Biology subjects

Song, G.

Publications and source records attributed to Song, G..

At least 73 records · Page 4Linked to original sources

bioRxiv2021

iCn3D: from Web-based 3D Viewer to Structure Analysis Tool in Batch Mode

iCn3D was initially developed as a web-based 3D molecular viewer. It then evolved from visualization into a full-featured interactive structural analysis software. It became a collaborative research instrument through the sharing of permanent, shortened URLs that encapsulate not only annotated visual molecular scenes, but also all underlying data and analysis scripts in a FAIR manner. More recently, with the growth of structural databases, the need to analyze large structural datasets systematically led us to use Python scripts and convert the code to be used in Node.js scripts. We showed a few examples of Python scripts at https://github.com/ncbi/icn3d/tree/master/icn3dpython to export secondary structures or PNG images from iCn3D. Users just need to replace the URL in the Python scripts to export other annotations from iCn3D. Furthermore, any interactive iCn3D feature can be converted into a Node.js script to be run in batch mode, enabling an interactive analysis performed on one or a handful of protein complexes to be scaled up to analysis features of large ensembles of structures. Currently available Node.js analysis scripts examples are available at https://github.com/ncbi/icn3d/tree/master/icn3dnode. This development will enable ensemble analyses on growing structural databases such as AlphaFold or RoseTTAFold on one hand and Electron Microscopy on the other. In this paper, we also review new features such as DelPhi electrostatic potential, 3D view of mutations, alignment of multiple chains, assembly of multiple structures by realignment, dynamic symmetry calculation, 2D cartoons at different levels, interactive contact maps, and use of iCn3D in Jupyter Notebook as described at https://pypi.org/project/icn3dpy.

bioinformatics↗

bioRxiv2021

Crystal structure of a constitutive active mutant of adenosine A2A receptor

Previously we reported a common activation pathway of the class A G protein-coupled receptors (GPCRs) in which a series of conserved residues/motifs undergo conformational change during extracellular agonist binding and finally induce the coupling of intracellular G protein, and successfully predicted several novel constitutive active or inactive mutations for A2A adenosine receptor (A2AAR) through this mechanism (Zhou et al., 2019). Here we determined the crystal structure of a typical A2AAR constitutive active mutant I92N in complex with agonist UK-432097 to reveal the molecular mechanism of mutation-induced constitutive activity. The mutated I92N forms a hydrophilic interaction network with nearby residues including W6.48 of the CWxP motif, which is absent in the wild type A2AAR. Although the mutant structure is overall similar to the previously determined intermediate state A2AAR structure (PDB ID: 3QAK), the molecular dynamics simulations suggested that the I92N mutant stabilizes the metastable intermediate state through the hydrophilic interaction network and favors the receptor conformational transition towards the active state. This research provides a structural template toward the special pharmacological outcome triggered by conformational mutation and sheds light on future structural or pharmacological studies among class A GPCRs.

molecular biology↗

bioRxiv2021

Targeted isolation of panels of diverse human broadly neutralizing antibodies against SARS-like viruses

The emergence of current SARS-CoV-2 variants of concern (VOCs) and potential future spillovers of SARS-like coronaviruses into humans pose a major threat to human health and the global economy 1-7. Development of broadly effective coronavirus vaccines that can mitigate these threats is needed 8, 9. Notably, several recent studies have revealed that vaccination of recovered COVID-19 donors results in enhanced nAb responses compared to SARS-CoV-2 infection or vaccination alone 10-13. Here, we utilized a targeted donor selection strategy to isolate a large panel of broadly neutralizing antibodies (bnAbs) to sarbecoviruses from two such donors. Many of the bnAbs are remarkably effective in neutralization against sarbecoviruses that use ACE2 for viral entry and a substantial fraction also show notable binding to non-ACE2-using sarbecoviruses. The bnAbs are equally effective against most SARS-CoV-2 VOCs and many neutralize the Omicron variant. Neutralization breadth is achieved by bnAb binding to epitopes on a relatively conserved face of the receptor binding domain (RBD) as opposed to strain-specific nAbs to the receptor binding site that are commonly elicited in SARS-CoV-2 infection and vaccination 14-18. Consistent with targeting of conserved sites, select RBD bnAbs exhibited in vivo protective efficacy against diverse SARS-like coronaviruses in a prophylaxis challenge model. The generation of a large panel of potent bnAbs provides new opportunities and choices for next-generation antibody prophylactic and therapeutic applications and, importantly, provides a molecular basis for effective design of pan-sarbecovirus vaccines.

immunology↗

bioRxiv2021

Broadly neutralizing antibodies to SARS-related viruses can be readily induced in rhesus macaques

