Biology subjects

Gu, Y.

Publications and source records attributed to Gu, Y..

At least 127 records · Page 7Linked to original sources

bioRxiv2023

Structure of scavenger receptor SCARF1 and its interaction with lipoproteins

SCARF1 (Scavenger receptor class F member 1, SREC-1 or SR-F1) is a type I transmembrane protein that recognizes multiple endogenous and exogenous ligands such as modified low-density lipoproteins (LDL) and is important for maintaining homeostasis and immunity. But the structural information and the mechanisms of ligand recognition of SCARF1 are largely unavailable. Here we solve the crystal structures of the N-terminal fragments of human SCARF1, which show that SCARF1 forms homodimers and its epidermal growth factor (EGF)-like domains adopt a long-curved conformation. Then we examine the interactions of SCARF1 with lipoproteins and are able to identify a region on SCARF1 for recognizing modified LDLs. The mutagenesis data show that the positively charged residues in the region are crucial for the interaction of SCARF1 with modified LDLs, which is confirmed by making chimeric molecules of SCARF1 and SCARF2. In addition, teichoic acids, a cell wall polymer expressed on the surface of gram-positive bacteria, are able to inhibit the interactions of modified LDLs with SCARF1, suggesting the ligand binding sites of SCARF1 might be shared for some of its scavenging targets. Overall, these results provide mechanistic insights into SCARF1 and its interactions with the ligands, which are important for understanding its physiological roles in homeostasis and the related diseases.

biophysics↗

bioRxiv2023

Mechanisms of ligand recognition and activation of melanin-concentrating hormone receptors

Melanin-concentrating hormone (MCH) is a cyclic neuropeptide that regulates food intake, energy balance, and other physiological functions by stimulating MCHR1 and MCHR2 receptors, both of which are class A G protein-coupled receptors. MCHR1 can couple with multiple G-proteins, including Gi/o, Gq/11, and Gs, while MCHR2 only couple to Gq/11. Here we present cryo-electron microscopy structures of MCH-activated MCHR1 with Gi and MCH-activated MCHR2 with Gq complexes, at the global resolutions of 3.01 [A] and 2.40 [A], respectively. These structures reveal that MCH adopts a consistent cysteine-mediated hairpin loop configuration in both receptors. A central arginine from the LGRVY core motif between the two cysteines of MCH penetrates deeply into the transmembrane pocket, triggering receptor activation. Integrated with mutational and functional insights, our findings elucidate the molecular underpinnings of ligand recognition and MCH receptor activation, offering a structured foundation for targeted drug design.

molecular biology↗

bioRxiv2023

A consistent map in the medial entorhinal cortex supports spatial memory

The medial entorhinal cortex (MEC) is hypothesized to function as a cognitive map for memory-guided navigation. How this map develops during learning and influences memory remains unclear. By imaging MEC calcium dynamics while mice successfully learned a novel virtual environment over ten days, we discovered that the dynamics gradually became more spatially consistent and then stabilized. Additionally, grid cells in the MEC not only exhibited improved spatial tuning consistency, but also maintained stable phase relationships, suggesting a network mechanism involving synaptic plasticity and rigid recurrent connectivity to shape grid cell activity during learning. Increased c-Fos expression in the MEC in novel environments further supports the induction of synaptic plasticity. Unsuccessful learning lacked these activity features, indicating that a consistent map is specific for effective spatial memory. Finally, optogenetically disrupting spatial consistency of the map impaired memory-guided navigation in a well-learned environment. Thus, we demonstrate that the establishment of a spatially consistent MEC map across learning both correlates with, and is necessary for, successful spatial memory.

neuroscience↗

bioRxiv2023

IL-1B-mediated inflammatory signaling drives ineffective erythropoiesis in early-stage myelodysplastic syndromes

Myelodysplastic syndromes (MDS) are a group of incurable hematopoietic stem cell (HSC) neoplasms characterized by peripheral blood cytopenias and a high risk of progression to acute myeloid leukemia. MDS represent the final stage in a continuum of HSCs genetic and functional alterations and are preceded by a premalignant phase, clonal cytopenia of undetermined significance (CCUS). Dissecting the mechanisms of CCUS maintenance may uncover therapeutic targets to delay or prevent malignant transformation. Here, we demonstrate that DNMT3A and TET2 mutations, the most frequent mutations in CCUS, induce aberrant HSCs differentiation towards the myeloid lineage at the expense of erythropoiesis by upregulating IL-1{beta}-mediated inflammatory signaling and that canakinumab rescues red blood cell transfusion dependence in early-stage MDS patients with driver mutations in DNMT3A and TET2. This study illuminates the biological landscape of CCUS and offers an unprecedented opportunity for MDS intervention during its initial phase, when expected survival is prolonged.

