Biology subjects

Zheng, F.

Publications and source records attributed to Zheng, F..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

MiR-574-5p activates human TLR8 to promote autoimmune signaling and lupus

Endosomal single-stranded RNA-sensing Toll-like receptor-7/8 (TLR7/8) plays a pivotal role in inflammation and immune responses and autoimmune diseases. However, the mechanisms underlying the initiation of the TLR7/8-mediated autoimmune signaling remain to be fully elucidated. Here, we demonstrate that miR-574-5p is aberrantly upregulated in tissues of lupus prone mice and in the plasma of lupus patients, with its expression levels correlating with the disease activity. miR-574-5p binds to and activates human hTLR8 or its murine ortholog mTlr7 to elicit a series of MyD88-dependent immune and inflammatory responses. These responses include the overproduction of cytokines and interferons, the activation of STAT1 signaling and B lymphocytes, and the production of autoantigens. In a transgenic mouse model, the induction of miR-574-5p overexpression is associated with increased secretion of antinuclear and anti-dsDNA antibodies, increased IgG and C3 deposit in the kidney, elevated expression of inflammatory genes in the spleen. In lupus-prone mice, lentivirus-mediated silencing of miR-574-5p significantly ameliorates major symptoms associated with lupus and lupus nephritis. Collectively, these results suggest that the miR-574-5p-hTLR8/mTlr7 signaling is an important axis of immune and inflammatory responses, contributing significantly to the development of lupus and lupus nephritis.

immunology↗

bioRxiv2023

A KDELR-mediated ER retrieval system guides the signaling pathways of UPR and AMPK to maintain cellular homeostasis

KDELR (Erd2 in yeasts) mediates the retrieval of ER-resident proteins from the Golgi apparatus, yet how the KDELR-mediated ER retrieval system is involved in regulating cellular homeostasis has been unclear. Here we report that the loss of the Erd2-mediated ER retrieval system induces the unfolded protein response (UPR) and increases mitochondrial respiration and reactive oxygen species (ROS) in an UPR-dependent manner. Moreover, transcriptomic analysis revealed that expression of the genes involved in mitochondrial respiration and the tricarboxylic acid cycle is enhanced in an UPR-dependent manner in cells lacking Erd2. In cells lacking Erd2, the enhancement of mitochondrial respiration and ROS is required for maintaining cell viability. The loss of the Erd2-mediated ER retrieval system also activates AMPK, and consequently derepresses carbon catabolite repression. Hence, our work establishes a role of the KDELR/Erd2-mediated ER retrieval system in guiding the signalling pathways of AMPK and UPR and underscores the crucial role of the KDELR/Erd2-mediated ER retrieval system in the maintenance of cellular homeostasis.

cell biology↗

bioRxiv2023

Structures of 9-1-1 DNA checkpoint clamp loading at gaps from start to finish and ramification to biology

Recent structural studies show the Rad24-RFC loads the 9-1-1 checkpoint clamp onto a recessed 5' end by binding the 5' DNA on Rad24 at an external surface site and threading the 3' ssDNA into the well-established internal chamber and into 9-1-1. We find here that Rad24-RFC loads 9-1-1 onto DNA gaps in preference to a recessed 5' DNA end, thus presumably leaving 9-1-1 on a 3' ss/ds DNA after Rad24-RFC ejects from the 5' gap end and may explain reports of 9-1-1 directly functioning in DNA repair with various TLS polymerases, in addition to signaling the ATR kinase. To gain a deeper understanding of 9-1-1 loading at gaps we report high-resolution structures of Rad24-RFC during loading of 9-1-1 onto 10-nt and 5-nt gapped DNAs. At a 10-nt gap we captured five Rad24-RFC-9-1-1 loading intermediates in which the 9-1-1 DNA entry gate varies from fully open to fully closed around DNA using ATP{gamma}S, supporting the emerging view that ATP hydrolysis is not needed for clamp opening/closing, but instead for dissociation of the loader from the clamp encircling DNA. The structure of Rad24-RFC-9-1-1 at a 5-nt gap shows a 180{degrees} axially rotated 3'-dsDNA which orients the template strand to bridge the 3'- and 5'- junctions with a minimum 5-nt ssDNA. The structures reveal a unique loop on Rad24 that limits the length of dsDNA in the inner chamber, and inability to melt DNA ends unlike RFC, thereby explaining Rad24-RFCs preference for a preexisting ssDNA gap and suggesting a direct role in gap repair in addition to its checkpoint role.