To prepare for future coronavirus (CoV) pandemics, it is desirable to generate vaccines capable of eliciting neutralizing antibody responses against multiple CoVs. Because of the phylogenetic similarity to humans, rhesus macaques are an animal model of choice for many virus-challenge and vaccine-evaluation studies, including SARS-CoV-2. Here, we show that immunization of macaques with SARS-CoV-2 spike (S) protein generates potent receptor binding domain cross- neutralizing antibody (nAb) responses to both SARS-CoV-2 and SARS-CoV-1, in contrast to human infection or vaccination where responses are typically SARS-CoV-2-specific. Furthermore, the macaque nAbs are equally effective against SARS-CoV-2 variants of concern. Structural studies show that different immunodominant sites are targeted by the two primate species. Human antibodies generally target epitopes strongly overlapping the ACE2 receptor binding site (RBS), whereas the macaque antibodies recognize a relatively conserved region proximal to the RBS that represents another potential pan-SARS-related virus site rarely targeted by human antibodies. B cell repertoire differences between the two primates appear to significantly influence the vaccine response and suggest care in the use of rhesus macaques in evaluation of vaccines to SARS-related viruses intended for human use. ONE SENTENCE SUMMARYBroadly neutralizing antibodies to an unappreciated site of conservation in the RBD in SARS- related viruses can be readily induced in rhesus macaques because of distinct properties of the naive macaque B cell repertoire that suggest prudence in the use of the macaque model in SARS vaccine evaluation and design.

immunology↗

bioRxiv2021

New distributional opportunities with niche innovation in Eurasian snowfinches

This study explores the evolutionary history of ecological niche characters in the Eurasian snowfinch lineage. Specifically, we use new analytical approaches to reconstruct ecological niche evolution, taking uncertainty in knowledge of the ecological niche limits into account. We found an overall pattern of niche conservatism in relation to both temperature and precipitation characteristics of niches, but for one dramatic niche evolution event, in Montifringilla nivalis. Interestingly, this species is also that which has by far the broadest geographic distribution among snowfinches. We conclude that an evolutionary change in niche characteristics perhaps within M. nivalis (i.e., present in some and not all of its populations) made possible the broad, westward range expansion of that species, thus changing the distributional potential of the snowfinch lineage dramatically.

evolutionary biology↗

bioRxiv2021

A protective broadly cross-reactive human antibody defines a conserved site of vulnerability on beta-coronavirus spikes

Broadly neutralizing antibodies (bnAbs) to coronaviruses (CoVs) are valuable in their own right as prophylactic and therapeutic reagents to treat diverse CoVs and, importantly, as templates for rational pan-CoV vaccine design. We recently described a bnAb, CC40.8, from a coronavirus disease 2019 (COVID-19)-convalescent donor that exhibits broad reactivity with human beta-coronaviruses ({beta}-CoVs). Here, we showed that CC40.8 targets the conserved S2 stem-helix region of the coronavirus spike fusion machinery. We determined a crystal structure of CC40.8 Fab with a SARS-CoV-2 S2 stem-peptide at 1.6 [A] resolution and found that the peptide adopted a mainly helical structure. Conserved residues in {beta}-CoVs interacted with CC40.8 antibody, thereby providing a molecular basis for its broad reactivity. CC40.8 exhibited in vivo protective efficacy against SARS-CoV-2 challenge in two animal models. In both models, CC40.8-treated animals exhibited less weight loss and reduced lung viral titers compared to controls. Furthermore, we noted CC40.8-like bnAbs are relatively rare in human COVID-19 infection and therefore their elicitation may require rational structure-based vaccine design strategies. Overall, our study describes a target on {beta}-CoV spike proteins for protective antibodies that may facilitate the development of pan-{beta}-CoV vaccines. SUMMARYA human mAb isolated from a COVID-19 donor defines a protective cross-neutralizing epitope for pan-{beta}-CoV vaccine design strategies

immunology↗

bioRxiv2021

Site-specific steric control of SARS-CoV-2 spike glycosylation

A central tenet in the design of vaccines is the display of native-like antigens in the elicitation of protective immunity. The abundance of N-linked glycans across the SARS-CoV-2 spike protein is a potential source of heterogeneity between the many different vaccine candidates under investigation. Here, we investigate the glycosylation of recombinant SARS-CoV-2 spike proteins from five different laboratories and compare them against infectious virus S protein. We find patterns which are conserved across all samples and this can be associated with site-specific stalling of glycan maturation which act as a highly sensitive reporter of protein structure. Molecular dynamics (MD) simulations of a fully glycosylated spike support s a model of steric restrictions that shape enzymatic processing of the glycans. These results suggest that recombinant spike-based SARS-CoV-2 immunogen glycosylation reproducibly recapitulates signatures of viral glycosylation.

