Biology subjects

Hu, S.

Publications and source records attributed to Hu, S..

At least 109 records · Page 6Linked to original sources

bioRxiv2023

Stanniocalcin 2 (STC2) is a potent biomarker of hepatocellular carcinoma with its expression being augmented in Nrf1α-deficient cells, but diminished in Nrf2-deficient cells

For insights into the fact that liver-specific knockout of Nrf1 leads to development of non-alcoholic steatohepatitis and spontaneous hepatoma, we previously found that loss of Nrf1 (i.e., a full-length isoform encoded by Nfe2l1) promotes HepG2-derived tumor growth in xenograft mice, but malgrowth of the xenograft tumor is significantly suppressed by knockout of Nrf2 (encoded by Nfe2l2). The mechanism underlying such marked distinctions in their pathologic phenotypes remains elusive, however, to date. Herein, we mined the transcriptome data of liver cancer from the TCGA database to establish a prognostic model of liver cancer and then calculated the predicted risk score of each cell line. The results indicated that knockout of Nrf1 significantly increased the risk score in HepG2 cells, whereas the risk score was reduced by knockout of Nrf2. Of note, stanniocalcin 2 (STC2, a biomarker of liver cancer, that is up-expressed in hepatocellular carcinoma (HCC) tissues with a reduction in the overall survival ratio of those patients) was augmented in Nrf1Nrf2-/- cells, but diminished in Nrf2-/- cells. Thereby, it is inferable that STC2 is likely involved in mediating the distinction between Nrf1Nrf2-/- and Nrf2-/-. Further investigation revealed that HIF1A is an upstream regulator of STC2 in caNrf2{Delta}N, rather than Nrf1Nrf2-/-, cells, and regulation of STC2 and HIF1A in Nrf1Nrf2-/- is determined by Nrf2, but the regulation of STC2 by Nrf2 may be independent on HIF1A. In turn, STC2 can regulate Nrf2 via the putative calcium-mediated Keap1-p62 signaling so to form a feedback regulatory loop. Such potential function of STC2 was further corroborated by a series of experiments combined with transcriptomic sequencing. The results unraveled that STC2 manifests as a dominant tumor-promoter, because the STC2-leading increases in clonogenicity of hepatoma cells and malgrowth of relevant xenograft tumor were almost completely abolished in STC2-/-cells. Together, these demonstrate that STC2 could be paved as a novel potent therapeutic target, albeit as a diagnostic marker, for hepatocellular carcinoma.

cell biology↗

bioRxiv2023

Global Marine Cold Seep Metagenomes Reveal Diversity of Taxonomy, Metabolic Function, and Natural Products

Cold seeps in the deep sea are closely linked to energy exploration as well as global climate change. The alkane-dominated chemical energy-driven model makes cold seeps an oasis of deep-sea life, showcasing an unparalleled reservoir of microbial genetic diversity. By analyzing 113 metagenomes collected from 14 global sites across 5 cold seep types, we present a comprehensive Cold Seep Microbiomic Database (CSMD) to archive the genomic and functional diversity of cold seep microbiome. The CSMD includes over 49 million non-redundant genes and 3175 metagenome-assembled genomes (MAGs), which represent 1897 species spanning 106 phyla. In addition, beta diversity analysis indicates that both sampling site and cold seep type have substantial impact on the prokaryotic microbiome community composition. Heterotrophic and anaerobic metabolisms are prevalent in microbial communities, accompanied by considerable mixotrophs and facultative anaerobes, indicating the versatile metabolic potential in cold seeps. Furthermore, secondary metabolic gene cluster analysis indicates that at least 98.81% of the sequences encode potentially novel natural products. These natural products are dominated by ribosomal processing peptides, which are widely distributed in archaea and bacteria. Overall, the CSMD represents a valuable resource which would enhance the understanding and utilization of global cold seep microbiomes.

microbiology↗

bioRxiv2023

Stricturing Crohn's disease single-cell RNA sequencing reveals fibroblast heterogeneity and intercellular interactions

