Biology subjects

He, A.

Publications and source records attributed to He, A..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Elucidating the assembly of gas vesicles by systematic protein-protein interaction analysis

Gas vesicles (GVs) are gas-filled microbial organelles formed by unique 3-nm thick, amphipathic, force-bearing protein shells, which can withstand multiple atmospheric pressures and maintain a physically stable air bubble with megapascal surface tension. However, the molecular process to assemble this shell remains elusive: while 6-8 assembly factor proteins were identified as essential, none of them have a defined function. As one of the first steps to elucidate the assembly mechanism, we devise a high-throughput in vivo assay to determine the interactions of all 11 proteins in a GV operon. Complete or partial deletions of the operon establish the interdependence relationship of the interaction on the background GV proteins with additional information on assembly tolerance and cellular burden. Clusters of GV protein interactions are revealed, which establishes the plausible protein complexes important for the assembly process of these protein organelles. We anticipate our findings will set the stage for solving the molecular mechanism of GV assembly and designing GVs that efficiently assemble in heterologous hosts during biomedical applications.

synthetic biology↗

bioRxiv2023

A Cell Cycle-Dependent Ferroptosis Sensitivity Switch Governed by EMP2

Ferroptosis is a non-apoptotic form of cell death characterized by iron-dependent lipid peroxidation. Ferroptosis can be induced by system xc- cystine/glutamate antiporter inhibition or by direct inhibition of the phospholipid hydroperoxidase glutathione peroxidase 4 (GPX4). The regulation of ferroptosis in response to system xc- inhibition versus direct GPX4 inhibition may be distinct. Here, we show that cell cycle arrest enhances sensitivity to ferroptosis triggered by GPX4 inhibition but not system xc- inhibition. Arrested cells have increased levels of oxidizable polyunsaturated fatty acid-containing phospholipids, which drives sensitivity to GPX4 inhibition. Epithelial membrane protein 2 (EMP2) expression is reduced upon cell cycle arrest and is sufficient to enhance ferroptosis in response to direct GPX4 inhibition. An orally bioavailable GPX4 inhibitor increased markers of ferroptotic lipid peroxidation in vivo in combination with a cell cycle arresting agent. Thus, responses to different ferroptosis-inducing stimuli can be regulated by cell cycle state.

cancer biology↗

bioRxiv2023

Deep spatial proteomic exploration of severe COVID-19-related pulmonary injury in post-mortem specimens

The lung, as a primary target of SARS-CoV-2, exhibits heterogeneous microenvironment accompanied by various histopathological changes following virus infection. However, comprehensive insight into the protein basis of COVID-19-related pulmonary injury with spatial resolution is currently deficient. Here, we generated a region-resolved quantitative proteomic atlas of seven major pathological structures within the lungs of COVID-19 victims by integrating histological examination, laser microdissection, and ultrasensitive proteomic technologies. Over 10,000 proteins were quantified across 71 dissected FFPE post-mortem specimens. By comparison with control samples, we identified a spectrum of COVID-19-induced protein and pathway dysregulations in alveolar epithelium, bronchial epithelium, and pulmonary blood vessels, providing evidence for the proliferation of transitional-state pneumocytes. Additionally, we profiled the region-specific proteomes of hallmark COVID-19 pulmonary injuries, including bronchiole mucus plug, pulmonary fibrosis, airspace inflammation, and hyperplastic alveolar type 2 cells. Bioinformatic analysis revealed the enrichment of cell-type and functional markers in these regions (e.g. enriched TGFBI in fibrotic region). Furthermore, we identified the up-regulation of proteins associated with viral entry, host restriction, and inflammatory response in COVID-19 lungs, such as FURIN and HGF. Collectively, this study provides spatial proteomic insights for understanding COVID-19-caused pulmonary injury, and may serve as a valuable reference for improving therapeutic intervention for severe pneumonia.

systems biology↗

bioRxiv2023

Fully automated and integrated proteomics sample preparation platform for high-throughput drug target identification