biochemistry↗

bioRxiv2021

Disassembly of HIV envelope glycoprotein trimer immunogens is driven by antibodies elicited via immunization

Rationally designed protein subunit vaccines are being developed for a variety of viruses including influenza, RSV, SARS-CoV-2 and HIV. These vaccines are based on stabilized versions of the primary targets of neutralizing antibodies on the viral surface, namely viral fusion glycoproteins. While these immunogens display the epitopes of potent neutralizing antibodies, they also present epitopes recognized by non or weakly neutralizing ("off-target") antibodies. Using our recently developed electron microscopy epitope mapping approach, we have uncovered a phenomenon wherein off-target antibodies elicited by HIV trimer subunit vaccines cause the otherwise highly stabilized trimeric proteins to degrade into cognate protomers. Further, we show that these protomers expose an expanded suite of off-target epitopes, normally occluded inside the prefusion conformation of trimer, that subsequently elicit further off-target antibody responses. Our study provides critical insights for further improvement of HIV subunit trimer vaccines for future rounds of the iterative vaccine design process.

immunology↗

bioRxiv2021

Interplay between brassinosteroids and TORC signaling in Arabidopsis revealed by integrated multi-dimensional analysis

Brassinosteroids (BR) and Target of Rapamycin Complex (TORC) are two major processes coordinating plant growth and stress responses. BRs function through a signaling pathway to extensively regulate gene expression and TORC is known to regulate translation and autophagy. Recent studies revealed that these two pathways crosstalk, but a system-wide view of their interplay is still missing. Thus, we performed transcriptome, proteome, and phosphoproteome profiling of Arabidopsis mutants with altered levels of either BIN2 or RAPTOR1B, two key players in BR and TORC signaling, respectively. We found that perturbation of BIN2 or RAPTOR1B levels affects a common set of gene-products involved in growth and stress responses. Additionally, we performed Multiplexed Assay for Kinase Specificity (MAKS), which provided a system-wide view of direct BIN2 substrates. Furthermore, phosphoproteomic data was used to reconstruct a kinase-signaling network and to identify novel proteins dependent on BR and/or TORC signaling pathways. Loss of function mutants of many of these proteins led to an altered BR response and/or modulated autophagy activity. Altogether, these results provide genome-wide evidence for crosstalk between BR and TORC signaling and established a kinase signaling network that defines the molecular mechanisms of BR and TORC interactions in the regulation of plant growth/stress balance.

plant biology↗

bioRxiv2021

Bridging Between Material Properties Of Proteins And The Underlying Molecular Interactions

In this work, we develop a novel method that bridges between material properties of proteins, particularly the modulus of elasticity, and the underlying molecular interactions. To this end, we employ both an all-atom normal mode analysis (NMA) model with the CHARMM force field and an elastic solid model for proteins and protein interfaces. And the "bridge" between the two models is a common physical property that they both can predict: the magnitude of thermal vibrations. This connection allows one to calibrate the Youngs moduli of proteins and protein interface regions. We find that the elastic moduli of proteins are mostly in the range of a few Gpa to 10 Gpa, while the elastic moduli of the interface regions are about an order smaller. The work is significant as it represents the first attempt to systematically compute the moduli of elasticity of proteins from molecular interactions.

molecular biology↗

bioRxiv2021

Quantitative proteomics reveals extensive lysine ubiquitination in the Arabidopsis root proteome and uncovers novel transcription factor stability states

Protein activity, abundance, and stability can be regulated by posttranslational modification including ubiquitination. Ubiquitination is conserved among eukaryotes and plays a central role in modulating cellular function and yet we lack comprehensive catalogs of proteins that are modified by ubiquitin in plants. In this study, we describe an antibody-based approach to enrich peptides containing the di-glycine (diGly) remnant of ubiquitin and coupled that with isobaric labeling to enable quantification, from up to 16-multiplexed samples, for plant tissues. Collectively, we identified 7,130 diGly-modified lysine residues sites arising from 3,178 proteins in Arabidopsis primary roots. These data include ubiquitin proteasome dependent ubiquitination events as well as ubiquitination events associated with auxin treatment. Gene Ontology analysis indicated that ubiquitinated proteins are associated with numerous biological processes including hormone signaling, plant defense, protein homeostasis, and root morphogenesis. We determined the ubiquitinated lysine residues that directly regulate the stability of the transcription factors CRYPTOCHROME-INTERACTING BASIC-HELIX-LOOP-HELIX 1 (CIB1), CIB1 LIKE PROTEIN 2 (CIL2), and SENSITIVE TO PROTON RHIZOTOXICITY (STOP1) using site directed mutagenesis and in vivo degradation assays. These comprehensive site-level ubiquitinome profiles provide a wealth of data for future studies related to modulation of biological processes mediated by this posttranslational modification in plants.

