Biology subjects

Peng, C.

Publications and source records attributed to Peng, C..

At least 109 records · Page 6Linked to original sources

bioRxiv2022

Resolving clonal substructure from single cell genomic data using CopyKit

High-throughput methods for single cell copy number sequencing have enabled the profiling of thousands of cells in parallel, yet there remains a significant bottleneck for data analysis. Here we present CopyKit, a comprehensive set of computational methods for the pre-processing and analysis of single cell copy number data to resolve clonal substructure and reconstruct genetic lineages in tumors. We performed single cell DNA sequencing of 2977 cells from multiple spatial regions in two liver metastasis and 7365 cells from three primary tumors with matched metastatic tissues. In the liver metastases, CopyKit resolved clonal substructure in different spatial regions, which revealed both clonal intermixing and spatial segregation in the tumor mass. In the matched metastatic colorectal and breast cancers, CopyKit resolved metastatic lineages and identified subclones and genomic events that were associated with metastases. These applications show that CopyKit is comprehensive tool for resolving copy number substructure in tumors.

genomics↗

bioRxiv2022

Structural analogue-based protein structure domain assembly assisted by deep learning

MotivationWith the breakthrough of AlphaFold2, the protein structure prediction problem has made a remarkable progress through end-to-end deep learning techniques, in which correct folds could be built for nearly all single-domain proteins. However, the full-chain modelling appears to be lower on average accuracy than that for the constituent domains and requires higher demand on computing hardware, indicating the performance of full-chain modelling still needs to be improved. In this study, we investigate whether the predicted accuracy of full-chain model can be further improved by domain assembly assisted by deep learning. ResultsIn this article, we developed a structural analogue-based protein structure domain assembly method assisted by deep learning, named SADA. In SADA, a multi-domain protein structure database (MPDB) was constructed for the full-chain analogue detection using individual domain models. Starting from the initial model constructed from the analogue, the domain assembly simulation was performed to generate the full-chain model through a two-stage differential evolution algorithm guided by the physics-based force field and an inter-residue distance potential predicted by deep learning. SADA was compared with the state-of-the-art domain assembly methods on 356 benchmark proteins, and the average TM-score of SADA models is 8.1% and 27.0% higher than that of DEMO and AIDA, respectively. We also assembled full-chain models of 20 human multi-domain proteins using individual domain models independently predicted by AlphaFold2, where the SADA full-chain models obtained a 4.8% higher average TM-score than full-chain models directly predicted by AlphaFold2 and fewer computing resources were required. In addition, we also find that the domains often interact in the similar way in the quaternary orientations if the domains have similar tertiary structures. Furthermore, homologous templates and structural analogues are complementary for multi-domain protein full-chain modelling. AvailabilityThe SADA web server are freely available at http://zhanglab-bioinf.com/SADA.

bioinformatics↗

bioRxiv2022

Proteomics and Functional Analysis Revealed TaGSTU6/TaCBSX3 Enhances Wheat Resistance to Powdery Mildew

Wheat stripe rust and powdery mildew are important worldwide diseases of wheat (Triticum aestivum). The wheat cultivar Xingmin318 (XM318) is resistant to both wheat stripe rust and powdery mildew, which are caused by Puccinia striiformis f. sp. tritici (Pst) and Blumeria graminis f. sp. tritici (Bgt), respectively. To explore the molecular mechanisms of wheat defenses against Pst and Bgt, quantitative proteomic analyses of XM318 inoculated with Pst and Bgt, respectively, were performed using tandem mass tags (TMT) technology. A total of 741 proteins were identified as differentially accumulated proteins (DAPs). Bioinformatics analyses indicated that some functional categories, including antioxidant activity, exhibited obvious differences between Pst and Bgt infections. Intriguingly, only 42 DAPs responded to both Pst and Bgt infections. Twelve DAPs were randomly selected for RT-qPCR analysis, and the mRNA expression levels of eleven were consistent with their protein expression. Furthermore, gene silencing using the virus-induced gene silencing system indicated that glutathione S-transferase (TaGSTU6) has an important role in resistance to Bgt but not to Pst. TaGSTU6 was shown to interact with the cystathionine beta-synthase (CBS) domain-containing protein (TaCBSX3). Knockdown of TaCBSX3 expression only reduced wheat resistance to Bgt infection. Overexpression of TaGSTU6 and TaCBSX3 in Arabidopsis (Arabidopsis thaliana) promoted plant resistance to Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). Our results indicated that the TaGSTU6 interacting with TaCBSX3 only confers wheat resistance to Bgt, suggesting that wheat has different response mechanisms to Pst and Bgt stress. One-sentence summaryProteomics revealed a difference in the wheat resistance response to Pst and Bgt, and the TaGSTU6/TaCBSX3 interaction plays an important role only in wheat resistance to Bgt.