With the increased demand of large-cohort proteomic analysis, fast and reproducible sample preparation has become the critical issue that needs to be solved. Herein, we developed a fully automated and integrated proteomics sample preparation workflow (autoSISPROT), enabling the simultaneous processing of 96 samples in less than 2.5 hours. Benefiting from its 96-channel all-in-tip operation, protein digestion, peptide desalting, and TMT labeling could be achieved in a fully automated manner. The autoSISPROT demonstrated good sample preparation performances, including >94% of digestion efficiency, nearly 100% of alkylation efficiency, >98% of TMT labeling efficiency, and >0.9 of intra- and inter-batch Pearson correlation coefficients. Furthermore, by combining with cellular thermal shift assay-coupled to mass spectrometry (CETSA-MS), the autoSISPROT was able to process and TMT-label 40 samples automatically and accurately identify the known target of methotrexate. Importantly, taking advantage of the data independent acquisition and isothermal CETSA-MS, the autoSISPROT was well applied for identifying known targets and potential off-targets of 20 kinase inhibitors by automatedly processing 87 samples, affording over a 10-fold improvement in throughput when compared to classical CETSA-MS. Collectively, we developed a fully automated and integrated workflow for high-throughput proteomics sample preparation and drug target identification.

systems biology↗

bioRxiv2023

Massively parallel single-cell profiling of transcriptome and multiple epigenetic proteins in cell fate regulation

Sculpting the epigenome with a combination of histone modifications and transcription factor (TF) occupancy determines gene transcription and cell fate specification. Here we first develop uCoTarget, utilizing a split-pool barcoding strategy for realizing ultra-high throughput single-cell joint profiling of multiple epigenetic proteins. Through extensive optimization for sensitivity and multimodality resolution, we demonstrate that uCoTarget enables simultaneous detection of five histone modifications (H3K27ac, H3K4me3, H3K4me1, H3K36me3 and H3K27me3) in 19,860 single cells. We applied uCoTarget to the in vitro generation of hematopoietic stem/progenitor cells (HSPCs) from human embryonic stem cells, presenting multimodal epigenomic profiles in 26,418 single cells. uCoTarget with high sensitivity per modality reveals establishment of pairing of HSPC enhancers (H3K27ac) and promoters (H3K4me3) along the differentiation trajectory and RUNX1 engagement priming for the H3K27ac activation along the HSPC path. We then develop uCoTargetX, an expansion of uCoTarget to simultaneously measure transcriptome and multiple epigenome targets. Together, our methods enable generalizable, versatile multi-modal profiles for reconstructing comprehensive epigenome and transcriptome landscapes and analyzing the regulatory interplay at single-cell level.

genomics↗

bioRxiv2023

Improved in situ Characterization of Proteome-wide Protein Complex Dynamics with Thermal Proximity Co-Aggregation

Vast majority of cellular activities are carried out by protein complexes that assembled dynamically in response to cellular needs and environmental cues. Large scale efforts had uncovered a large repertoire of functionally uncharacterized protein complexes which necessitate new strategies to delineate their roles in various cellular activities and diseases. Thermal proximity co-aggregation profiling could be readily deployed to simultaneously characterize the dynamics for hundreds to thousands of protein complexes in situ across different cellular conditions. Toward this goal, we had optimized the original method both experimentally and computationally. In this new iteration termed Slim-TPCA, fewer temperatures are used which increase throughputs by over 3X, while coupled with new scoring metrics and statistical evaluation resulted in minimal compromise in coverage and the detection of more relevant protein complexes. Overall, less samples are needed, false positives from batch effects are minimized and statistical evaluation time is reduced by two orders of magnitude. We applied Slim-TPCA to profile state of protein complexes in K562 cells under different duration of glucose deprivation. More protein complexes are found dissociated based on TPCA signature in accordance with expected downregulation of most cellular activities. These complexes include 55S ribosome and various respiratory complexes in mitochondria revealing the utility of TPCA to study protein complexes in organelles. On other hand, protein complexes involved in protein transport and degradation are found increasingly associated revealing their involvement in metabolic reprogramming during glucose deprivation. In summary. Slim-TPCA is an efficient strategy for proteome-wide characterization of protein complexes. The various algorithmic improvement of Slim-TPCA is available as Python package at https://pypi.org/project/Slim-TPCA/

bioinformatics↗

bioRxiv2023

VHL Mutation Drives Human Clear Cell Renal Cell Carcinoma Progression Through PI3K/AKT-Dependent Cholesteryl Ester Accumulation