plant biology↗

bioRxiv2020

Cross-reactive serum and memory B cell responses to spike protein in SARS-CoV-2 and endemic coronavirus infection

Pre-existing immune responses to seasonal endemic coronaviruses could have profound consequences for antibody responses to SARS-CoV-2, either induced in natural infection or through vaccination. Such consequences are well established in the influenza and flavivirus fields. A first step to establish whether pre-existing responses can impact SARS-CoV-2 infection is to understand the nature and extent of cross-reactivity in humans to coronaviruses. We compared serum antibody and memory B cell responses to coronavirus spike (S) proteins from pre-pandemic and SARS-CoV-2 convalescent donors using a series of binding and functional assays. We found weak evidence of pre-existing SARS-CoV-2 cross-reactive serum antibodies in pre-pandemic donors. However, we found stronger evidence of pre-existing cross-reactive memory B cells that were activated on SARS-CoV-2 infection. Monoclonal antibodies (mAbs) isolated from the donors showed varying degrees of cross-reactivity with betacoronaviruses, including SARS and endemic coronaviruses. None of the cross-reactive mAbs were neutralizing except for one that targeted the S2 subunit of the S protein. The results suggest that pre-existing immunity to endemic coronaviruses should be considered in evaluating antibody responses to SARS-CoV-2.

immunology↗

bioRxiv2020

Integrated omics networks reveal the temporal signaling events of brassinosteroid response in Arabidopsis

Brassinosteroids (BRs) are plant steroid hormones that are known to regulate cell division and stress response. We used a systems biology approach to integrate multi-omic datasets and unravel the molecular signaling events of BR response in Arabidopsis. We profiled the levels of 32,549 transcripts, 9,035 protein groups, and 26,950 phosphorylation sites from Arabidopsis seedlings treated with brassinolide (BL, most active BR) for six different lengths of time. We then constructed a network inference pipeline called Spatiotemporal Clustering and Inference of Omics Networks (SC-ION) that was able to integrate these multi-omic data into one, cohesive regulatory network. Our network illustrates the signaling cascade of BR response, starting with kinase-substrate phosphorylation and ending with transcriptional regulation. We used our network predictions to identify putative, relevant phosphorylation sites on the TF BRI1-EMS-SUPPRESSOR 1 (BES1); the importance of which we experimentally validated. Additionally, we identified an uncharacterized TF, which we named BRONTOSAURUS (BRON), that regulates cell division. Further, we show that bron mutant roots are hypersensitive to BL. Together, this work demonstrates the power of integrative network analysis applied to multiomic data and provides fundamental insights into the molecular signaling events occurring during BR response.

plant biology↗

bioRxiv2020

Rapid isolation of potent SARS-CoV-2 neutralizing antibodies and protection in a small animal model

The development of countermeasures to prevent and treat COVID-19 is a global health priority. In under 7 weeks, we enrolled a cohort of SARS-CoV-2-recovered participants, developed neutralization assays to interrogate serum and monoclonal antibody responses, adapted our high throughput antibody isolation, production and characterization pipeline to rapidly screen over 1000 antigen-specific antibodies, and established an animal model to test protection. We report multiple highly potent neutralizing antibodies (nAbs) and show that passive transfer of a nAb provides protection against high-dose SARS-CoV-2 challenge in Syrian hamsters. The study suggests a role for nAbs in prophylaxis, and potentially therapy, of COVID-19. The nAbs define protective epitopes to guide vaccine design.

immunology↗

bioRxiv2020

Optimized sgRNA design by deep learning to balance the off-target effects and on-target activity of CRISPR/Cas9

The CRISPR/Cas9 system derived from bacteria especially Streptococcus pyogenes (SpyCas9) is currently considered as the most advanced tool used for numerous areas of biological study in which it is useful to target or modify specific DNA sequences. However, low on-target cleavage efficiency and off-target effects impede its wide application. Several different sgRNA design tools for SpyCas9 by using various algorithms have been developed, including linear regression model, support vector machine (SVM) model and convolutional neuron network model. While the deep insight into the sgRNA features contributing for both on-target activity and off-target still remains to be determined. Here, with public large-scale CRISPR screen data, we evaluated contribution of different features influence sgRNA activity and off-target effects, and developed models for sgRNA off-target evaluation and on-target activity prediction. In addition, we combined both activity and off-target prediction models and packaged them as an online sgRNA design tool, OPT-sgRNA.

