Biology subjects

Sun, F.

Publications and source records attributed to Sun, F..

At least 55 records · Page 3Linked to original sources

bioRxiv2024

Discovery of Alkenyl Oxindole as a Novel PROTAC Moiety for Targeted Protein Degradation via CRL4DCAF11 Recruitment

Alkenyl oxindoles have been characterized as autophagosome-tethering compounds (ATTECs), which can target mutant huntingtin protein (mHTT) for lysosomal degradation. In order to expand the application of alkenyl oxindoles for targeted protein degradation, we designed and synthesized a series of hetero-bifunctional compounds by conjugating different alkenyl oxindoles with the BRD4 inhibitor JQ1. Through structure-activity relationship study, we successfully developed JQ1-alkenyl oxindole conjugates that potently degrade BRD4. Unexpectedly, we found that these molecules degrade BRD4 through the ubiquitin-proteasome system, rather than the autophagy-lysosomal pathway. Using pooled CRISPR interference (CRISPRi) screening, we revealed that JQ1-alkenyl oxindole conjugates recruit the E3 ubiquitin ligase complex CRL4DCAF11 for substrate degradation. Furthermore, we validated the most potent hetero-bifunctional molecule HL435 as a promising drug-like lead compound to exert antitumor activity both in vitro and in vivo. Our research provides new employable PROTAC moieties for targeted protein degradation, providing new possibilities for drug discovery.

biochemistry↗

bioRxiv2023

Structural elucidation of how ARF small GTPases induce membrane tubulation for vesicle fission

The ADP-Ribosylation Factor (ARF) small GTPases have been found to act in vesicle fission through a direct ability to tubulate membrane. Here, we have used cryo-electron microscopy (EM) to solve the structure of an ARF6 protein lattice assembled on tubulated membrane to 3.9 [A] resolution. ARF6 forms tetramers that polymerize into helical arrays to form this lattice. We identify, and confirm functionally, protein contacts critical for this lattice formation. The solved structure also suggests how the ARF amphipathic helix is positioned in the lattice for membrane insertion, and how a GTPase-activating protein (GAP) docks onto the lattice to catalyze ARF-GTP hydrolysis in completing membrane fission. As ARF1 and ARF6 are structurally conserved, we have also modeled ARF1 onto the ARF6 lattice, which has allowed us to pursue the reconstitution of Coat Protein I (COPI) vesicles to confirm more definitively that the ARF lattice acts in vesicle fission. Our findings are notable for having achieved the first detailed glimpse of how a small GTPase bends membrane and having provided a molecular understanding of how an ARF protein acts in vesicle fission.

cell biology↗

bioRxiv2023

Parallel Molecular Data Storage by Printing Epigenetic Bits on DNA

DNA storage has shown potential to transcend current silicon-based data storage technologies in storage density, lifetime and energy consumption. However, writing large-scale data directly into DNA sequences by de novo synthesis remains uneconomical in time and cost. Inspired by the natural genomic modifications, in this work, we report an alternative, parallel strategy that enables the writing of arbitrary data on DNA using premade nucleic acids. With epigenetic modifications as information bits, our system employed DNA self-assembly guided enzymatic methylation to perform movable-type printing on universal DNA templates. By programming with a finite set of 700 DNA movable types and 5 templates, we achieved the synthesis-free writing of [~]270000 bits on an automated platform with 350 bits written per reaction. The data encoded in complex epigenetic patterns were retrieved high-throughput by nanopore sequencing, and algorithms were developed to finely resolve 240 modification patterns per sequencing reaction. Our framework presents a new modality of DNA-based data storage that is parallel, programmable, stable and scalable. Such a modality opens up avenues towards practical data storage and dual-mode data functions in biomolecular systems.

biochemistry↗

bioRxiv2023

Xanthomonas euvesicatoria effector XeAvrRxo1 triggers a Rxo1-mediated defense response in Nicotiana benthamiana and its chaperone Xe4429 functions as an antitoxin, an expression repressor, and an enhancer of XeAvrRxo1 secretion