BackgroundCholesteryl ester (CE) accumulation in intracellular lipid droplets (LDs) is an essential signature of clear cell renal cell carcinoma (ccRCC), but its molecular mechanism and pathological significance remain elusive. MethodsEnabled by the label-free Raman spectromicroscopy, which integrated stimulated Raman scattering microscopy with confocal Raman spectroscopy on the same platform, we quantitatively analyzed LD distribution and composition at the single cell level in intact ccRCC cell and tissue specimens in situ without any processing or exogenous labeling. Since we found that commonly used ccRCC cell lines actually did not show the CE-rich signature, primary cancer cells were isolated from human tissues to retain the lipid signature of ccRCC with CE level as high as the original tissue, which offers a preferable cell model for the study of cholesterol metabolism in ccRCC. Moreover, we established a patient-derived xenograft (PDX) mouse model that retained the CE-rich phenotype of human ccRCC. FindingsSurprisingly, our results revealed that CE accumulation was induced by tumor suppressor VHL mutation, the most common mutation of ccRCC. Moreover, VHL mutation was found to promote CE accumulation by upregulating HIF and subsequent PI3K/AKT/mTOR/SREBPs pathway. Inspiringly, inhibition of cholesterol esterification remarkably suppressed ccRCC aggressiveness in vitro and in vivo with negligible toxicity, through the reduced membrane cholesterol-mediated downregulations of integrin and MAPK signaling pathways. InterpretationCollectively, our study improves current understanding of the role of CE accumulation in ccRCC and opens up new opportunities for treatment. FundingThis work was supported by National Natural Science Foundation of China (No. U23B2046 and No. 62027824), National Key R&D Program of China (No. 2023YFC2415500), Fundamental Research Funds for the Central Universities (No. YWF-22-L-547), PKU-Baidu Fund (No. 2020BD033), Peking University First Hospital Scientific and Technological Achievement Transformation Incubation Guidance Fund (No.2022CX02), and Beijing Municipal Health Commission (No. 2020-2Z-40713).

cancer biology↗

bioRxiv2021

Novel low-avidity glypican-3 specific CARTs resist exhaustion and mediate durable antitumor effects against hepatocellular carcinoma

Chimeric antigen receptor engineered T cells (CARTs) are being developed to treat solid tumors including hepatocellular carcinoma (HCC). However, thus far, CARTs have not been as effective against solid tumors as compared to blood cancers. A main reason is that, once infiltrating into a solid tumor mass, CARTs are surrounded and chronically stimulated by persistent target antigens, which may drive them to exhaustion. We hypothesize that, due to weak engagement, low-avidity CARTs will resist the antigen-driven exhaustion and apoptosis and maintain effector functions in solid tumors, generating durable antitumor effects. To test this idea, we developed a novel human glypican-3 (hGPC3) specific antibody (8F8) that binds an epitope close to that of GC33 (the frequently used high-affinity antibody), but with ~17 folds lower affinity. In vitro, the low-avidity 8F8 CART killed tumor cells and produced effector cytokines to the same extent as high-avidity GC33 CART. Remarkably, however, 8F8 CART expanded and persisted to a greater extent than GC33 CART in vivo, resulting in durable responses against HCC xenografts. Compared to GC33 CARTs, there were significantly more (5 times) 8F8-BBz CART detected in the tumor mass. Importantly, the tumor infiltrating 8F8 CARTs were less apoptotic and more resistant to exhaustion, revealed by their enhanced and durable effector functions overtime. We predict that this novel low-avidity 8F8-BBz CART has a greater potential than mainstream high-avidity CARTs in effectively treating patients with HCC or other hGPC3+ solid tumors.

immunology↗

bioRxiv2021

Regulation Network of Colorectal Cancer Specific Enhancers in Progression of Colorectal Cancer