BackgroundFibroblasts play a key role in stricture formation in Crohns disease (CD) but understanding its pathogenesis requires a systems-level investigation to uncover new treatment targets. We studied full thickness CD tissues to characterize fibroblast heterogeneity and function by generating the first single cell RNA sequencing (scRNAseq) atlas of strictured bowel and providing proof of principle for therapeutic target validation. MethodsWe performed scRNAseq of 13 fresh full thickness CD resections containing non-involved, inflamed non-strictured, and strictured segments as well as 7 normal non-CD bowel segments. Each segment was separated into mucosa/submucosa or muscularis propria and analyzed separately for a total of 99 tissue samples and 409,001 cells. We validated cadherin-11 (CDH11) as a potential therapeutic target by using whole tissues, isolated intestinal cells, NanoString nCounter, next generation sequencing, proteomics and animal models. ResultsOur integrated dataset revealed fibroblast heterogeneity in strictured CD with the majority of stricture-selective changes detected in the mucosa/submucosa, but not the muscle layer. Cell-cell interaction modeling revealed CXCL14+ as well as MMP/WNT5A+ fibroblasts displaying a central signaling role in CD strictures. CDH11, a fibroblast cell-cell adhesion molecule, was broadly expressed and upregulated, and its pro-fibrotic function was validated by NanoString nCounter, RNA sequencing, tissue target expression, in vitro gain- and loss-of-function experiments, proteomics, and two animal models of experimental colitis. ConclusionA full-thickness bowel scRNAseq atlas revealed previously unrecognized fibroblast heterogeneity and interactions in CD strictures and CDH11 was validated as a potential therapeutic target. These results provide a new resource for a better understanding of CD stricture formation and opens potential therapeutic developments.

immunology↗

bioRxiv2023

Temporal colonization of the gut microbiome in neonatal Bos taurus at single nucleotide resolution

BackgroundThe rumen of neonatal calves is underdeveloped and exhibits limited functionality during early life. Thus, the acquisition and colonization of microbes in the gut are key to establishing a healthy host-microbiome symbiosis for neonatal calves. Microbiome-linked health outcomes appear to be the consequences of individual strains of specific microbes. However, the temporal colonization of pioneering microbial strains and their linkages to the health and growth of neonatal calves are poorly understood. ResultsTo address this, we longitudinally profiled the gut microbiome of 36 neonatal calves from birth up to 2 months postpartum and carried out microbial transplantation (MT) to reshape their gut microbiome. Genomic reconstruction of deeply sequenced fecal samples resulted in a total of 3,931 metagenomic assembled genomes (MAGs), of which 397 were identified as new species when compared with existing databases of Bos taurus. Single nucleotide level metagenomic profiling shows a rapid influx of microbes after birth, followed by strong selection during the first few weeks of life. MT was found to reshape the genetic makeup of 33 MAGs (FDR<0.05), mainly from Prevotella and Bacteroides species. We further linked over 20 million microbial single nucleotide variations (SNVs) to 736 plasma metabolites, which enabled us to characterize 24 study-wide significant associations (P < 4.4x10-9) that identify the potential microbial genetic regulation of host immune and neuro-related metabolites, including glutathione and L-dopa. Our integration analyses further revealed that microbial genetic variations may influence the health status and growth performance of neonatal calves by modulating metabolites via structural regulation of their encoded proteins. For instance, we found that the albumin levels and total antioxidant capacity in neonatal calves were correlated with L-dopa, which was determined by SNVs via structural regulations of metabolic enzymes. ConclusionsThe current results indicate that the temporal colonization of microbial strains and MT-induced strain replacement are integral in the development of the gut microbiome of neonatal calves and may help to develop strategies that can improve the health status and growth performance of neonatal calves.

microbiology↗

bioRxiv2023

Early microbial intervention reduces diarrhea while improves later milk production in newborn calves