Xanthomonas euvesicatoria (X. euvesicatoria) is the causal agent of bacterial spot disease that threatens pepper and tomato production around the globe. X. euvesicatoria gene Xe4428 encodes a type III effector (T3E) that shares 89.67% amino acid identity with Xanthomonas oryzae pv. oryzicola (Xoc) T3E AvrRxo1. Deletion of Xe4428 in the genome of X. euvesicatoria (strain Xcv85-10) compromised its virulence to infect pepper and Nicotiana benthamiana plants. Transient co-expression of Xe4428 and Rxo1 on pepper and N. benthamiana plant leaves results in a robust hypersensitive reaction. Thus, Xe4428, renamed as XeAvrRxo1, is a bona fide orthologue of XocAvrRxo1 that possesses both virulence and avirulence functions. Expression of XeAvrRxo1 in E. coli and X. euvesicatoria is toxic to both bacterial cells. Another X. euvesicatoria gene Xe4429, encodes a putative chaperone of XeAvrRxo1, which can interact with XeAvrRxo1 to suppress its toxicity in X. euvesicatoria and E. coli bacterial cells. Xe4429 also binds to the promoter region of XeavrRxo1 and represses its transcription/translation in X. euvesicatoria bacterial cells. In addition, expression of Xe4429 can enhance the secretion and translocation of XeAvrRxo1 into plant cells. Therefore, Xe4429 functions as an antitoxin, a transcription repressor, and a type III chaperone that is capable of enhancing the secretion and translocation of XeAvrRxo1 during pathogenesis.

pathology↗

bioRxiv2023

EMformer: Transformer-Based Isotropic Reconstruction for Volume Electron Microscopy

Volume electron microscopy (vEM) has become a rapidly developing technique for studying the 3D architecture of biological specimens, such as cells, tissues, and organs at nanometer resolution; this technique involves collecting a series of electron micrographs of axial sequential sections and reconstructing the 3D volume, providing useful information on the cellular ultrastructural spectrum. This technique currently suffers from anisotropic resolution between the lateral (x, y) and axial (z) directions and the loss/damage of sections. Here, we develop a new algorithm, IsoVEM, based on a video transformer model to boost the axial resolution and achieve isotropic reconstruction of vEM. By learning high-resolution axial structures and utilizing the 3D continuity of biological structures, IsoVEM can recover axial information and repair random lost/damaged sections based on a self-supervision strategy, achieving a higher resolution than existing methods, which has been validated for both simulated FIB-SEM datasets and experimental ssTEM datasets. In addition to visual validation, the segmentation efficiency and statistical precision of various ultrastructures, e.g., neurons, mitochondria, vesicles, and membrane bilayers, also prove the better performance of IsoVEM. Therefore, using IsoVEM, we achieve isotropic reconstruction via anisotropic axial sampling, which increases the vEM throughput for studying large-scale biological architectures.

biophysics↗

bioRxiv2023

Comparison of the effectiveness of different normalization methods for metagenomic cross-study phenotype prediction under heterogeneity

The human microbiome, comprising microorganisms residing within and on the human body, plays a crucial role in various physiological processes and has been linked to numerous diseases. To analyze microbiome data, it is essential to account for inherent heterogeneity and variability across samples. Normalization methods have been proposed to mitigate these variations and enhance comparability. However, the performance of these methods in predicting binary phenotypes remains understudied. This study systematically evaluates different normalization methods in microbiome data analysis and their impact on disease prediction. Our findings highlight the strengths and limitations of scaling, compositional data analysis, transformation, and batch correction methods. Scaling methods like TMM and RLE show consistent performance, while compositional data analysis methods exhibit mixed results. Transformation methods, such as Blom and NPN, demonstrate promise in capturing complex associations. Batch correction methods, including BMC and Limma, consistently outperform other approaches. However, the influence of normalization methods is constrained by population effects, disease effects, and batch effects. These results provide insights for selecting appropriate normalization approaches in microbiome research, improving predictive models, and advancing personalized medicine. Future research should explore larger and more diverse datasets and develop tailored normalization strategies for microbiome data analysis.

bioinformatics↗

bioRxiv2023

Cryo-EM structures of human SID-1 transmembrane family proteins and implications for their low-pH-dependent RNA transport activity