biochemistry↗

bioRxiv2023

Disulfiram Exerts anti-pulmonary Fibrosis Effect by Activating PGE2 Synthesis

Idiopathic pulmonary fibrosis (IPF) is marked with the replacement of normal alveolar tissue by thicker and harder fibrous material, damaged exchange ability. Currently, nintedanib and pirfenidone, are the only FDA-approved drugs with limited efficacy for IPF, which indicated an urgent need to explore new therapies. Disulfiram (DSF), an acetaldehyde dehydrogenase inhibitor, used as anti-alcohol treatment. Despite reported with anti-hepatic fibrosis effect of DSF, the underlying mechanism remains unclear. In our study, DSF exhibited regulative impact on abnormal proliferation, EMT and ECM production in cell models of IPF including primary DHLF-IPF cells and TGF-{beta}1-stimulated A549 cells. The absence of COX-2 was restored by DSF treatment, together with elevated prostaglandin biosynthesis both in vitro and in vivo models of IPF. Furthermore, the anti-fibrotic effect of DSF was impeded with COX-2 knockdown or pharmacological inhibition in TGF-{beta}1-stimulated A549 cells, however, exogenous PGE2 reclaimed with anti-EMT function. In established animal model of IPF, DSF ameliorated declined lung function and histopathological changes, and restrained the lung hydroxyproline content. Together, these findings suggest that the anti-fibrotic effect of DSF was achieved through re-activation of COX-2 mediated PGE2 biosynthesis. The above results suggest that DSF can be applied therapeutically in fibrotic conditions.

cell biology↗

bioRxiv2023

Optimization of Cas12a for multiplexed genome-scale transcriptional activation

Cas12a CRISPR technology, unlike Cas9, allows for facile multiplexing of guide RNAs from a single transcript, simplifying combinatorial perturbations. While Cas12a has been implemented for multiplexed knockout genetic screens, it has yet to be optimized for CRISPR activation (CRISPRa) screens in human cells. Here we develop a new Cas12a-based transactivation domain (TAD) recruitment system using the ALFA nanobody and demonstrate simultaneous activation of up to four genes. We screen a genome-wide library to identify modulators of growth and MEK inhibition and we compare these results to those obtained with open reading frame (ORF) overexpression and Cas9-based CRISPRa. We find that the activity of multiplexed arrays is largely predictable from the best-performing guide and provide criteria for selecting active guides. We anticipate that these results will greatly accelerate the exploration of gene function and combinatorial phenotypes at scale.

genomics↗

bioRxiv2023

BCM PDX Portal: An Intuitive Web-based Tool for Patient-Derived Xenograft Collection Management, as well as Visual Integration of Clinical and Omics Data

ObjectiveMouse Patient-Derived Xenograft (PDX) models are essential tools for evaluating experimental therapeutics. Baylor College of Medicine (BCM) established a PDX Core to provide technical support and infrastructure for PDX-based research. To manage PDX collections effectively, de-identified patient clinical and omics data, as well as PDX-related information and omics data, must be curated and stored. Data must then be analyzed and visualized for each case. To enhance PDX collection management and data dissemination, the BCM Biomedical Informatics Core created the BCM PDX Portal (https://pdxportal.research.bcm.edu/). Materials and MethodsPatient clinical data are abstracted from medical records for each PDX and stored in a central database. Annotations are reviewed by a clinician and de-identified. PDX development method and biomarker expression are annotated. DNAseq, RNAseq, and proteomics data are processed through standardized pipelines and stored. PDX gene expression (mRNA/protein), copy number alterations, and mutations can be searched in combination with clinical markers to identify models potentially useful as a PDX cohort. ResultsPDX collection management and PDX selection of models for drug evaluation are facilitated using the PDX Portal. DiscussionTo improve the translational effectiveness of PDX models, it is beneficial to use a tool that captures and displays multiple features of the patient clinical and molecular data. Selection of models for studies should be representative of the patient cohort from which they originated. ConclusionThe BCM PDX Portal is a highly effective PDX collection management tool allowing data access in a visual, intuitive manner thereby enhancing the utility of PDX collections.