The rumen of neonatal calves is not well-developed and exhibits limited functionality. Therefore, the establishment of intestinal microbiota may play an instrumental role in their health and performance, but it has been rarely explored. Thus, we aim to explore the temporal colonization of the gut microbiome and the potential benefits of early microbial intervention in newborn calves. We followed up on the temporal dynamics of the gut microbiome and plasma metabolome in 36 newborn calves during the first two months of life and established their relationships with their health status and performance. We also evaluated whether microbiota transplantation (MT) could influence their phenotypes by modulating metabolism and its impact on later milk production performance.We showed that the composition and ecological interactions of the gut microbiome are likely to reach maturity one month after birth. Temporal changes in the gut microbiome of newborn calves are widely associated with changes in their physiological statuses, such as growth and fiber digestion. Importantly, we observed that MT reshapes the gut microbiome of newborns by altering the abundance and interaction of Bacteroides species, as well as amino acid pathways, such as arginine biosynthesis. Two-year follow-up of those calves further showed that MT improves their later milk production. Notably, MT improves fiber digestion, antioxidant capacity of newborns while reducing diarrhea. MT also contributes to significant changes in the metabolomic landscape, and with putative causal mediation analysis, we suggest that altered gut microbial composition in newborns may influence physiological status through microbial-derived metabolites. The data from the study may help develop strategies to manipulate the gut microbiota during early life, which may be significantly relevant to the health and production of newborn calves. HIGHLIGHTSO_LIThe gut microbial composition and ecological interaction in newborn calves reach maturity one month after birth C_LIO_LITemporal shifts in the gut microbiome of newborn calves are associated with phenotypic changes, including growth, digestion, and antioxidative capacity C_LIO_LIEarly microbial intervention in newborn calves reduces diarrhea while improving later milk production C_LIO_LIThe gut microbial impact on newborn calves is mediated by plasma metabolites C_LI

microbiology↗

bioRxiv2023

Degenerated CRISPRs widely regulate Cas expression to balance immunity and cost

CRISPR RNAs (crRNAs) and Cas proteins together provide prokaryotes with adaptive immunity against genetic invaders. How Cas expression is fine-tuned to avoid energy burden while satisfying the dynamic need of crRNAs remains poorly understood. Here we experimentally demonstrated widespread degenerated mini-CRISPRs encode CreR (Cas-regulating) RNAs to mediate autorepression of type I-B, I-E and V-A Cas proteins, based on their partial complementarity to cas promoters. This autorepression decreases energy burden and autoimmune risks, thus mitigating the fitness cost on host cell, and remarkably, senses and responds to alterations in the volume of canonical crRNAs, which compete with CreR for Cas proteins. Moreover, CreR-guided Cas autorepression can be subverted by diverse anti-CRISPR (Acr) proteins that destruct Cas proteins, which in turn replenishes the weapon depot. Our data unveil a general degenerated crRNA-guided autorepression paradigm for diverse Cas effectors, which highlights the intricate (self-)regulation of CRISPR-Cas and its transcriptional counterstrategy against Acr attack.

microbiology↗

bioRxiv2023

Dynamical modeling reveals RNA decay mediates the effect of matrix stiffness on aged muscle stem cell fate

Loss of muscle stem cell (MuSC) self-renewal with aging reflects a combination of influences from the intracellular (e.g., post-transcriptional modifications) and extracellular (e.g., matrix stiffness) environment. Whereas conventional single cell analyses have revealed valuable insights into factors contributing to impaired self-renewal with age, most are limited by static measurements that fail to capture nonlinear dynamics. Using bioengineered matrices mimicking the stiffness of young and old muscle, we showed that while young MuSCs were unaffected by aged matrices, old MuSCs were phenotypically rejuvenated by young matrices. Dynamical modeling of RNA velocity vector fields in silico revealed that soft matrices promoted a self-renewing state in old MuSCs by attenuating RNA decay. Vector field perturbations demonstrated that the effects of matrix stiffness on MuSC self-renewal could be circumvented by fine-tuning the expression of the RNA decay machinery. These results demonstrate that post-transcriptional dynamics dictate the negative effect of aged matrices on MuSC self-renewal. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/529950v2_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1a840corg.highwire.dtl.DTLVardef@d354cforg.highwire.dtl.DTLVardef@650556org.highwire.dtl.DTLVardef@41587c_HPS_FORMAT_FIGEXP M_FIG Graphical abstract description: The balance of self-renewal and differentiation in young muscle stem cells (MuSCs) is robust to perturbations of the biophysical microenvironment. In contrast, aged MuSCs are highly sensitive to extrinsic perturbations, and exposure to a youthful microenvironment rejuvenates the self-renewing potential of aged MuSCs by modulating post-transcriptional RNA dynamics. C_FIG

cell biology↗

bioRxiv2023

Spatiotemporal reconstruction of the origin and assembly of smooth muscles in the intestinal villus