Human SIDT1 and SIDT2 are closely related members of the systemic RNA interference (RNAi)-defective (SID-1) transmembrane family. Both mediate RNA internalization and intracellular transport and are involved in various biological processes. However, the molecular basis of RNA uptake, especially for exogenous small RNAs, remains elusive. Here, we present the cryo-electron microscopy (cryo-EM) structures of human SIDT1 and SIDT2. Both structures reveal the overall architecture of a dimeric arrangement contributed by the {beta}-strand-rich extracellular domain (ECD) and the transmembrane domain (TMD) with 11 passes, highlighting the remarkable structural congruence. The in situ assays confirm that SIDT1 and SIDT2 exist as dimers or higher-order oligomers. We demonstrate that for both SIDT1 and SIDT2, the ECD binds small RNAs, such as dietary plant-derived miRNA, only under acidic conditions. In addition, RNA binding under low pH can trigger higher-order assembly of the ECD dimer, suggesting the potential importance of oligomerization during RNA uptake. Our results illustrate the molecular features of the conserved SID-1 family proteins to elucidate the mechanism of the low pH-dependent activation of RNA uptake mediated by SIDT1 and SIDT2. This study provides a promising understanding of the molecular basis of the nucleic acid delivery platform, which may potentially open new avenues for the design and optimization of RNA-based therapies.

biochemistry↗

bioRxiv2023

Co-evolution of spheroid fluidization and mechanical fracture in a morphogenic cascade

Cells migrate and invade tissues during development, immune responses, and cancer. Collective invasion is generally understood to be driven by invading cells unjamming and pushing through barriers such as the extracellular matrix and surrounding tissues. Whether these barriers actively contribute to invasion remains unclear. Using ovarian adenocarcinoma spheroids invading mesothelium derived from benign pleural effusions as an experimental model, combined with modelling, we examine invasion across molecular to multicellular scales. We identify intercellular integrin adhesions linking invasive leader cells to the tissue barrier, triggering apical constrictions within the barrier. This constriction shrinks cell-cell contacts, leading to barrier rupture. Thus, the tissue barrier plays a mechanically active role in invasion. Rather than cells pushing through, we find that coordinated subcellular contractility between the invading leader cell and the barrier drives barrier tensile rupture and invasion, independent of a jamming transition. Together, our findings challenge prevailing paradigms of collective cell invasion.

cell biology↗

bioRxiv2023

Timing-dependent modulation of working memory by VTA dopamine release in medial prefrontal cortex

Dopamine significantly modulates working memory (WM)1-8, a fundamental cognitive function that maintains information during a brief delay period9-11. The temporal precision of dopaminergic modulation in WM and related neural mechanisms remain elusive. Here we unveiled the pivotal role of dopamine timing in WM performance with head-fixed mice engaged in learning an olfactory WM task. Through electrophysiology and optogenetics, we found that dopaminergic neurons in the ventral tegmental area (VTA) could encode WM information during the delay period, and manipulating dopaminergic neuronal activity bidirectionally modulated WM performance in an inverted U-shaped manner. Optogenetic manipulation of VTA activity also induced bidirectional changes in WM-related neural activity in the medial prefrontal cortex (mPFC). Imaging with a dopamine-sensitive GPCR-activation-based sensor (GRABDA)12,13 revealed transient dopamine peaks in the mPFC specifically during the early-delay period. Optogenetic stimulation of VTA-to-mPFC dopaminergic projections during the early- and late-delay period enhanced and impaired WM performance, respectively. Manipulations outside these periods or involving the nucleus accumbens had no effect. Single-unit recordings demonstrated that optogenetic excitation of VTA-to-mPFC projections could modulate mPFC neuronal activity in a manner consistent with behavioral modulation. Thus, the timing of dopamine release is critical in modulation of neuronal activity and behavior.

neuroscience↗

bioRxiv2023

In situ structural insights into the excitation contraction coupling mechanism of skeletal muscle