Enhancers regulate multiple genes through higher-order chromatin structure and further affect cancer progression. Epigenetic changes in cancer cells activate several cancer specific enhancers that are silenced in normal cells. These cancer specific enhancers are potential therapeutic targets of cancer. However, functions and regulation network of colorectal cancer specific enhancers are still unknown. Here in this study, we profile colorectal cancer specific enhancers and reveal the regulation network of these enhancers by analysis of HiChIP, Hi-C and RNA-seq data. We propose the regulation network of colorectal cancer specific enhancers plays important role in progression of colorectal cancer.

genetics↗

bioRxiv2021

Single-cell epigenomic tracing of lifelong endothelial cell plasticity across mouse organs

Endothelial cells (ECs) across ages and tissues are highly heterogeneous in developmental origins, structures, functions, and cellular plasticity. Here, we applied CoBATCH for single-cell epigenomic tracing of dynamic EC lineage histories in five mouse organs from development to ageing. Our analyses showed that epigenomic memory reflects both developmental origins and tissue-restricted specialization of EC sublineages but with varying time lengths across organs. To gain insights into cellular plasticity of ECs, we identified bivalent chromatin occupancy of otherwise mutually exclusive EC- (ERG) and mesenchymal-specific (TWIST1/SNAI1) transcription factors promoting endothelial-to-mesenchymal transition. We further revealed that pseudotime trajectories by histone modifications H3K36me3 and H3K27ac faithfully recapitulate short- and long-range EC fate change over senescence, respectively. Together, our data provide a unique exploration of chromatin-level cell fate regulation of organotypic EC lineages across the lifespan. One-Sentence SummarySingle-cell chromatin binding is examined for tracing endothelial cell lineages in mouse organs across the lifespan.

genomics↗

bioRxiv2021

Anti-GD2 antibody disrupts GD2:Siglec-7 interactions and synergizes with CD47 blockade to mediate tumor eradication

The disialoganglioside GD2 is consistently overexpressed in neuroblastoma and osteosarcoma, and is variably expressed in other sarcomas, gliomas, neuroendocrine tumors, and epithelial cancers. Anti-GD2 antibodies have improved the survival rates of patients with neuroblastoma only when administered as part of intense chemotherapy-based cytotoxic regimens, which are associated with debilitating late effects including hearing loss, growth retardation, and secondary leukemias. Despite broad expression of GD2 on osteosarcoma, anti-GD2 antibody has not mediated significant antitumor activity in that disease or any other GD2+ cancers. CD47 is a checkpoint molecule overexpressed on tumor cells that inhibits macrophage activity, and CD47 blockade has demonstrated promising clinical activity in early human trials. We investigated whether anti-CD47 antibody could enhance the efficacy of anti-GD2 antibody in neuroblastoma and other GD2+ malignancies. We demonstrate substantial synergy of these two agents, resulting in the recruitment of tumor associated macrophages (TAMs) to mediate robust and durable anti-tumor responses. The responses are driven by GD2-specific factors that reorient the balance of macrophage activity towards phagocytosis of tumor cells, including disruption of a newly described GD2:Siglec-7 axis. These results demonstrate the unique synergy of combining anti-GD2 with anti-CD47, which has the potential to significantly enhance outcomes for children with neuroblastoma and osteosarcoma and will soon be investigated in a first-in-human clinical trial.

immunology↗

bioRxiv2021

Selective inhibition reveals the regulatory function of DYRK2 in protein synthesis and calcium entry

The dual-specificity tyrosine phosphorylation-regulated kinase DYRK2 has emerged as a key regulator of cellular processes such as proteasome-mediated protein degradation. To gain further insights into its function, we took a chemical biology approach and developed C17, a potent small-molecule DYRK2 inhibitor, through multiple rounds of structure-based optimization guided by a number of co-crystallized structures. C17 displayed an effect on DYRK2 at a single-digit nanomolar IC50 and showed outstanding selectivity for the human kinome containing 467 other human kinases. Using C17 as a chemical probe, we further performed quantitative phosphoproteomic assays and identified several novel DYRK2 targets, including eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and stromal interaction molecule 1 (STIM1). DYRK2 phosphorylated 4E-BP1 at multiple sites, and the combined treatment of C17 with AKT and MEK inhibitors showed synergistic 4E-BP1 phosphorylation suppression. The phosphorylation of STIM1 by DYRK2 substantially increased the interaction of STIM1 with the ORAI1 channel, and C17 impeded the store-operated calcium entry process. Collectively, these studies further expand our understanding of DYRK2 and provide a valuable tool to further pinpoint its biological function.

