that it is due in part to a loss of proteostasis during aging. We also found that without daf-16, dramatic gene expression changes occur as early as on adult day 2, indicating that DAF-16 acts to stabilize the transcriptome during normal aging. Our results thus reveal that normal aging is not simply a process in which the gene expression program descends into chaos due to loss of regulatory activities; rather, there is active transcriptional regulation during aging.
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http://dx.doi.org/10.1111/acel.12896DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6516157PMC
June 2019

The USTC co-opts an ancient machinery to drive piRNA transcription in .

Genes Dev 2019 01 19;33(1-2):90-102. Epub 2018 Dec 19.

Wellcome Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge CB2 1QN, United Kingdom; Department of Genetics, University of Cambridge, Cambridge CB2 3EH, United Kingdom.

Piwi-interacting RNAs (piRNAs) engage Piwi proteins to suppress transposons and nonself nucleic acids and maintain genome integrity and are essential for fertility in a variety of organisms. In , most piRNA precursors are transcribed from two genomic clusters that contain thousands of individual piRNA transcription units. While a few genes have been shown to be required for piRNA biogenesis, the mechanism of piRNA transcription remains elusive. Here we used functional proteomics approaches to identify an upstream sequence transcription complex (USTC) that is essential for piRNA biogenesis. The USTC contains piRNA silencing-defective 1 (PRDE-1), SNPC-4, twenty-one-U fouled-up 4 (TOFU-4), and TOFU-5. The USTC forms unique piRNA foci in germline nuclei and coats the piRNA cluster genomic loci. USTC factors associate with the Ruby motif just upstream of type I piRNA genes. USTC factors are also mutually dependent for binding to the piRNA clusters and forming the piRNA foci. Interestingly, USTC components bind differentially to piRNAs in the clusters and other noncoding RNA genes. These results reveal the USTC as a striking example of the repurposing of a general transcription factor complex to aid in genome defense against transposons.
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http://dx.doi.org/10.1101/gad.319293.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6317315PMC
January 2019

Author Correction: Cryo-EM structure of the exocyst complex.

Nat Struct Mol Biol 2018 Dec;25(12):1137

Department of Biology, University of Pennsylvania, Philadelphia, PA, USA.

In the version of this article originally published, the value given for electron dose in Table 1 was incorrect. This value was originally stated as 4.8 but should have been 50. The error has been corrected in the HTML and PDF versions of the article.
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http://dx.doi.org/10.1038/s41594-018-0157-yDOI Listing
December 2018

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

Connective tissue growth factor (CCN2) is a matricellular preproprotein controlled by proteolytic activation.

J Biol Chem 2018 11 27;293(46):17953-17970. Epub 2018 Sep 27.

From the Institute for Surgical Research, Oslo University Hospital and University of Oslo, NO-0424 Oslo, Norway; Center for Heart Failure Research, University of Oslo, NO-0316 Oslo, Norway. Electronic address:

Connective tissue growth factor (CTGF; now often referred to as CCN2) is a secreted protein predominantly expressed during development, in various pathological conditions that involve enhanced fibrogenesis and tissue fibrosis, and in several cancers and is currently an emerging target in several early-phase clinical trials. Tissues containing high CCN2 activities often display smaller degradation products of full-length CCN2 (FL-CCN2). Interpretation of these observations is complicated by the fact that a uniform protein structure that defines biologically active CCN2 has not yet been resolved. Here, using DG44 CHO cells engineered to produce and secrete FL-CCN2 and cell signaling and cell physiological activity assays, we demonstrate that FL-CCN2 is itself an inactive precursor and that a proteolytic fragment comprising domains III (thrombospondin type 1 repeat) and IV (cystine knot) appears to convey all biologically relevant activities of CCN2. In congruence with these findings, purified FL-CCN2 could be cleaved and activated following incubation with matrix metalloproteinase activities. Furthermore, the C-terminal fragment of CCN2 (domains III and IV) also formed homodimers that were ∼20-fold more potent than the monomeric form in activating intracellular phosphokinase cascades. The homodimer elicited activation of fibroblast migration, stimulated assembly of focal adhesion complexes, enhanced RANKL-induced osteoclast differentiation of RAW264.7 cells, and promoted mammosphere formation of MCF-7 mammary cancer cells. In conclusion, CCN2 is synthesized and secreted as a preproprotein that is autoinhibited by its two N-terminal domains and requires proteolytic processing and homodimerization to become fully biologically active.
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http://dx.doi.org/10.1074/jbc.RA118.004559DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6240875PMC
November 2018

Author Correction: Structure and mechanogating mechanism of the Piezo1 channel.

Nature 2018 11;563(7730):E19

School of Pharmaceutical Sciences or Life Sciences, Tsinghua University, Beijing, China.

In Extended Data Fig. 9a of this Article, the bottom micrographs of mPiezo1-ΔL3-4-IRES-GFP and mPiezo1-ΔL7-8-IRES-GFP (labelled 'permeabilized') are inadvertently the same images. The corrected figure panels are shown in the accompanying Amendment.
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http://dx.doi.org/10.1038/s41586-018-0513-4DOI Listing
November 2018

Mapping disulfide bonds from sub-micrograms of purified proteins or micrograms of complex protein mixtures.

Biophys Rep 2018 23;4(2):68-81. Epub 2018 Apr 23.

1National Institute of Biological Sciences, Beijing, Beijing, 102206 China.

Disulfide bonds are vital for protein functions, but locating the linkage sites has been a challenge in protein chemistry, especially when the quantity of a sample is small or the complexity is high. In 2015, our laboratory developed a sensitive and efficient method for mapping protein disulfide bonds from simple or complex samples (Lu . in Nat Methods 12:329, 2015). This method is based on liquid chromatography-mass spectrometry (LC-MS) and a powerful data analysis software tool named pLink. To facilitate application of this method, we present step-by-step disulfide mapping protocols for three types of samples-purified proteins in solution, proteins in SDS-PAGE gels, and complex protein mixtures in solution. The minimum amount of protein required for this method can be as low as several hundred nanograms for purified proteins, or tens of micrograms for a mixture of hundreds of proteins. The entire workflow-from sample preparation to LC-MS and data analysis-is described in great detail. We believe that this protocol can be easily implemented in any laboratory with access to a fast-scanning, high-resolution, and accurate-mass LC-MS system.
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http://dx.doi.org/10.1007/s41048-018-0050-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5937861PMC
April 2018

Cryo-EM structure of an early precursor of large ribosomal subunit reveals a half-assembled intermediate.

Protein Cell 2019 02 19;10(2):120-130. Epub 2018 Mar 19.

Graduate School of Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, 100730, China.

Assembly of eukaryotic ribosome is a complicated and dynamic process that involves a series of intermediates. It is unknown how the highly intertwined structure of 60S large ribosomal subunits is established. Here, we report the structure of an early nucleolar pre-60S ribosome determined by cryo-electron microscopy at 3.7 Å resolution, revealing a half-assembled subunit. Domains I, II and VI of 25S/5.8S rRNA pack tightly into a native-like substructure, but domains III, IV and V are not assembled. The structure contains 12 assembly factors and 19 ribosomal proteins, many of which are required for early processing of large subunit rRNA. The Brx1-Ebp2 complex would interfere with the assembly of domains IV and V. Rpf1, Mak16, Nsa1 and Rrp1 form a cluster that consolidates the joining of domains I and II. Our structure reveals a key intermediate on the path to establishing the global architecture of 60S subunits.
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http://dx.doi.org/10.1007/s13238-018-0526-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6340896PMC
February 2019