cancer biology↗

bioRxiv2023

A phage nucleus-associated RNA-binding protein is required for jumbo phage infection

Large-genome bacteriophages (jumbo phages) of the Chimalliviridae family assemble a nucleus-like compartment bounded by a protein shell that protects the replicating phage genome from host-encoded restriction enzymes and CRISPR/Cas nucleases. While the nuclear shell provides broad protection against host nucleases, it necessitates transport of mRNA out of the nucleus-like compartment for translation by host ribosomes, and transport of specific proteins into the nucleus-like compartment to support DNA replication and mRNA transcription. Here we identify a conserved phage nuclear shell-associated protein that we term Chimallin C (ChmC), which adopts a nucleic acid-binding fold, binds RNA with high affinity in vitro, and binds phage mRNAs in infected cells. ChmC also forms phase-separated condensates with RNA in vitro. Targeted knockdown of ChmC using mRNA-targeting dCas13d halts infections at an early stage. Taken together, our data suggest that the conserved ChmC protein acts as a chaperone for phage mRNAs, potentially stabilizing these mRNAs and driving their translocation through the nuclear shell to promote translation and infection progression.

microbiology↗

bioRxiv2023

PVN microglia via P2Y12 transmit hemodynamic signal to promote sympathetic excitation in hypertension

Hypertension is usually accompanied with an elevated sympathetic tonicity, but how sympathetic hyperactivity is triggered is not fully understood. Recent advances reveal that microglia-centered neuroinflammation contributes to sympathetic excitation in hypertension. In this study, we performed a temporospatial analysis of microglia at both morphological and transcriptomic levels, and found that microglia in the hypothalamic paraventricular nucleus (PVN) were early responders to hypertensive challenges. PVN is the central hub for maintaining cardiovascular function via regulation of fluid balance and sympathetic outflow. Comprehensive vasculature analyses unveiled that PVN was characterized by high capillary density, thin vessel diameter, and complex vascular topology among brain regions. As such, PVN is susceptible to the penetration of ATP released from the vasculature in response to hemodynamic disturbance after blood pressure increase. ATP ligation to microglial P2Y12 receptor is responsible for the microglial accumulation and activation in the PVN. Furthermore, either pharmacological blockade or genetic ablation of microglial P2Y12 could substantially restrain blood pressure increase under hypertensive challenge. Together, these findings disclose that a unique vasculature pattern results in the vulnerability of PVN pre-sympathetic neurons to hypertension-associated insults, which is mediated by microglia.

neuroscience↗

bioRxiv2023

Bacterial Shedu immune nucleases share a common enzymatic core regulated by diverse sensor domains

Prokaryotes encode diverse anti-bacteriophage immune systems, including the single-protein Shedu nuclease. Here we reveal the structural basis for activation of Bacillus cereus Shedu. In the inactive homotetramer, a key catalytic residue in Shedus nuclease domain is sequestered away from the catalytic site. Activation involves a conformational change that completes the active site and promotes assembly of a homo-octamer for coordinated double-strand DNA cleavage. Removal of Shedus N-terminal domain ectopically activates the enzyme, suggesting that this domain allosterically inhibits Shedu in the absence of infection. Bioinformatic analysis of nearly 8,000 Shedu homologs reveals remarkable diversity in their N-terminal regulatory domains: we identify 79 domain families falling into eight functional classes, including diverse nucleic acid binding, enzymatic, and other domains. Together, these data reveal Shedu as a broad family of immune nucleases with a common nuclease core regulated by diverse N-terminal domains that likely respond to a range of infection-related signals.