Intestinal smooth muscles are the workhorse of the digestive system. Inside the millions of finger-like intestinal projections called villi, strands of smooth muscle cells contract to propel absorbed dietary fats through the adjacent lymphatic vessel, called the lacteal, sending fats into the blood circulation for energy production. Despite this vital function, how villus smooth muscles form, how they assemble alongside lacteals, and how they repair throughout life remain unknown. Here we combine single-cell RNA sequencing of the mouse intestine with quantitative lineage tracing to reveal the mechanisms of formation and differentiation of villus smooth muscle cells. Within the highly regenerative villus, we uncover a local hierarchy of subepithelial fibroblast progenitors that progress to become mature smooth muscle fibers, via an intermediate contractile myofibroblast-like phenotype. This continuum persists in the adult intestine as the major source of renewal of villus smooth muscle cells during adult life. We further found that the NOTCH3-DLL4 signaling axis governs the assembly of villus smooth muscles alongside their adjacent lacteal, and we show that this is necessary for gut absorptive function. Overall, our data shed light on the genesis of a poorly defined class of intestinal smooth muscle and pave the way for new opportunities to accelerate recovery of digestive function by stimulating muscle repair.

developmental biology↗

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↗

bioRxiv2022

MIDAS: a deep generative model for mosaic integration and knowledge transfer of single-cell multimodal data

AO_SCPLOWBSTRACTC_SCPLOWRapidly developing single-cell multi-omics sequencing technologies generate increasingly large bodies of multimodal data. Integrating multimodal data from different sequencing technologies, i.e. mosaic data, permits larger-scale investigation with more modalities and can help to better reveal cellular heterogeneity. However, mosaic integration involves major challenges, particularly regarding modality alignment and batch effect removal. Here we present a deep probabilistic framework for the mosaic integration and knowledge transfer (MIDAS) of single-cell multimodal data. MIDAS simultaneously achieves dimensionality reduction, imputation, and batch correction of mosaic data by employing self-supervised modality alignment and information-theoretic latent disentanglement. We demonstrate its superiority to other methods and reliability by evaluating its performance in full trimodal integration and various mosaic tasks. We also constructed a single-cell trimodal atlas of human peripheral blood mononuclear cells (PBMCs), and tailored transfer learning and reciprocal reference mapping schemes to enable flexible and accurate knowledge transfer from the atlas to new data. Applications in mosaic integration, pseudotime analysis, and cross-tissue knowledge transfer on bone marrow mosaic datasets demonstrate the versatility and superiority of MIDAS.

bioinformatics↗

bioRxiv2022

Histone H1.0 Couples Cellular Mechanical Behaviors to Chromatin Structure

Tuning of genome structure and function is accomplished by chromatin binding proteins, which determine the transcriptome and phenotype of the cell. We sought to investigate how communication between extracellular stress and chromatin structure may regulate cellular mechanical behaviors. We demonstrate that the linker histone H1.0, which compacts nucleosomes into higher order chromatin fibers, controls genome organization and cellular stress response. Histone H1.0 has privileged expression in fibroblasts across tissue types in mice and humans, and modulation of its expression is necessary and sufficient to mount a myofibroblast phenotype in these cells. Depletion of histone H1.0 prevents transforming growth factor beta (TGF-{beta})-induced fibroblast contraction, proliferation and migration in a histone H1 isoform-specific manner via inhibition of a transcriptome comprised of extracellular matrix, cytoskeletal and contractile genes. Histone H1.0 is associated with local regulation of gene expression via mechanisms involving chromatin fiber compaction and reprogramming of histone acetylation, rendering the cell stiffer in response to cytokine stimulation. Knockdown of histone H1.0 prevented locus-specific histone H3 lysine 27 acetylation by TGF-{beta} and decreased levels of both HDAC1 and the chromatin reader BRD4, thereby preventing transcription of a fibrotic gene program. Transient depletion of histone H1.0 in vivo decompacts chromatin and prevents fibrosis in cardiac muscle, thereby linking chromatin structure with fibroblast phenotype in response to extracellular stress. Our work identifies an unexpected role of linker histones to orchestrate cellular mechanical behaviors, directly coupling cellular force generation, nuclear organization and gene transcription. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=166 SRC="FIGDIR/small/518399v1_ufig1.gif" ALT="Figure 1"> View larger version (103K): org.highwire.dtl.DTLVardef@11a38e1org.highwire.dtl.DTLVardef@8c4ab4org.highwire.dtl.DTLVardef@7c822aorg.highwire.dtl.DTLVardef@45d02b_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