cancer biology↗

bioRxiv2022

Genetically encoded photocatalytic protein labeling enables spatially-resolved profiling of intracellular proteome

Mapping the subcellular organization of proteins is crucial for understanding their biological functions. Herein, we report a reactive oxygen species induced protein labeling and identification (RinID) method for profiling subcellular proteome in the context of living cells. Our method capitalizes on a genetically encoded photocatalyst, miniSOG, to locally generate singlet oxygen that reacts with proximal proteins. Labeled proteins are conjugated in situ with an exogenously supplied nucleophilic probe, which serves as a functional handle for subsequent affinity enrichment and mass spectrometry-based protein identification. From a panel of nucleophilic compounds, we identify biotin-conjugated aniline and propargyl amine as highly reactive probes. As a demonstration of the spatial specificity and depth of coverage in mammalian cells, we apply RinID in the mitochondrial matrix, capturing 394 mitochondrial proteins with 97% specificity. We further demonstrate the broad applicability of RinID in various subcellular compartments, including the nucleus and the endoplasmic reticulum.

biochemistry↗

bioRxiv2022

Accounting for small variations in the tracrRNA sequence improves sgRNA activity predictions for CRISPR screening

CRISPR technology is a powerful tool for studying genome function. To aid in picking sgRNAs that have maximal efficacy against a target of interest from many possible options, several groups have developed models that predict sgRNA on-target activity. Although multiple tracrRNA variants are commonly used for screening, no existing models account for this feature when nominating sgRNAs. Here we develop an on-target model, Rule Set 3, that makes optimal predictions for multiple tracrRNA variants. We validate Rule Set 3 on a new dataset of sgRNAs tiling essential and non-essential genes, demonstrating substantial improvement over prior prediction models. By analyzing the differences in sgRNA activity between tracrRNA variants, we show that Pol III transcription termination is a strong determinant of sgRNA activity. We expect these results to improve the performance of CRISPR screening and inform future research on tracrRNA engineering and sgRNA modeling.

bioengineering↗

bioRxiv2022

Community assessment of methods to deconvolve cellular composition from bulk gene expression

We evaluate deconvolution methods, which infer levels of immune infiltration from bulk expression of tumor samples, through a community-wide DREAM Challenge. We assess six published and 22 community-contributed methods using in vitro and in silico transcriptional profiles of admixed cancer and healthy immune cells. Several published methods predict most cell types well, though they either were not trained to evaluate all functional CD8+ T cell states or do so with low accuracy. Several community-contributed methods address this gap, including a deep learning-based approach, whose strong performance establishes the applicability of this paradigm to deconvolution. Despite being developed largely using immune cells from healthy tissues, deconvolution methods predict levels of tumor-derived immune cells well. Our admixed and purified transcriptional profiles will be a valuable resource for developing deconvolution methods, including in response to common challenges we observe across methods, such as sensitive identification of functional CD4+ T cell states.

bioinformatics↗

bioRxiv2022

Cadmium is detrimental to Caenorhabditis elegans via anetwork involving circRNA, lncRNA and phosphorylatedprotein