pathology↗

bioRxiv2021

DomBpred: protein domain boundary predictor using inter-residue distance and domain-residue level clustering

Domain boundary prediction is one of the most important problems in the study of protein structure and function, especially for large proteins. At present, most domain boundary prediction methods have low accuracy and limitations in dealing with multi-domain proteins. In this study, we develop a sequence-based protein domain boundary predictor, named DomBpred. In DomBpred, the input sequence is firstly classified as either a single-domain protein or a multi-domain protein through a designed effective sequence metric based on a constructed single-domain sequence library. For the multi-domain protein, a domain-residue level clustering algorithm inspired by Ising model is proposed to cluster the spatially close residues according inter-residue distance. The unclassified residues and the residues at the edge of the cluster are then tuned by the secondary structure to form potential cut points. Finally, a domain boundary scoring function is proposed to recursively evaluate the potential cut points to generate the domain boundary. DomBpred is tested on a large-scale test set of FUpred comprising 2549 proteins. Experimental results show that DomBpred better performs than the state-of-the-art methods in classifying whether protein sequences are composed by single or multiple domains, and the Matthews correlation coefficient is 0.882. Moreover, on 849 multi-domain proteins, the domain boundary distance and normalised domain overlap scores of DomBpred are 0.523 and 0.824, respectively, which are 5.0% and 4.2% higher than those of the best comparison method, respectively. Comparison with other methods on the given test set shows that DomBpred outperforms most state-of-the-art sequence-based methods and even achieves better results than the top-level template-based method.

bioinformatics↗

bioRxiv2021

MPDB: a unified multi-domain protein structure database integrating structural analogue detection

With the development of protein structure prediction methods and biological experimental determination techniques, the structure of single-domain proteins can be relatively easier to be modeled or experimentally solved. However, more than 80% of eukaryotic proteins and 67% of prokaryotic proteins contain multiple domains. Constructing a unified multi-domain protein structure database will promote the research of multi-domain proteins, especially in the modeling of multi-domain protein structures. In this work, we develop a unified multi-domain protein structure database (MPDB). Based on MPDB, we also develop a server with two functional modules: (1) the culling module, which filters the whole MPDB according to input criteria; (2) the detection module, which identifies structural analogues of the full-chain according to the structural similarity between input domain models and the protein in MPDB. The module can discover the potential analogue structures, which will contribute to high-quality multi-domain protein structure modeling.

bioinformatics↗

bioRxiv2021

Spatiotemporal transcriptome at single-cell resolution reveals key radial glial cell population in axolotl telencephalon development and regeneration

Brain regeneration requires a precise coordination of complex responses in a time- and region-specific manner. Identifying key cell types and molecules that direct brain regeneration would provide potential targets for the advance of regenerative medicine. However, progress in the field has been hampered largely due to limited regeneration capacity of the mammalian brain and understanding of the regeneration process at both cellular and molecular level. Here, using axolotl brain with extrodinary regeneration ability upon injury, and the SpaTial Enhanced REsolution Omics-sequencing (Stereo-seq), we reconstructed the first architecture of axolotl telencephalon with gene expression profiling at single-cell resolution, and fine cell dynamics maps throughout development and regeneration. Intriguingly, we discovered a marked heterogeneity of radial glial cell (RGC) types with distinct behaviors. Of note, one subtype of RGCs is activated since early regeneration stages and proliferates while other RGCs remain dormant. Such RGC subtype appears to be the major cell population involved in early wound healing response and gradually covers the injured area before presumably transformed into the lost neurons. Altogether, our work systematically decoded the complex cellular and molecular dynamics of axolotl telencephalon in development and regeneration, laying the foundation for studying the regulatory mechanism of brain regeneration in the future.