bioinformatics↗

bioRxiv2020

An Integrated Systems Biology Approach Identifies the Proteasome as a Critical Host Machinery for ZIKV and DENV Replication

The Zika (ZIKV) and dengue (DENV) flaviviruses exhibit similar replicative processes but distinct clinical outcomes. A systematic understanding of virus-host protein-protein interaction networks can reveal cellular pathways critical to viral replication and disease pathogenesis. Here we employed three independent systems biology approaches toward this goal. First, protein array analysis of direct interactions between individual ZIKV/DENV viral proteins and 20,240 human proteins revealed multiple conserved cellular pathways and protein complexes, including proteasome complexes. Second, an RNAi screen of 10,415 druggable genes to identify host proteins required for ZIKV infection uncovered proteasome proteins. Third, a high-throughput screening of 6,016 bioactive compounds for ZIKV inhibitors yielded 134 effective compounds, including six proteasome inhibitors that suppress both ZIKV and DENV replication. Integrative analyses of these orthogonal datasets pinpoints proteasome as critical host machinery for ZIKV/DENV replication. Our study provides multi-omics datasets for further studies of flavivirus-host interactions, disease pathogenesis, and new drug targets.

microbiology↗

bioRxiv2019

Role of Chemokine and TNF signaling pathway in oral squamous cell carcinoma: A RNA deep sequencing analysis of oral buccal mucosa squamous carcinoma model of Chinese hamster

Oral cancer is one of the most common cancers in the world, meanwhile, differentially expressed genes are thought to regulate the development and progression of oral squamous cell carcinomas (OSCC). In this study we screened RNA transcripts from the oral buccal mucosa of healthy male Chinese hamster, divided into 3 groups: a control group with no disposal, a solvent control group coated with acetone solvent, and an experimental group coated with 0.5% DMBA acetone solution by high-throughput RNA sequencing. Tophat and Bowtie were used to align the high-quality reads into transcripts, DEseq was used to analysis the expression of differential gene. Then, the Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted. The chemokine and TNF signaling pathway were differentially expression and the mRNA expression of CXCL1, CXCL2, CXCL3, CCL7, MMP9, monitored by qRT-PCR, increased remarkably in the cancer group and coincided with the result of RNA-Sequencing. Meanwhile, the CXCL1, CXCL2, CXCL3, and CCL7 are significantly enriched in the chemokine signaling pathway, and CXCL1, CXCL2, CXCL3, and MMP9 are significantly enriched in the tumor necrosis factor (TNF) signaling pathway. The differentially expression of the chemokine and TNF signaling pathway was a response to the invasion of the organism immune system due to oral buccal mucosa squamous carcinoma. All the findings provided novel insights for further molecular researches of oral cancer.

molecular biology↗

bioRxiv2019

Receptor-Like Kinase Phosphorylation of Arabidopsis Heterotrimeric G-Protein Gα - Subunit AtGPA1

As molecular on-off switches, heterotrimeric G protein complexes, comprised of a G subunit and an obligate G{beta}{gamma} dimer, transmit extracellular signals received by G protein- coupled receptors (GPCRs) to cytoplasmic targets that respond to biotic and abiotic stimuli. Signal transduction is modulated by phosphorylation of GPCRs and G protein complexes. In Arabidopsis thaliana, the G subunit AtGPA1 is phosphorylated by the receptor-like kinase (RLK) BRI1-ASSOCIATED Kinase 1 (BAK1), but the extent that other RLKs phosphorylates AtGPA1 is unknown. We mapped 22 trans-phosphorylation sites on AtGPA1 by 12 RLKs hypothesized to act in the Arabidopsis G protein signaling pathway. Cis-phosphorylation sites on these RLKs were also identified. BRI1, BAK1, and SERK1 have been reported as Ser/Thr and Tyr dual specificity kinases. We identified 4 more dual specificity kinases: IOS1, PSY1R, PEPR1, and AT2G37050. Multiple sites are present in the core AtGPA1 functional units, including pSer52 and pThr53 of the conserved P-loop that directly binds nucleotide/phosphate, pThr164 and pSer175 from E helix in the intramolecular domain interface for nucleotide exchange and GTP hydrolysis, and pThr193 or pThr194 in Switch I (SwI) that coordinates nucleotide exchange and protein partner binding. Several AtGPA1 S/T phosphorylation sites are nucleotide-dependent phosphorylation patterns, such as S52/T53 in the P-loop and T193 and/or T194 in SwI.

plant biology↗