Cadmium (Cd) as a heavy metal causes serious environmental pollution and multiple organ and system damage in human. However, little is known about the specific molecular mechanisms of the associated regulatory networks. In this study, we selected Caenorhabditis elegans (C. elegans) to investigate the effects of Cd exposure as it acts as an acknowledged and established genetic model organism. A total of 26 differentially-expressed circular RNA (DEcircRNAs), 143 lncRNAs (DElncRNAs), 69 microRNAs (DEmiRNAs) and 6209 mRNAs (DEmRNAs) were found and identified, which might influence reproductive function, aging processes and nervous system functions through regulating the levels of circRNAs and lncRNAs and the controlling of regulatory networks of circRNA/lncRNA-miRNA-mRNA. Based on quantitative PCR, four DEcircRNAs and three DElncRNAs were confirmed to have different expression levels between the Cd-treated and control group. Further, 5 protein-coding genes might be regulated by DElnRNAs through cis-acting and 114 by trans-acting elements. Additionally, 42 differentially regulative phosphopeptides were detected and 4 novel pairs of transcription factors (TFs)-kinase-substrate that might be influenced by Cd exposure were constructed by phosphoproteomics. Our findings suggest that Cd might influence multi-functions and the aging process of C. elegans and may inhibit the expression of TFs to reduce phosphorylated levels of the corresponding protein. SynopsisCadmium exists widely in soil, water and air. This study manifested the regulatory network involving circRNA, lncRNA and phosphorylated protein in C.elegans after Cd exposure, which revealing the potential molecular mechanism underlying the toxic effect caused by Cd. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/486470v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1a012bborg.highwire.dtl.DTLVardef@2dc128org.highwire.dtl.DTLVardef@1e43ebaorg.highwire.dtl.DTLVardef@1d236b8_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2022

The production of diverse brGDGTs by an Acidobacterium allows a direct test of temperature and pH controls on their distribution

Microbial lipid biomarkers preserved in geological archives can be used to explore past climate changes. Branched glycerol dialkyl glycerol tetraethers (brGDGTs) are unique bacterial biomarkers that have been used as molecular tools for the quantitative determination of terrestrial temperatures and the pH of depositional environments over a range of geological timescales. However, the exact biological source organisms - especially of the entire suite of brGDGTs found in the environment - remains unclear; by extension, so do the mechanisms that govern these proxies. Here, we identified a brGDGT-producing strain Candidatus Solibacter usitatus Ellin6076, by identifying archaeal tetraether synthase homologs in bacterial genomes. This strain synthesizes diverse brGDGTs, including regular C5-methylated and cyclic brGDGTs, and brGDGTs comprise up to 66% of the major lipids, far exceeding the proportions found in previous studies. The degree of C5-methylation in cultured strain Ellin6076 is primarily determined by temperature, whereas cyclization appears to be influenced by multiple factors. Consequently, culture-derived paleoclimate indices are in agreement with the global soil-derived MBT5ME (methylation index of C5-methyl brGDGTs) proxy for temperature but not the CBT5ME (cyclization index of C5-methyl brGDGTs) proxy for pH. Our findings provide important insights from a physiological perspective into the underlying mechanism of brGDGT-based proxies. Significance StatementBranched glycerol dialkyl glycerol tetraethers (brGDGTs) are biomarkers widely used for the quantitative estimation of past climatic changes due to their ubiquitous occurrence in the environment and the relationships between their distributions and temperature and pH. However, the ecophysiology of brGDGT-producing bacteria and the mechanistic basis for brGDGT-based climate proxies remain unknown. Here, we identify a brGDGT-producing Acidobacterium and present a physiological study of brGDGTs in response to cultivation variables, which provides pivotal insights into how brGDGT producers modulate methylation and cyclization under different culturing conditions. Our study represents a significant advancement in understanding the physiological role of lipid structures in microbial adaptation and helps us interpret the relationships between brGDGT-based proxies and environmental conditions of the geological environment.

microbiology↗

bioRxiv2021

Rad24-RFC loads the 9-1-1 clamp by inserting DNA from the top of a wide-open ring, opposite the mechanism of RFC/PCNA