Excitation-contraction coupling (ECC) is a fundamental mechanism in control of skeletal muscle contraction and occurs at triad junctions, where dihydropyridine receptors (DHPRs) on transverse tubules sense excitation signals and then cause calcium release from the sarcoplasmic reticulum via coupling to type 1 ryanodine receptors (RyR1s), inducing the subsequent contraction of muscle filaments. However, the molecular mechanism remains unclear due to the lack of structural details. Here, we explored the nanometre-resolution architecture of triad junction by cryo-electron tomography, solved the in situ structure of RyR1 in complex with FKBP12 and calmodulin, and discovered the intact RyR1-DHPR supercomplex. RyR1s arrange into two rows on the terminal cisternae membrane by forming right-hand corner-to-corner contacts, and tetrads of DHPRs bind to RyR1s in an alternating manner, forming another two rows on the transverse tubule membrane. Such unique arrangement is important for synergistic calcium release and provides direct evidence of physical coupling in ECC.

biophysics↗

bioRxiv2023

Longer mandible or nose? Co-evolution of feeding organs in early elephantiforms

The long-trunked elephantids underwent a significant evolutionary stage characterized by an exceptionally elongated mandible. The initial elongation and subsequent regression of the long mandible, along with its co-evolution with the trunk, present an intriguing issue that remains incompletely understood. Through comparative functional and eco-morphological investigations, as well as feeding preference analysis, we reconstructed the feeding behavior of major groups of longirostrine elephantiforms. In the Platybelodon clade, the rapid evolutionary changes observed in the narial region, strongly correlated with mandible and tusk characteristics, suggest a crucial evolutionary transition where feeding function shifted from the mandible to the trunk, allowing proboscideans to expand their niches to more open regions. This functional shift further resulted in elephantids relying solely on their trunks for feeding. Our research provides insights into how unique environmental pressures shape the extreme evolution of organs, particularly in large mammals that developed various peculiar adaptations during the late Cenozoic global cooling trends.

paleontology↗

bioRxiv2023

NanoPDLIM2 enhanced efficacy of PD-1 blockade and chemotherapy in mouse lung cancers

BackgroundImmune checkpoint inhibitors (ICIs) and their combination with other therapies such as chemotherapy, fail in most cancer patients. We previously identified the PDZ-LIM domain-containing protein 2 (PDLIM2) as a bona fide tumor suppressor that is repressed in lung cancer to drive cancer and its chemo and immunotherapy resistance, suggesting a new target for lung cancer therapy improvement. MethodsHuman clinical samples and data were used to investigate PDLIM2 genetic and epigenetic changes in lung cancer. Using an endogenous mouse lung cancer model faithfully recapitulating refractory human lung cancer and a clinically feasible nano-delivery system, we investigated the therapeutic efficacy, action mechanism, and safety of systemically administrated PDLIM2 expression plasmids encapsulated in nanoparticles (nanoPDLIM2) and its combination with PD-1 antibody and chemotherapeutic drugs. ResultsPDLIM2 repression in human lung cancer involves both genetic deletion and epigenetic alteration. NanoPDLIM2 showed low toxicity, high tumor specificity, antitumor activity, and greatly improved the efficacy of anti-PD-1 and chemotherapeutic drugs, with complete tumor remission in most mice and substantial tumor reduction in the remaining mice by their triple combination. Mechanistically, nanoPDLIM2 increased major histocompatibility complex class I (MHC-I) expression, suppressed multi-drug resistance 1 (MDR1) induction and survival genes and other tumor-related genes expression in tumor cells, and enhanced lymphocyte tumor infiltration, turning the cold tumors hot and sensitive to ICIs and rendering them vulnerable to chemotherapeutic drugs and activated tumor-infiltrating lymphocytes (TILs) including those unleashed by ICIs. ConclusionsThese studies established a clinically applicable PDLIM2-based combination therapy with great efficacy for lung cancer and possibly other cold cancers.

cancer biology↗

bioRxiv2023

EHMT2/G9a-Inhibition Reprograms Cancer-Associated Fibroblasts (CAFs) to a More Differentiated, Less Proliferative and Invasive State