A novel crosstalk between Nrf2 and Smad2/3 bridged by two nuanced Keap1 isoforms

The Keap1-Nrf2 signalling to transcriptionally regulate antioxidant response element (ARE)-driven target genes has been accepted as key redox-sensitive pathway governing a vast variety of cellular stresses during healthy survival and disease development. Herein, we identified two nuanced isoforms and {beta} of Keap1, arising from its first and another in-frame translation starting codons, respectively. In identifying those differential expression genes monitored by Keap1 and/or Keap1{beta}, an unusual interaction of Keap1 with Smad2/3 was discovered by parsing transcriptome sequencing, Keap1-interacting protein profiling and relevant immunoprecipitation data. Further examination validated that Smad2/3 enable physical interaction with Keap1, as well as its isoforms and {beta}, by both EDGETSD and DLG motifs in the linker regions between their MH1 and MH2 domains, such that the stability of Smad2/3 and its transcriptional activity are enhanced with the prolonged half-lives and signalling responses from the cytoplasmic to nuclear compartments. The activation of Smad2/3 by Keap1, Keap1 or Keap1{beta} was likely contributable to a coordinative or another competitive effect of Nrf2, particularly in distinct Keap1-based cellular responses to its cognate growth factor or redox stress. Overall, this discovery presents a novel functional bridge crossing both the Keap1-Nrf2 redox signalling and the TGF-{beta}1-Smad2/3 pathways in healthy growth and development.

cell biology↗

bioRxiv2022

Robust identification of extrachromosomal DNA and genetic variants using multiple genetic abnormality sequencing (MGA-Seq)

Genomic abnormalities, including structural variation (SV), copy number variation (CNV), single-nucleotide polymorphism (SNP), homogenously staining regions (HSR) and extrachromosomal DNA (ecDNA), are strongly associated with cancer, rare diseases and infertility. A robust technology to simultaneously detect these genomic abnormalities is highly desired for clinical diagnosis and basic research. In this study, we developed a simple and cost-effective method - multiple genetic abnormality sequencing (MGA-Seq) - to simultaneously detect SNPs, CNVs, SVs, ecDNA and HSRs in a single tube. This method has been successfully applied in both cancer cell lines and clinical tumour samples and revealed that focal amplification in tumour tissue is substantially heterogeneous. Notably, we delineated the architecture of focal amplification and the ecDNA network by MGA-Seq, which facilitated the exploration of the regulation of gene expression in ecDNA. This method could be extensively applied for diagnosis and may greatly facilitate the investigation of the genomic mechanism for genetic diseases.

genomics↗

bioRxiv2022

Essential role of the amino-terminal region of Drosha for the Microprocessor function

The ribonuclease (RNase) III enzyme Drosha enables processing of microRNA (miRNA) primary transcripts (pri-miRNAs) and control of ribosomal protein (RP) biogenesis by the Microprocessor. The extensively studied carboxyl-terminal half of Drosha is sufficient to crop pri-miRNAs in vitro, but the function of the evolutionarily conserved Drosha amino-terminal region (NTR) is unknown, despite it harboring mutations linked to a hereditary vascular disorder. Here, we provide evidence that, when replacing the endogenous Drosha, a mutant missing the NTR ({Delta}N-Drosha) fails to associate with endogenous pri-miRNAs and to support the expression of all miRNAs, except the miR-183 cluster. Surprisingly, Argonaute2 (Ago2) associates with and cleaves pri-miR-183 in {Delta}N-Drosha cells and in Drosha-depleted cells. {Delta}N-Drosha is also unable to inhibit RP biogenesis upon serum starvation. Thus, Drosha NTR is essential for pri-miRNA processing and RP biogenesis in vivo, and Ago2 can process the miR-183 cluster in the absence of Drosha.

molecular biology↗

bioRxiv2022

The roles and evolution of the four LEAFY homologues in floral patterning and leaf development in woodland strawberry