developmental biology↗

bioRxiv2021

Mod3D: A Low-Cost, Flexible Modular System of Live-Cell Microscopy Chambers and Holders

Live-cell microscopy imaging typically involves the use of high-quality glass-bottom chambers that allow cell culture, gaseous buffer exchange and optical properties suitable for microscopy applications. However, commercial sources of these chambers can add significant annual costs to cell biology laboratories. Consumer products in three-dimensional printing technology, for both Filament Deposition Modeling (FDM) and Masked Stereo Lithography (MSLA), have resulted in more biomedical research labs adopting the use of these devices for prototyping and manufacturing of lab plastic-based items, but rarely consumables. Here we describe a modular, live-cell chamber with multiple design options that can be mixed per experiment. Single reusable carriers and the use of biodegradable plastics, in a hybrid of FDM and MSLA manufacturing methods, reduce plastic waste. The system is easy to adapt to bespoke designs, with concept-to-prototype in a single day, offers significant cost savings to the users over commercial sources, and no loss in dimensional quality or reliability.

cell biology↗

bioRxiv2021

BRET-based self-cleaving biosensors for SARS-CoV-2 3CLpro Inhibitor Discovery

The 3C-like protease (3CLpro) of SARS-CoV-2 is an attractive drug target for developing antivirals against SARS-CoV-2. A few small molecule inhibitors of 3CLpro are in clinical trials for COVID-19 treatments and more inhibitors are being developed. One limiting factor for 3CLpro inhibitors development is that the cellular activities of such inhibitors have to be evaluated in a Biosafety Level 3 (BSL-3) or BSL-4 laboratory. Here, we design genetically encoded biosensors that can be used in BSL-2 laboratories to set up cell-based assays for 3CLpro inhibitor discovery. The biosensors were constructed by linking a green fluorescent protein (GFP2) to the N-terminus and a Renilla luciferase (RLuc8) to the C-terminus of SARS-CoV-2 3CLpro, with the linkers derived from the cleavage sequences of 3CLpro. After over-expression of the biosensors in HEK293 cells, 3CLpro can be released from GFP2 and RLuc by self-cleavage, resulting in a decrease of the bioluminescence resonance energy transfer (BRET) signal. Using one of these biosensors, pBRET-10, we evaluated the cellular activities of several 3CLpro inhibitors. These inhibitors restored the BRET signal by blocking the proteolysis of pBRET-10, and their relative activities measured using pBRET-10 were consistent with their anti-SARS-CoV-2 activities reported previously. We conclude that the biosensor pBRET-10 is a useful tool for SARS-CoV-2 3CLpro inhibitor discovery. Furthermore, our strategy can be used to design biosensors for other viral proteases that share the same activation mechanism as 3CLpro, such as HIV protease PR and HCV protease NS3. HighlightsO_LISensitive cell-based biosensors for 3CLpro inhibitor discovery in BSL-2 laboratories. C_LIO_LIThe BRET-based self-cleaving biosensors mimic the in vivo autoproteolytic activation of 3CLpro. C_LIO_LISimilar biosensors can be designed for other self-cleaving proteases, such as HIV protease PR and HCV protease NS3. C_LI

synthetic biology↗

bioRxiv2021

The cryo-EM structure of the chloroplast ClpP complex reveals an interaction with the co-chaperonin complex that inhibits ClpP proteolytic activity

Protein homeostasis in plastids is strategically regulated by the protein quality control system involving multiple chaperones and proteases, among them the Clp protease. We determined the structure of the chloroplast ClpP complex from Chlamydomonas reinhardtiiby cryo-EM. ClpP contains two heptameric catalytic rings without any symmetry. The top ring contains one ClpR6, three ClpP4 and three ClpP5 subunits while the bottom ring is composed of three ClpP1C subunits and one each of the ClpR1-4 subunits. ClpR3, ClpR4 and ClpT4 subunits connect the two rings and stabilize the complex. The chloroplast Cpn11/20/23 co-chaperonin, a co-factor of Cpn60, forms a cap on the top of ClpP by protruding mobile loops into hydrophobic clefts at the surface of the top ring. The co-chaperonin repressed ClpP proteolytic activity in vitro. By regulating Cpn60 chaperone and ClpP protease activity, the co-chaperonin may play a role in coordinating protein folding and degradation in the chloroplast.