microbiology↗

bioRxiv2023

Genome-wide identification of silencers in human cells

Transcriptional regulation is a complex process that is controlled by a variety of factors, including enhancers and silencers. Silencers, also known as repressor elements, play a crucial role in the fine-tuning of gene expression by inhibiting or suppressing transcription in the human genome. Although significant progresses have been made, genome-wide silencer research is still in its early stages. Here, we used a genome-wide method called massively parallel reporter assays (MPRAs) to identify silencers in three human cell lines: K562, LNCap, and HEK293T. We identified 739,434, 643,484, and 491,952 silencer regions in these cell lines, respectively. We found that most of the silencers we identified had inhibitory activity and significantly enriched inhibitory motifs. These results confirm that silencers are ubiquitous in the human genome and play an important role in regulating gene expression. Therefore, our study provides a general strategy for genome-wide functional identification of silencer elements. This information could be used to better understand the mechanisms of gene regulation and to develop new therapeutic strategies for diseases that are caused by dysregulation of gene expression.

genomics↗

bioRxiv2023

Deciphering the genetic interactions between Pou4f3, Gfi1 and Rbm24 in maintaining cochlear hair cell survival

Mammals harbor a limited number of sound-receptor hair cells (HCs) that cannot be regenerated after damage. Thus, investigating the underlying molecular mechanisms that maintain HC survival is crucial for preventing hearing impairment. Intriguingly, Pou4f3-/- or Gfi1-/-HCs form initially but then rapidly degenerate, whereas Rbm24-/-HCs degenerate considerably later. However, the transcriptional cascades involving Pou4f3, Gfi1, and Rbm24 remains undescribed. Here, we demonstrate that Rbm24 expression is completely repressed in Pou4f3-/- HCs but unaltered in Gfi1-/- HCs, and further that the expression of both POU4F3 and GFI1 is intact in Rbm24-/- HCs. Moreover, by using in vivo mouse transgenic reporter assays, we identify three Rbm24 enhancers to which POU4F3 binds. Lastly, through in vivo genetic testing of whether Rbm24 restoration alleviates the degeneration of Pou4f3-/-HCs, we show that ectopic Rbm24 alone cannot prevent Pou4f3-/-HCs from degenerating. Collectively, our findings provide new molecular and genetic insights into how HC survival is regulated.

developmental biology↗

bioRxiv2023

A conserved and druggable pocket in class B G protein coupled receptors for orally active small molecule agonists

Class B G protein-coupled receptors (GPCRs), including glucagon-like receptor 1 (GLP-1R) and parathyroid hormone receptor 1 (PTH1R), are peptide hormone receptors and important drug targets. Injectable peptide drugs targeting class B GPCRs have been developed for the treatment of many diseases, including type 2 diabetes, obesity, and osteoporosis, but orally available small molecule drugs are hotly pursued in the field, especially small molecule agonists of GLP-1R and PTH1R. Here we report the first high-resolution structure of the human PTH1R in complex with the stimulatory G protein (Gs) and a small molecule agonist, PCO371, which reveals an unexpected binding mode of PCO371 at the interface of PTH1R and Gs. The binding site of PCO371 is totally different from all binding sites previously reported for small molecules or peptide ligands in GPCRs. Residues that make up the PCO371 binding pocket are mostly conserved in class B GPCRs and a single mutation in PTH type 2 receptor (PTH2R) and two residue mutations in GLP-1R convert these receptors to respond to PCO371 activation. Functional assays reveal that PCO371 is a G-protein biased agonist that is defective in promoting PTH1R-mediated arrestin signaling. Together, these results uncover a distinct binding site for designing small molecule agonists for PTH1R and possible other members of class B GPCRs and define a receptor conformation that is only specific for G protein activation but not arrestin signaling. These insights should facilitate the design of distinct types of class B GPCR small molecule agonists for various therapeutic indications.

biochemistry↗

bioRxiv2023

A non-image-forming visual circuit mediates the innate fear of heights

The neural basis of fear of heights remains largely unknown. In this study, we investigated the fear response to heights in mice and observed characteristic aversive behaviors resembling human height vertigo. We identified visual input as a critical factor in mouse reactions to heights, while peripheral vestibular input was found to be nonessential for fear of heights. Unexpectedly, we found that fear of heights in naive mice does not rely on image-forming visual processing by the primary visual cortex. Instead, a subset of neurons in the ventral lateral geniculate nucleus (vLGN), which connects to the lateral/ventrolateral periaqueductal gray (l/vlPAG), drives the expression of fear associated with heights. Additionally, we observed that a subcortical visual pathway linking the superior colliculus to the lateral posterior thalamic nucleus inhibits the defensive response to height threats. These findings highlight a rapid fear response to heights threat through a subcortical visual and defensive pathway from the vLGN to the l/vlPAG.