The plant-specific transcription factor LEAFY (LFY), generally maintained as a single copy gene in most angiosperm species, plays critical roles in flower and leaf development. However, wild strawberry Fragaria vesca possesses four LFY homologues in the genome, their respective functions and evolution remain unknown. Through chemical mutagenesis screen, we identified two allelic mutations in one of the four LFY homologues, FveLFYa, in F. vesca, causing homeotic conversion of floral organs and reiterative outgrowth of ectopic florets. Both CRISPR-knockout and transgenic rescue confirmed the identity of FveLFYa. Ectopic expression in Arabidopsis lfy-5 mutant revealed that only FveLFYa and FveLFYb can rescue the flower defects and induce solitary flowers in leaf axils. Disruption of FveLFYc, the second abundantly expressed LFY homologue, caused no obvious morphology phenotypes in F. vesca. FveLFYb and FveLFYd are barely expressed. Expression of FveAP1, homologue of the well-known LFY target AtAP1, is not changed in the fvelfya flowers, possibly caused by an absence of any FveLFYa binding site in its promoter. Loss of Axillary Meristems encodes a GRAS transcription factor essential for stamen initiation. The ectopic florets are eliminated in fvelfya lam, suggesting that LAM is required for floret production. Moreover, approximately 30% of mature leaves have smaller or fewer leaflets in fvelfya. Among these homologues, only FveLFYa is syntenic to the homologues in other species. Overall, the detailed analyses of the four LFY homologues in woodland strawberry demonstrate that only FveLFYa plays crucial roles in floral patterning with rewired gene network.

plant biology↗

bioRxiv2022

Arabidopsis CPK6 regulates drought tolerance under high nitrogen by the phosphorylation of NRT1.1

Nitrogen is an essential macronutrient for plant growth and development, and its availability to some extent is regulated by drought stress. Calcium-dependent protein kinases (CPKs) are a unique family of Ca2+ sensors with diverse functions in nitrogen and drought signaling pathways. However, which and how CPKs involve in the crosstalk between drought stress and nitrogen transportation remains largely unknown. Here we identified the drought tolerant function of Arabidopsis CPK6 under high nitrogen condition. The CPK6 expression is induced by the treatments of ABA and drought. The mutant cpk6 is insensitive to the ABA treatment, but sensitive to drought only under high nitrogen condition. CPK6 interacts with and phosphorylates the Thr571 in NRT1.1 protein, and thus represses its NO3- transporting activity under drought stress. Taken together, we showed the evidences that CPK6 regulates Arabidopsis drought tolerance through the phosphorylation of NRT1.1, and that enriches the knowledge of nitrogen uptake in plants during drought stress.

genetics↗

bioRxiv2022

Substrate-binding Glycine Residues are Major Determinants for Hydrolase and Ligase Activity of Plant Legumains

Peptide asparaginyl ligases (PALs) are Asn/Asp(Asx)-specific ligases that are useful for precision modifications of proteins and live-cell surfaces. However, PALs share high structural similarity to the far more common asparaginyl endopeptidases (AEPs), also known as legumains that hydrolyze peptide bonds after Asx, thus making it challenging to identify PALs in a sea of AEPs. Previously we identified sequences flanking the catalytic site as ligase activity determinants (LADs) for legumains. Here we show that two conserved substrate-binding Gly residues are critical, but negative determinants for ligase activity, based on a combined bioinformatics analysis of 1,500 plant legumains, mutagenesis and functional study of 16 novel legumains, plus identification of seven new PALs. We also show that PALs are rare and AEPs are much more common, accounting for about 1% and 88%, respectively. Our results suggest that specific glycine residues are molecular determinants to identify PALs and AEPs as two different legumain subfamilies.

plant biology↗

bioRxiv2022

A spatial atlas of Wnt and Frizzled receptor expression in adult mouse liver

Hepatic zonation is critical for most metabolic functions in liver. Wnt signaling plays an important role in establishing and maintaining liver zonation. Yet, the anatomic expression of Wnt signaling components, including all 10 Frizzled receptors (Fzds), has not been characterized in adult liver. To address this, we quantitatively mapped the spatial expression of Wnt/Fzd pathway components in adult mouse liver via multiplex fluorescent in situ hybridization. While all 10 Fzds are expressed within a metabolic unit, Fzds 1, 4, and 6 are the highest expressed. Though the majority of Wnt signaling occurs in zone 3, expression of most Fzds is not zonated. In contrast, Fzd6 is preferentially expressed in zone 1. We also discovered that Wnt2 and Wnt9b expression is highly zonated and primarily found in zone 3. Therefore, our results suggest that zonated Wnt expression is critical for zonation maintenance in healthy adult liver. Finally, we showed that Fzds and Wnts are not uniformly expressed by all hepatic cell types. Rather, there is broad distribution among both hepatocytes and non-parenchymal cells, including endothelial cells. Overall, our establishment of a definitive mRNA expression atlas of Wnt/Fzd pathway components opens the door to future functional characterization in healthy and disease states.

cell biology↗