plant biology↗

bioRxiv2021

The extracellular ATP receptor P2RX7 imprints a pro-memory transcriptional signature in effector CD8+ T cells

Development of central memory (Tcm) and resident memory (Trm) CD8+ T cells, which respectively promote immunity in the circulation and in barrier tissues, is not completely understood. Tcm and Trm cells may arise from common precursors, however their fate-inducing signals are elusive. We found that virus-specific effector CD8+ T cells display heterogeneous expression of the extracellular ATP sensor P2RX7. P2RX7-high expression is confined, at peak effector phase, to CD62L+ memory precursors which preferentially form Tcm cells. Among early effector CD8+ T cells, asymmetrical P2RX7 distribution correlated with distinct transcriptional signatures, with P2RX7-high cells enriched for memory and tissue-residency sets. P2RX7-high early effectors preferentially form both Tcm and Trm cells. Defective Tcm and Trm formation in P2RX7 deficiency is significantly reverted when the transcriptional repressor Zeb2 is ablated. Our study indicates that unequal P2RX7 upregulation in effector CD8+ T cells is a foundational element of the early Tcm/Trm fate.

immunology↗

bioRxiv2021

Integrative molecular and clinical profiling of acral melanoma identifies LZTR1 as a key tumor promoter and therapeutic target

Acral melanoma, the most common melanoma subtype among non-Caucasian individuals, is associated with poor prognosis. However, its key molecular drivers remain obscure. Here, we performed integrative genomic and clinical profiling of acral melanomas from a cohort of 104 patients treated in North America or China. We found that recurrent, late-arising amplifications of cytoband chr22q11.21 are a leading determinant of inferior survival, strongly associated with metastasis, and linked to downregulation of immunomodulatory genes associated with response to immune checkpoint blockade. Unexpectedly, LZTR1 - a known tumor suppressor in other cancers - is a key candidate oncogene in this cytoband. Silencing of LZTR1 in melanoma cell lines caused apoptotic cell death independent of major hotspot mutations or melanoma subtypes. Conversely, overexpression of LZTR1 in normal human melanocytes initiated processes associated with metastasis, including anchorage-independent growth, formation of spheroids, and increased levels of MAPK and SRC activities. Our results provide new insights into the etiology of acral melanoma and implicate LZTR1 as a key tumor promoter and therapeutic target.

cancer biology↗

bioRxiv2021

Selective Chemical Labeling and Sequencing of 5-Hydroxymethylcytosine in DNA at Single-Base Resolution

5-Hydroxymethylcytosine (5hmC), the oxidative product of 5-methylcytosine (5mC) catalyzed by ten-eleven translocation (TET) enzymes, plays an important role in many biological processes as an epigenetic mediator. Prior studies have shown that 5hmC can be selectively labeled with chemically-modified glucose moieties and enriched using click chemistry with biotin affinity approaches. Besides, DNA deaminases of the AID/APOBEC family can discriminate modified 5hmC bases from cytosine (C)or 5-methylcytosine (5mC). Herein, we developed a method based on ESC whole-genome analysis which could enrich 5hmC-containing DNA by selective chemical labeling and locate 5hmC sites at single-base resolution with enzyme-based deamination. The combination experimental design is an extension of previous methods, and we hope that this cost-effective single base resolution 5hmC sequencing method could be used to promote the mechanism and diagnosis research of 5hmC.

genomics↗

bioRxiv2021

Tumor protein D52 (TPD52) affects cancer cell metabolism by negatively regulating AMPK.

The AMP-activated protein kinase (AMPK) is a central regulator of energy homeostasis, with deregulation leading to cancer and other diseases. When intracellular ATP levels decrease during energy stress, AMPK is phosphorylated and activated through AMP binding. However, how this pathway is dysregulated in cancer remains unclear. Here, we find that tumor protein D52 (TPD52), initially identified to be overexpressed in many human cancers, forms a stable complex with AMPK in cancer cells. TPD52 directly interacts with AMPK and inhibits AMPK kinase activity in vitro and in vivo. We generated TPD52 transgenic mice, and found that overexpression of TPD52 leads to AMPK inhibition and multiple metabolic defects in mice. Together, our results shed new light on AMPK regulation and on our understanding of the etiology of cancers with TPD52 overexpression.