biochemistry↗

bioRxiv2020

Long-range gene regulation network of the MGMT enhancer in modulating glioma cell sensitivity to temozolomide

Acquired resistance to temozolomide (TMZ) is a major obstacle in glioblastoma treatment. MGMT, a DNA repair protein, and the methylation at its gene promoter, plays an important role in TMZ resistance. However, some evidences have suggested a MGMT-independent mechanisms underlying TMZ resistance. Here, we used MGMT enhancer as a model and discovered that its deletion in glioma cells of low MGMT expression induced increased sensitivity to temozolomide. Analysis of a combination of RNA-seq and Capture Hi-C further suggested multiple long-range target genes regulated by the MGMT enhancer and that interactions may play important roles in glioma cell sensitivity to TMZ. This study reveals a novel mechanism of regulation of TMZ sensitivity in glioma cells.

genetics↗

bioRxiv2020

Single-cell joint detection of chromatin occupancy and transcriptome enables higher-dimensional epigenomic reconstructions

Deciphering mechanisms in cell fate decisions requires single-cell holistic reconstructions of multi-dimensional epigenome in transcriptional regulation. Here we develop CoTECH, a combinatorial barcoding method allowing for high-throughput single-cell joint detection of chromatin occupancy and transcriptome. First, we used CoTECH to examine bivalent histone marks (H3K4me3 and H3K27me3) with transcription from naive to primed mouse embryonic stem cells. Concurrent bivalent marks in pseudo-single cells linked via transcriptome were computationally derived, resolving pseudotemporal bivalency trajectories and disentangling a context-specific interplay between H3K4me3/H3K27me3 and transcription level. Next, CoTECH with H3K27ac, an active enhancer marker, revealed the regulatory basis of endothelial-to-hematopoietic transition in two waves of hematopoietic cells and distinctive enhancer-gene linking schemes guiding hemogenic endothelial cell (HEC) emergence, indicating a unique epigenetic control of transcriptional regulation for hematopoietic stem cell priming. Together, CoTECH provides an efficient framework for single-cell co-assay of chromatin occupancy and transcription, thus, enabling higher-dimensional epigenomic reconstructions.

genomics↗

bioRxiv2020

Generation and Validation of Versatile Inducible CRISPRi Embryonic Stem Cell and Mouse Models

CRISPR-Cas9 has been widely used far beyond genome editing. Fusions of deactivated Cas9 (dCas9) to transcription effectors enable interrogation of the epigenome and controlling of gene expression. However the large transgene size of dCas9-fusion hinders its applications especially in somatic tissues. Here, we develop a robust CRISPR interference (CRISPRi) system by transgenic expression of doxycycline (Dox) inducible dCas9-KRAB in mouse embryonic stem cells (iKRAB ESC). After introduction of specific gRNAs, the induced dCas9-KRAB efficiently maintains gene inactivation, though it exerts modest effects on active gene expression. Proper timing of Dox addition during cell differentiation or reprogramming allows us to study or screen spatiotemporally activated promoters or enhancers and thereby the gene functions. Furthermore, taking the ESC for blastocyst injection, we generate an iKRAB knockin (KI) mouse model that enables shut-down of gene expression and loss-of-function studies ex vivo and in vivo by a simple transduction of gRNAs. Thus, our inducible CRISPRi ESC line and KI mouse provide versatile and convenient platforms for functional interrogation and high-throughput screens of specific genes and potential regulatory elements in the setting of development or diseases.

genetics↗

bioRxiv2020

Recombinant SARS-CoV-2 spike S1-Fc fusion protein induced high levels of neutralizing responses in nonhuman primates