In response to DNA damage, the ring-shaped 9-1-1 clamp is loaded onto 5 recessed DNA to arrest the cell cycle and activate the DNA damage checkpoint. The 9-1-1 clamp is a heterotrimeric ring that is loaded in S. cerevisiae by Rad24-RFC, an alternative clamp loader in which Rad24 replaces the Rfc1 subunit in the RFC1-5 clamp loader of PCNA. Unlike RFC that loads the PCNA ring onto a 3-ss/ds DNA junction, Rad24-RFC loads the 9-1-1 ring onto a 5-ss/ds DNA junction, a consequence of DNA damage. The underlying 9-1-1 clamp loading mechanism has been a mystery. Here we report two 3.2-[A] cryo-EM structures of Rad24-RFC bound to DNA and either a closed or 27 [A] open 9-1-1 clamp. The structures reveal a completely unexpected mechanism by which a clamp can be loaded onto DNA. The Rad24 subunit specifically recognizes the 5-DNA junction and holds ds DNA outside the clamp loader and above the plane of the 9-1-1 ring, rather than holding DNA inside and below the clamp as in RFC. The 3 ssDNA overhang is required to obtain the structure, and thus confers a second DNA binding site. The bipartite DNA binding by Rad24-RFC suggests that ssDNA may be flipped into the open 9-1-1 ring, similar to ORC-Cdc6 that loads the Mcm2-7 ring on DNA. We propose that entry of ssDNA through the 9-1-1 ring triggers the ATP hydrolysis and release of the Rad24-RFC. The key DNA binding residues are conserved in higher eukaryotes, and thus the 9-1-1 clamp loading mechanism likely generalizes.

biochemistry↗

bioRxiv2021

Structure and computation-guided design of a mutation-integrated trimeric RBD candidate vaccine with broad neutralization against SARS-CoV-2

The spike (S) protein receptor-binding domain (RBD) of SARS-CoV-2 is an attractive target for COVID-19 vaccine developments, which naturally exists in a trimeric form. Here, guided by structural and computational analyses, we present a mutation-integrated trimeric form of RBD (mutI tri-RBD) as a broadly protective vaccine candidate, in which three RBDs were individually grafted from three different circulating SARS-CoV-2 strains including the prototype, Beta (B.1.351) and Kappa (B.1.617). The three RBDs were then connected end-to-end and co-assembled to possibly mimic the native trimeric arrangements in the natural S protein trimer. The recombinant expression of the mutI tri-RBD, as well as the homo-tri-RBD where the three RBDs were all truncated from the prototype strain, by mammalian cell exhibited correct folding, strong bio-activities, and high stability. The immunization of both the mutI tri-RBD and homo-tri-RBD plus aluminum adjuvant induced high levels of specific IgG and neutralizing antibodies against the SARS-CoV-2 prototype strain in mice. Notably, regarding to the "immune-escape" Beta (B.1.351) variant, mutI tri-RBD elicited significantly higher neutralizing antibody titers than homo-tri-RBD. Furthermore, due to harboring the immune-resistant mutations as well as the evolutionarily convergent hotspots, the designed mutI tri-RBD also induced strong broadly neutralizing activities against various SARS-CoV-2 variants, especially the variants partially resistant to homo-tri-RBD. Homo-tri-RBD has been approved by the China National Medical Products Administration to enter clinical trial (No. NCT04869592), and the superior broad neutralization performances against SARS-CoV-2 support the mutI tri-RBD as a more promising vaccine candidate for further clinical developments.

immunology↗

bioRxiv2021

Proteome plasticity in response to persistent environmental change

Temperature is a variable component of the environment and all organisms must deal with or adapt to temperature change. Acute temperature change activates cellular stress responses resulting in the refolding or removal of damaged proteins. However, how organisms adapt to long-term temperature change remains largely unexplored. Here, we report that budding yeast responds to long-term high temperature challenge by switching from chaperone induction to the reduction of temperature sensitive proteins and re-localizing a portion of its proteome. Surprisingly, we also find many proteins adopt an alternative conformation. Using Fet3p as an example, we find that the temperature-dependent conformational difference is accompanied by distinct thermostability, subcellular localization, and importantly, cellular functions. We postulate that in addition to the known mechanisms of adaptation, conformational plasticity allows some polypeptides to acquire new biophysical properties and functions when environmental change endures.

molecular biology↗

bioRxiv2021

The intestinal microbiota-driven dopamine level influences social interaction and innate color preference in zebrafish larvae