cell biology↗

bioRxiv2021

Discovery of a molecular glue that enhances UPRmt to restore proteostasis via TRKA-GRB2-EVI1-CRLS1 axis

Lowering proteotoxicity is a potentially powerful approach for the treatment of neurological disorders, such as Parkinsons disease. The unfolded protein response (UPR) is a major mechanism that preserves the network maintaining cellular proteostasis. In the present study, we developed the screening strategy to discover compounds that significantly enhanced the activation of mitochondrial UPR (UPRmt) through increasing cardiolipin content. We identified that ginsenoside Rg3 (Rg3) increased cardiolipin depending on cardiolipin synthase 1 (CRLS1) in both worms and in human neural cells. Using LiP-SMap (limited proteolysis-mass spectrometry) strategy, we identified GRB2 (growth factor receptor bound protein 2) as a direct target of Rg3 in human neural cells. Rg3 enhances the binding between GRB2 and TRKA, that transduces signals via phosphrorylation of ERK. We provide bioinformatic and experimental evidence that EVI1, the critical oncogenic transcriptional regulator in leukemia, binds to CRLS1 promoter region and stimulated CRLS1 expression and subsequently increased cardiolipin content in the presence of Rg3. In a Parkinsons disease mouse model, Rg3 restores motor function by protecting nigral dopaminergic neurons dependent on Grb2. Our data recapitulate the TRKA-GRB2-EVI1-CRLS1 axis in maintaining proteostasis in Parkinsons disease via UPRmt.

pharmacology and toxicology↗

bioRxiv2021

MVA Vector Vaccines Inhibit SARS CoV-2 Replication in Upper and Lower Respiratory Tracts of Transgenic Mice and Prevent Lethal Disease

Replication-restricted modified vaccinia virus Ankara (MVA) is a licensed smallpox vaccine and numerous clinical studies investigating recombinant MVAs (rMVAs) as vectors for prevention of other infectious diseases have been completed or are in progress. Two rMVA COVID-19 vaccine trials are at an initial stage, though no animal protection studies have been reported. Here, we characterize rMVAs expressing the S protein of CoV-2. Modifications of full length S individually or in combination included two proline substitutions, mutations of the furin recognition site and deletion of the endoplasmic retrieval signal. Another rMVA in which the receptor binding domain (RBD) flanked by the signal peptide and transmembrane domains of S was also constructed. Each modified S protein was displayed on the surface of rMVA-infected human cells and was recognized by anti-RBD antibody and by soluble hACE2 receptor. Intramuscular injection of mice with the rMVAs induced S-binding and pseudovirus-neutralizing antibodies. Boosting occurred following a second homologous rMVA but was higher with adjuvanted purified RBD protein. Weight loss and lethality following intranasal infection of transgenic hACE2 mice with CoV-2 was prevented by one or two immunizations with rMVAs or by passive transfer of serum from vaccinated mice. One or two rMVA vaccinations also prevented recovery of infectious CoV-2 from the lungs. A low amount of virus was detected in the nasal turbinates of only one of eight rMVA-vaccinated mice on day 2 and none later. Detection of subgenomic mRNA in turbinates on day 2 only indicated that replication was abortive in immunized animals. SignificanceVaccines are required to control COVID-19 during the pandemic and possibly afterwards. Recombinant nucleic acids, proteins and virus vectors that stimulate immune responses to the CoV-2 S protein have provided protection in experimental animal or human clinical trials, though questions remain regarding their ability to prevent spread and the duration of immunity. The present study focuses on replication-restricted modified vaccinia virus Ankara (MVA), which has been shown to be a safe, immunogenic and stable smallpox vaccine and a promising vaccine vector for other infectious diseases and cancer. In a transgenic mouse model, one or two injections of recombinant MVAs that express modified forms of S inhibited CoV-2 replication in the upper and lower respiratory tracts and prevented severe disease.

microbiology↗

bioRxiv2020

A Sequential Niche Multimodal Conformation Sampling Algorithm for Protein Structure Prediction