Cancer-associated fibroblasts (CAFs) have previously been shown to play a pivotal role in multiple cancer dynamics, including mediating tumor cell invasion: their pro-invasive secretory profile and ability to remodel the extracellular matrix (ECM) architecture particularly promote tumor progression through tumor cell invasion into surrounding tissue areas and beyond. Given that reduced CAF abundance in tumors correlates with improved outcomes in various cancers, we set out to identify epigenetic targets involved in CAF activation in the tumor-stromal margin to reduce overall tumor aggressiveness. Using the GLAnCE (Gels for Live Analysis of Compartmentalized Environments) co-culture platform, we performed an image-based, phenotypic screen and identified EHMT2 (also known as G9a), an epigenetic enzyme that targets the methylation of histone 3 lysine 9 (H3K9), as the most potent modulator of CAF abundance and CAF-mediated tumor cell invasion. Transcriptomic and functional analysis of EHMT2-inhibited CAFs revealed the involvement of EHMT2 in driving CAFs towards a pro-invasive phenotype. Further, EHMT2 signaling mediated CAF hyperproliferation, a feature that is typically associated with activated fibroblasts present in tumors, but the molecular basis for which has not thus far been identified. This study suggests a role for EHMT2 as a regulator of CAF hyperproliferation within the tumor mass, which in turn magnifies CAF-induced pro-invasive effects on tumor cells.

cancer biology↗

bioRxiv2023

DeepETPicker: Fast and accurate 3D particle picking for cryo-electron tomography using weakly supervised deep learning

Picking particles of biological macromolecules from their cryo-electron tomograms is critical to solving their 3D structures in situ. To reach sub-nanometre resolution, large numbers of particles often need to be picked, a laborious and time-consuming task if performed manually. To date, however, the adoption of automated particle-picking methods remains limited because of the limitations in their accuracy, processing speed and, for those based on learning models, manual annotation cost. To overcome the limitations, we develop DeepETPicker, a deep learning model for fast and accurate picking of 3D particles from cryo-electron tomograms. The training of DeepETPicker requires only weak supervision with low numbers of simplified Gaussian-type labels, reducing the burden of manual annotation of tomograms under very low signal-to-noise ratios. The simplified labels combined with the customized and lightweight model architecture of DeepETPicker as well as GPU-accelerated pooling enable substantially improved accuracy and accelerated processing speed. When tested on simulated as well as real tomograms, DeepETPicker outperforms the competing state-of-the-art methods by achieving the highest overall accuracy and speed, which translate into better quality of picked particles and higher resolutions of final reconstruction maps. DeepETPicker is provided in open source with a user-friendly interface to support automated particle picking for high-resolution cryo-electron tomography in situ.

bioinformatics↗

bioRxiv2023

CytoSIP: An annotated structural atlas for interactions involving cytokine or cytokine receptor

Cytokines primarily interact with specific cytokine receptors on the cell surface as essential signal transduction pathways in many physiological and pathological processes. Therapeutic agents targeting cytokine-cytokine receptor (CK-CKR) interactions lead to the disruption in cellular signaling function and have been demonstrated effective in the treatment of many diseases including tumors. However, a lack of universal and quick access to annotated structural surface regions on CK/CKR has limited the progress of a structure-driven approach to the development of targeted macromolecular drugs and precision medicine therapeutics. Herein we develop CytoSIP (Single nucleotide polymorphisms (SNPs), Interface, and Phenotype), a rich internet application based on a database of atomic interactions around hotspots in experimentally determined CK/CKR structural complexes. The content of the CytoSIP database includes the following key features: (1) SNPs on CK/CKR; (2) interactions involving CK/CKR at the domain level, including CK/CKR interfaces, oligomeric interfaces, epitopes, or other drug targeting surfaces; and (3) diseases and phenotypes associated with CK/CKR or SNPs. The database introduces a unique tri-level SIP data model to link genetic variants (atomic level) to disease phenotypes (organism level) using protein structure (complexes) as an underlying framework (molecule level). Moreover, CytoSIP implements screening criteria and tools to allow customized selection of relevant subset of CK/CKR for the study of interest. This reduces the time and resources needed to interrogate large datasets and allows rapid screening of cytokines and cytokine receptor proteins interfaces for hotspots targeted drug design and any other specific cellular signaling/function mechanisms and their correlation to pathologies. The CytoSIP framework crafted herein bridges CK/CKR genotype with phenotype, facilitating not only the panoramic investigation of the context-dependent crosstalk between CK/CKR but also the development of targeted therapeutic agents. CytoSIP portal website is publicly accessible at https://CytoSIP.biocloud.top.