Gut microbiota influence neurodevelopment of brain and programing of behaviors. However, the mechanism underlining the relationship between shoals behaviors and intestinal microbiota remain controversial and the roles of neurotransmitters are still unclear. Here we show that, shoaling behavior affected the innate color preference of shoals, indicating that shoals tended to choose a favorable color environment that benefits social contact. Meanwhile, administration of D1-R antagonist disrupted the social interaction which led to the deficits of color preference. More importantly, the altered microbiota caused by an antibiotic OTC decreased the sociability and weakened shoals color preference. When given a supplement of LGG after OTC exposure, fish exhibited an unexpectedly recovery capability in social cohesion and color preference. Our findings show that dopamine level of brain could mediate both social recognition and color preference, and highlight the pathway of microbial metabolites through the microbiota-gut-brain axis that coordinate the production of dopamine.

animal behavior and cognition↗

bioRxiv2021

BTBD9 is a novel component of IGF signaling and regulates manganese-induced dopaminergic dysfunction

Restless legs syndrome (RLS) is a common neurological disorder associated with iron deficiency and dopaminergic (DAergic) neuronal dysfunction. BTBD9 is a genetic risk factor for RLS. However, its molecular function remains largely unknown. Here, we report the interaction between BTBD9, manganese (Mn) and insulin/insulin-like growth factor (IGF) signaling in Caenorhabditis elegans, mouse Neuro2a cells and humans. We found that elevated Mn downregulated BTBD9 mRNA levels; in turn, BTBD9 expression attenuated Mn-induced cellular stress and dopaminergic neurodegeneration. As Mn is a known co-factor for insulin receptor and IGF-1 receptor, which activates IGF signaling, we posited that BTBD9 negatively regulates IGF signaling. Our results showed that the protective effects of BTBD9 against Mn toxicity were dependent on the forkhead box O (FOXO) protein. Furthermore, BTBD9 overexpression significantly elevated FOXO level and decreased PKB level, while phosphoinositide-dependent kinase-1 (PDK1) level remained unchanged. We conclude that BTBD9 acts as a key component in the IGF signaling pathway. Meanwhile, the roles of Mn in DAergic neurotoxicity and regulating BTBD9 shed new light on the etiology of RLS.

neuroscience↗

bioRxiv2020

Mapping cell structure across scales by fusing protein images and interactions

ABSTRACTThe eukaryotic cell is a multi-scale structure with modular organization across at least four orders of magnitude1,2. Two central approaches for mapping this structure – protein fluorescent imaging and protein biophysical association – each generate extensive datasets but of distinct qualities and resolutions that are typically treated separately3,4. Here, we integrate immunofluorescent images in the Human Protein Atlas5 with ongoing affinity purification experiments from the BioPlex resource6 to create a unified hierarchical map of eukaryotic cell architecture. Integration involves configuring each approach to produce a general measure of protein distance, then calibrating the two measures using machine learning. The evolving map, called the Multi-Scale Integrated Cell (MuSIC 1.0), currently resolves 69 subcellular systems of which approximately half are undocumented. Based on these findings we perform 134 additional affinity purifications, validating close subunit associations for the majority of systems. The map elucidates roles for poorly characterized proteins, such as the appearance of FAM120C in chromatin; identifies new protein assemblies in ribosomal biogenesis, RNA splicing, nuclear speckles, and ion transport; and reveals crosstalk between cytoplasmic and mitochondrial ribosomal proteins. By integration across scales, MuSIC substantially increases the mapping resolution obtained from imaging while giving protein interactions a spatial dimension, paving the way to incorporate many molecular data types in proteome-wide maps of cells.Competing Interest StatementThe authors have declared no competing interest.View Full Text

systems biology↗

bioRxiv2020

Decoding of persistent multiscale structures in complex biological networks

In any omics study, the scale of analysis can dramatically affect the outcome. For instance, when clustering single-cell transcriptomes, is the analysis tuned to discover broad or specific cell types? Likewise, protein communities revealed from protein networks can vary widely in sizes depending on the method. Here we use the concept of "persistent homology", drawn from mathematical topology, to identify robust structures in data at all scales simultaneously. Application to mouse single-cell transcriptomes significantly expands the catalog of identified cell types, while analysis of SARS-COV-2 protein interactions suggests hijacking of WNT. The method, HiDeF, is available via Python and Cytoscape.

systems biology↗