MotivationMassive local minima on the protein energy surface often causes traditional conformation sampling algorithms to be easily trapped in local basin regions, because they are difficult to stride over high-energy barriers. Also, the lowest energy conformation may not correspond to the native structure due to the inaccuracy of energy models. This study investigates whether these two problems can be alleviated by a sequential niche technique without loss of accuracy. ResultsA sequential niche multimodal conformation sampling algorithm for protein structure prediction (SNfold) is proposed in this study. In SNfold, a derating function is designed based on the knowledge learned from the previous sampling and used to construct a series of sampling-guided energy functions. These functions then help the sampling algorithm stride over high-energy barriers and avoid the re-sampling of the explored regions. In inaccurate protein energy models, the high- energy conformation that may correspond to the native structure can be sampled with successively updated sampling-guided energy functions. The proposed SNfold is tested on 300 benchmark proteins and 24 CASP13 FM targets. Results show that SNfold is comparable with Rosetta restrained by distance (Rosetta-dist) and C-QUARK. SNfold correctly folds (TM-score [≥] 0.5) 231 out of 300 proteins. In particular, compared with Rosetta-dist protocol, SNfold achieves higher average TM- score and improves the sampling efficiency by more than 100 times. On the 24 CASP13 FM targets, SNfold is also comparable with four state-of-the-art methods in the CASP13 server group. As a plugin conformation sampling algorithm, SNfold can be extended to other protein structure prediction methods. AvailabilityThe source code and executable versions are freely available at https://github.com/iobio-zjut/SNfold. Contactzgj@zjut.edu.cn

bioinformatics↗

bioRxiv2020

Sequential Measurements of Catalytic Activities of Multi-Drug-Resistance Transporters and Cytochrome P450 Enzymes by Cytometry of Reaction Rate Constant

Cytometry of reaction rate constant (CRRC) is an accurate and robust approach to characterize cell-population heterogeneity using rate constants of cellular processes for which kinetic mechanisms are known. We work on a CRRC-based method to develop predictors of tumor chemoresistance driven by two processes: drug extrusion by multi-drug-resistance (MDR) transporters and drug inactivation by cytochrome-P450 enzymes (CYP). Each of the two possess is studied with its specific substrate and the process activity is characterized by a corresponding unimolecular rate constant. Due to the incompatibility of MDR and CYP assays, MDR and CYP activities may be difficult to measure simultaneously suggesting that they may need to be measured sequentially. The sequential measurements may also impose a problem: the results of the second assay may be affected by artifacts exerted by the first assay. The goal of this work was to understand whether the cells have a memory of the first assay that significantly affects the results of the second assay. To achieve this goal, we compared CRRC results for two orders of sequential measurements: the MDR[->]CYP order in which MDR activity is measured before CYP activity and the CYP[->]MDR order in which CYP activity is measured before MDR activity. It was found that the results of the CYP assay were similar in both orders; on the contrary, the results of the MDR assay were significantly different. Our findings suggest that MDR and CYP activity can be studied sequentially provided that MDR activity is measured first and CYP activity second.

cancer biology↗

bioRxiv2020

In-depth analysis reveals complex molecular etiology of idiopathic cerebral palsy

Cerebral palsy (CP), the most prevalent physical disability in children, has long been ignored with regard to its inherent molecular mechanism. In this work, we performed in-depth clinical and molecular analysis on 120 idiopathic CP families, and identified in half of the patients the underlying risk factors. By a compilation of 117 CP-related genes, we recognized the characteristic features in terms of inheritance and function, and proposed a dichotomous classification system according to the expression patterns. In two patients with both CP and intellectual disability, we revealed that the defective TYW1, a tRNA hypermodification enzyme, caused microcephaly and problems in motion and cognition by hindering neuronal proliferation and migration. We developed an algorithm and proved in mice brain that this malfunctioning hypermodification specifically perturbed the translation of a subset of proteins involved in cell cycling. In a CP patient with normal intelligence, we identified a mitochondrial enzyme GPAM, the hypomorphic form of which led to hypomyelination of corticospinal tract. We confirmed that the aberrant Gpam in mice perturbed the lipid metabolism in astrocyte, resulting in suppressed astrocyte proliferation and a shortage of lipid contents supplied for oligodendrocyte myelination. This work broadened the scope of understanding of CP etiology and paved a way for innovative therapeutic strategies.

genetics↗