neuroscience↗

bioRxiv2023

Identification of the bacteriophage nucleus protein interaction network

In the arms race between bacteria and bacteriophages (phages), some large-genome jumbo phages have evolved a protein shell that encloses their replicating genome to protect it against DNA-targeting immune factors. By segregating the genome from the host cytoplasm, however, the "phage nucleus" introduces the need to specifically transport mRNA and proteins through the nuclear shell, and to dock capsids on the shell for genome packaging. Here, we use proximity labeling and localization mapping to systematically identify proteins associated with the major nuclear shell protein chimallin (ChmA) and other distinctive structures assembled by these phages. We identify six uncharacterized nuclear shell-associated proteins, one of which directly interacts with self-assembled ChmA. The structure and protein-protein interaction network of this protein, which we term ChmB, suggests that it forms pores in the ChmA lattice that serve as docking sites for capsid genome packaging, and may also participate in mRNA and/or protein transport.

biochemistry↗

bioRxiv2023

Neural and behavioral correlates of edible cannabis-induced poisoning: characterizing a novel preclinical model

Accidental exposure to {Delta}9-tetrahydrocannabinol (THC)-containing edible cannabis, leading to cannabis poisoning, is common in children and pets; however, the neural mechanisms underlying these poisonings remain unknown. Therefore, we examined the effects of acute edible cannabis-induced poisoning on neural activity and behavior. Adult Sprague-Dawley rats (6 males, 7 females) were implanted with electrodes in the prefrontal cortex (PFC), dorsal hippocampus (dHipp), cingulate cortex (Cg), and nucleus accumbens (NAc). Cannabis poisoning was then induced by exposure to a mixture of Nutella (6 g/kg) and THC-containing cannabis oil (20 mg/kg). Subsequently, cannabis tetrad and neural oscillations were examined 2, 4, 8, and 24 h after THC exposure. In another cohort (16 males, 15 females), we examined the effects of cannabis poisoning on learning and prepulse inhibition, and the serum and brain THC and 11-hydroxy-THC concentrations. Cannabis poisoning resulted in sex differences in brain and serum THC and 11-hydroxy-THC levels over a 24-h period. It also caused gamma power suppression in the Cg, dHipp, and NAc in a sex- and time-dependent manner. Cannabis poisoning also resulted in hypolocomotion, hypothermia, and anti-nociception in a time-dependent manner and impairments in learning and prepulse inhibition. Our results suggest that the impairments in learning and information processing may be due to the decreased gamma power in the dHipp and PFC. Additionally, most of the changes in neural activity and behavior appear 2 hours after ingestion, suggesting that interventions at or before this time might be effective in reversing or reducing the effects of cannabis poisoning.

neuroscience↗

bioRxiv2023

RNA Sensing and Innate Immunity Constitutes a Barrier for Interspecies Chimerism

Interspecies chimera formation with human pluripotent stem cells (PSCs) holds great promise to generate humanized animal models and provide donor organs for transplant. However, the approach is currently limited by low levels of human cells ultimately represented in chimeric embryos. Different strategies have been developed to improve chimerism by genetically editing donor human PSCs. To date, however, it remains unexplored if human chimerism can be enhanced in animals through modifying the host embryos. Leveraging the interspecies PSC competition model, here we discovered retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) signaling, an RNA sensor, in "winner" cells plays an important role in the competitive interactions between co-cultured mouse and human PSCs. We found that genetic inactivation of Ddx58/Ifih1-Mavs-Irf7 axis compromised the "winner" status of mouse PSCs and their ability to outcompete PSCs from evolutionarily distant species during co-culture. Furthermore, by using Mavs-deficient mouse embryos we substantially improved unmodified donor human cell survival. Comparative transcriptome analyses based on species-specific sequences suggest contact-dependent human-to-mouse transfer of RNAs likely plays a part in mediating the cross-species interactions. Taken together, these findings establish a previously unrecognized role of RNA sensing and innate immunity in "winner" cells during cell competition and provides a proof-of-concept for modifying host embryos, rather than donor PSCs, to enhance interspecies chimerism.

cell biology↗

bioRxiv2023

Peroxisomal compartmentalization of amino acid biosynthesis reactions imposes an upper limit on compartment size