immunology↗

bioRxiv2023

In-cell structural insight into the stability of sperm microtubule doublet

The propulsion for mammalian sperm swimming is generated by flagella beating. Microtubule doublets (DMTs) along with microtubule inner proteins (MIPs) are essential structural blocks of flagella. However, the intricate molecular architecture of sperm DMT remains elusive. Here, by in situ cryo-electron tomography, we solved the in-cell structure of mouse sperm DMT up to 3.6 [A] resolution and built its model with 36 kinds of MIPs in 48 nm periodicity. We identified multiple copies of Tektin5 that reinforce Tektin bundle, and multiple MIPs with different periodicities that anchor the Tektin bundle to tubulin wall. This architecture contributes to a superior stability of A-tubule than B-tubule of DMT, which was revealed by structural comparison of DMTs from the intact and deformed axonemes. Our work provides an overall molecular picture of sperm DMT that is essential to understand the molecular mechanism of sperm motility as well as the related ciliopathies.

cell biology↗

bioRxiv2023

A novel mouse allele of the DNA/RNA helicase senataxin (Setxspcar3) causing meiotic arrest of spermatocytes and male infertility

An unbiased screen for discovering novel genes for fertility identified the spcar3, spermatocyte arrest 3, mutant phenotype. The spcar3 mutation identified a new allele of the Setx gene, encoding senataxin, a DNA/RNA helicase that regulates transcription termination by resolving DNA/RNA hybrid R-loop structures. Although mutations in the human SETX gene cause neural disorders, Setxspcar3 mutant mice do not show any apparent neural phenotype, but instead exhibit male infertility and female subfertility. Histology of the Setxspcar3mutant testes revealed absence of spermatids and mature spermatozoa in the seminiferous tubules. Cytological analysis of chromosome spread preparations of the Setxspcar3 mutant spermatocytes revealed normal synapsis, but aberrant DNA damage in the autosomes, and defective formation of the sex body. Furthermore, Setxspcar3 testicular cells exhibited abnormal accumulation of R-loops compared to wild type testicular cells. Transient expression assays identified regions of the senataxin protein required for sub-nuclear localization. Together, these results not only confirm that senataxin is required for normal meiosis and spermatogenesis but also provide a new resource for determination of its role in maintaining R-loop formation and genome integrity.

cell biology↗

bioRxiv2023

COMT inhibitor tolcapone represses the migration and invasion of trophoblast and results in preeclampsia-like phenotypes in mice

Pregnancy in young-onset Parkinsons disease (YOPD) is rare; however, medication for this condition is critical for maternal and fetal health. Tolcapone is an effective antiparkinsonian drug. However, its safety studies on mothers and fetuses are limited. In this study, we aimed to investigate the effects of tolcapone on the mother and the developing fetus during different stages of gestation. Tolcapone was administered to pregnant mice at a dose of 60 (H-Tol) or 30 mg/kg/day (L-Tol) during early or mid-gestation. We observed that tolcapone administration during early gestation causes abortion and delays fetal development in a dose-dependent manner. During mid-gestation tolcapone barely caused embryo lethality; however, the mice developed preeclampsia-like phenotypes, including maternal hypertension, proteinuria, and fetal growth restriction. Histomorphological analysis of placentas from tolcapone treated mice revealed abnormalities in the trophoblast layer and the impaired trophoblast invasion in the decidua. Mechanistically, we revealed that tolcapone inhibits the invasion and migration of trophoblasts in vitro, with changes in the protein expression of Snail, Twist, and E-cadherin. In conclusion, tolcapone caused embryo lethality and growth restriction during early gestation, whereas it caused preeclampsia-like phenotypes in mice with defective trophoblast invasion in mid-gestation. Collectively, our study provides novel insights into the effects of tolcapone on pregnancy.

pharmacology and toxicology↗