The COVID-19 outbreak has become a global pandemic responsible for over 2,000,000 confirmed cases and over 126,000 deaths worldwide. In this study, we examined the immunogenicity of CHO-expressed recombinant SARS-CoV-2 S1-Fc fusion protein in mice, rabbits, and monkeys as a potential candidate for a COVID-19 vaccine. We demonstrate that the S1-Fc fusion protein is extremely immunogenic, as evidenced by strong antibody titers observed by day 7. Strong virus neutralizing activity was observed on day 14 in rabbits immunized with the S1-Fc fusion protein using a pseudovirus neutralization assay. Most importantly, in less than 20 days and three injections of the S1-Fc fusion protein, two monkeys developed higher virus neutralizing titers than a recovered COVID-19 patient in a live SARS-CoV-2 infection assay. Our data strongly suggests that the CHO-expressed SARS-CoV-2 S1-Fc recombinant protein could be a strong candidate for vaccine development against COVID-19. HighlightsO_LICHO-expressed S1-Fc protein is very immunogenic in various animals and can rapidly induce strong antibody production C_LIO_LIS1-Fc protein solicits strong neutralizing activities against live virus C_LIO_LIStable CHO cell line expressing 50 mg/L of S1-Fc and a 3,000 L Bioreactor can produce 3 million doses of human COVID-19 vaccine every 10 days, making it an accessible and affordable option for worldwide vaccination C_LI

immunology↗

bioRxiv2019

Vascular Progenitors Generated from Tankyrase Inhibitor-Regulated Diabetic Induced Pluripotent Stem Cells Potentiate Efficient Revascularization of Ischemic Retina

Vascular regenerative therapies with conventional human induced pluripotent stem cells (hiPSC) currently remain limited by high interline variability of differentiation and poor efficiency for generating functionally transplantable vascular progenitors (VP). Here, we report the advantage of tankyrase inhibitor-regulated naive hiPSC (N-hiPSC) for significantly improving vascular cell therapies. Conventional hiPSC reprogrammed from type-1 diabetic donor fibroblasts (DhiPSC) were stably reverted to naive epiblast-like state with high functional pluripotency with a cocktail of LIF and three small molecules inhibiting the tankyrase, MEK, and GSK3{beta} signaling pathways (LIF-3i). Naive diabetic VP (N-DVP) differentiated from naive DhiPSC (N-DhiPSC) expanded more efficiently, possessed higher proliferation, possessed more stable genomic integrity and displayed higher in vitro vascular functionality than primed diabetic VP (DVP) generated from isogenic conventional DhiPSC. Moreover, N-DVP survived, migrated, and engrafted in vivo into the deep vasculature of the neural retinal layers with significantly higher efficiencies than isogenic primed DVP in a murine model of ischemic retinopathy. Epigenetic analyses of CpG DNA methylation and histone configurations at developmental promoters of N-hiPSC revealed tight regulation of lineage-specific gene expression and a de-repressed naive epiblast-like epigenetic state that was highly poised for multi-lineage transcriptional activation. We propose that reprogramming of patient donor cells to a tankyrase inhibitor-regulated N-hiPSC may more effectively erase epigenetic aberrations sustained from chronic diseases such as diabetes for subsequent regenerative therapies. More broadly, tankyrase inhibitor-regulated N-hiPSC represent a new class of human stem cells with high epigenetic plasticity, improved multi-lineage functionality, and potentially high impact for regenerative medicine.

developmental biology↗

bioRxiv2019

CoBATCH for high-throughput single-cell epigenomic profiling

An efficient, generalizable method for genome-wide mapping of single-cell histone modifications or chromatin-binding proteins is so far lacking. Here we develop CoBATCH, combinatorial barcoding and targeted chromatin release, for single-cell profiling of genomic distribution of chromatin-binding proteins in cell culture and tissue. Protein A in fusion to Tn5 transposase is enriched through specific antibodies to genomic regions and Tn5 generates indexed chromatin fragments ready for the library preparation and sequencing. Importantly, through a combinatorial barcoding strategy, we are able to measure epigenomic features up to tens of thousands single cells per experiment. CoBATCH produces not only high signal-to-noise features, but also ~10,000 reads per cells, allowing for efficiently deciphering epigenetic heterogeneity of cell populations and subtypes and inferring developmental histories. Thus, obviating specialized device, CoBATCH is easily deployable for any laboratories in life science and medicine.

genomics↗