Cellular metabolism relies on just a few redox cofactors. Selective compartmentalization may prevent competition between metabolic reactions requiring the same cofactor. Is such compartmentalization necessary for optimal cell function? Is there an optimal compartment size? Here we probe these fundamental questions using peroxisomal compartmentalization of the last steps of lysine and histidine biosynthesis in the fission yeast Schizosaccharomyces japonicus. We show that compartmentalization of these NAD+ dependent reactions together with a dedicated NADH/NAD+ recycling enzyme supports optimal growth when an increased demand for anabolic reactions taxes cellular redox balance. In turn, compartmentalization constrains the size of individual organelles, with larger peroxisomes accumulating all the required enzymes but unable to support both biosynthetic reactions at the same time. We propose that compartmentalized biosynthetic reactions are sensitive to the size of the compartment, likely due to scaling-dependent changes within the system, such as enzyme packing density.

cell biology↗

bioRxiv2023

Chromatin binding by HORMAD proteins regulates meiotic recombination initiation

The meiotic chromosome axis coordinates chromosome organization and interhomolog recombination in meiotic prophase and is essential for fertility. In S. cerevisiae, the HORMAD protein Hop1 mediates enrichment of axis proteins at nucleosome-rich genomic islands through a central chromatin-binding region (CBR). Here, we use cryoelectron microscopy to show that the Hop1 CBR directly recognizes bent nucleosomal DNA through a composite interface in its PHD and winged helix-turn-helix domains. Targeted disruption of the Hop1 CBR-nucleosome interface causes loss of axis proteins from nucleosome-rich islands, reduces meiotic DNA double-strand breaks (DSBs), and leads to defects in chromosome synapsis. Synthetic effects with the disassemblase Pch2 suggest that nucleosome binding delays a conformational switch in Hop1 from a DSB-promoting, Pch2-inaccessible state to a DSB-inactive, Pch2-accessible state to regulate the extent of meiotic DSB formation. Phylogenetic analyses of meiotic HORMADs reveal an ancient origin of this domain, suggesting that these mechanisms are broadly conserved.

cell biology↗

bioRxiv2023

Spatial transcriptomic atlas of shoot organogenesis in tomato callus

Callus is a reprogrammed transitional cell mass during plant regeneration. Pluripotent callus cells develop into fertile shoots through de novo shoot organogenesis. This study represents a pioneering effort in exploring the spatial transcriptome of tomato callus during shoot regeneration, using technologies including BGI Stereo-seq, BMKMANU S1000, and 10x Visium. The results indicate that the callus comprises highly heterogeneous cells, classified into various cell types based on spatial gene expression and histological observation, including epidermis, shoot primordium, vascular tissue, inner callus, and outgrowth shoots. The developmental trajectories from shoot primordium to outgrowth shoot are traced, and vascular tissue development is characterized. The single-cell resolution spatial approach reveals the origin of shoot primordia from the sub-epidermis. The spatial full length RNA sequencing shows high incompletely spliced (IS) ratios in the shoot primordium cells. These findings enhance our knowledge of plant organogenesis and highlight the significance of spatial biology in plant research.

cell biology↗

bioRxiv2023

High-throughput screening of functional neo-antigens and their specific TCRs via the Jurkat reporter system combine with droplet microfluidics

T-cell receptor (TCR)-engineered T cells can precisely recognize a broad repertoire of targets derived from both intracellular and surface proteins of tumor cells. TCR-T adoptive cell therapy has shown safety and promising efficacy in solid tumor immunotherapy. However, antigen-specific functional TCR screening is time-consuming and expensive, which limits its application clinically. Here, we developed a novel integrated antigen-TCR screening platform based on droplet microfluidics technology, enabling high-throughput peptide-major histocompatibility complex (pMHC) library-to-TCR library screening with high sensitivity and low background signal. We introduced DNA barcoding technology to label peptide antigen candidate-loaded antigen-presenting cells (APCs) and Jurkat reporter cells to check the specificity of pMHC-TCR candidates. Coupled with the next-generation sequencing pipeline, interpretation of the DNA barcodes and the gene expression level of the Jurkat T-cell activation pathway provided a clear peptide-MHC-TCR recognition relationship. Our proof-of-principle study demonstrates that the platform could achieve unbiased pMHC-TCR library-on-library screening, which is expected to be used in the cross-reactivity and off-target testing of candidate pMHC-TCR libraries in clinical applications.

bioengineering↗