Biology subjects

Liu, J.-L.

Publications and source records attributed to Liu, J.-L..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Structural basis of bifunctional CTP/dCTP synthase

Nucleotides are important molecules of life. According to the sugar, nucleotides can be divided into nucleotides and deoxynucleotides, which are the basic components of RNA and DNA respectively. CTP synthase (CTPS) catalyzes the last step in the de novo synthesis of CTP, which can form cytoophidia in cells from all three domains of life. We have recently found that CTPS combines with NTPs to form filaments, and solved the structures of CTPS bound with NTPs. Previous biochemical studies have shown that CTPS can also serve as dCTPS, i.e. CTPS/dCTPS can not only bind UTP, ATP and GTP to generate CTP, but also bind deoxynucleotides to generate dCTP. However, the structural basis of the bifunctional enzyme CTPS/dCTPS binding deoxynucleotide is not clear. In this study, we find that CTPS/dCTPS can form filaments bound with deoxynucleotides. Biochemically, we compare the binding and reaction characteristics of the corresponding nucleotides/deoxynucleotides and CTPS/dCTPS. Using cryo-electron microscopy, we solve the the structure of CTPS/dCTPS bound with deoxynucleotides at near-atomic resolution. This study not only provides a structural basis for understanding the catalysis and regulation of bifunctional CTPS/dCTPS, but also opens a door for further exploration the compartmentation of CTPS/dCTPS inside a cell.

biochemistry↗

bioRxiv2023

Natural evolution of SARS-CoV-2 variants in K18-ACE2 mice gives rise to more virulent virus and variant alleles associated with treatment resistance.

The persistence of COVID-19 is partly due to viral evolution reducing vaccine and treatment efficacy. Serial infections of Wuhan-like SARS-CoV-2 in Balb/c mice yielded mouse-adapted strains with greater infectivity and mortality. We investigated if passaging unmodified B.1.351 (Beta) and B.1.617.2 (Delta) 20 times in K18-ACE2 mice, expressing human ACE2 receptor, in a BSL-3 laboratory without selective pressures, would drive human health-relevant evolution and if evolution was lineage-dependent. Late-passage virus caused more severe disease, at organism and lung tissue scales, with late-passage Delta demonstrating antibody resistance and interferon suppression. This resistance co-occurred with a de novo spike S371F mutation, linked with both traits. S371F, an Omicron-characteristic mutation, was co-inherited at times with spike E1182G per Nanopore sequencing, existing in different quasi-species at others. Both are linked to mammalian GOLGA7 and ZDHHC5 interactions, which mediate viral-cell entry and antiviral response. This study demonstrates SARS-CoV-2s tendency to evolve with phenotypic consequences, its evolution varying by lineage, and suggests non-dominant quasi-species contribute.

genetics↗

bioRxiv2022

eCRUIS captures RNA-protein interaction in vitro and in vivo

As an information bridge between DNA and protein, RNA regulates cellular processes and gene expression in a variety of ways. From synthesis to degradation, RNA is associated with a series of RNA-binding proteins. Therefore, it is very important to develop innovative methods to study the interaction between RNA and protein. Previously, we developed an RNA-centric method, called CRISPR-based RNA-United Interacting System (CRUIS), to capture RNA-protein interaction in cells. On this basis, here we develop an enhanced CRUIS (eCRUIS) by combining the power of dCas13d and the engineered promiscuous ligase TurboID. The new version allows us to label RNA-binding proteins on the target RNA within 30 minutes, which may be used in vivo. By introducing bait-assay with exogenous RNA, we confirm that eCRUIS can effectively label RNA-binding proteins on bait RNA in a short time. eCRUIS provides a wider range of in vitro and in vivo applications.

molecular biology↗

bioRxiv2022

Cytoophidia maintain the integrity of Drosophila follicle epithelium

CTP synthase (CTPS) forms a filamentous structure termed the cytoophidium in all three domains of life. The female reproductive system of Drosophila is an excellent model for studying the physiological function of cytoophidia. Here, we use CTPSH355A, a point mutation that destroys the cytoophidium-forming ability of CTPS, to explore the in vivo function of cytoophidia. In CTPSH355A egg chambers, we observe the ingression and increased heterogeneity of follicle cells. In addition, we find that the cytoophidium-forming ability of CTPS, rather than the protein level, is the cause of the defects observed in CTPSH355A mutants. To sum up, our data indicate that cytoophidia play an important role in maintaining the integrity of follicle epithelium.

developmental biology↗

bioRxiv2022

Cytoophidia safeguard binucleation of Drosophila male accessory gland cells

Although most cells are mononuclear, the nucleus can exist in the form of binucleate or even multinucleate to respond to different physiological processes. The male accessory gland of Drosophila is the organ that produces semen, and its main cells are binucleate. Here we observe that CTP synthase (CTPS) forms filamentous cytoophidia in binuclear main cells, primarily located at the cell boundary. In CTPSH355A, a point mutation that destroys the formation of cytoophidia, we find that the nucleation mode of the main cells changes, including mononucleates and vertical distribution of binucleates. Although the overexpression of CTPSH355A can restore the level of CTPS protein, it will neither form cytoophidia nor eliminate the abnormal nucleation pattern. Therefore, our data indicate that there is an unexpected functional link between the formation of cytoophidia and the maintenance of binucleation in Drosophila main cells.

developmental biology↗

bioRxiv2022

GTP as an enzymatic synchronizer

CTP synthase (CTPS), the sole enzyme for de novo CTP biosynthesis, requires GTP as an allosteric effector for efficient catalysis1. Although the labile intermediates and chemical reaction steps have been characterized, how CTPS coordinates its structural domains through interactions with GTP to continuously catalyze thus reactions and thereby synthesize CTP correctly remains elusive. Through cryo-EM analysis of 34 structural states (2.0-3.3 [A] resolution) spanning pre-catalytic, catalytic, and post-catalytic conformations, we delineate how GTP cycling--intact binding and dissociation--directly gates three sequential activation checkpoints. 1) Catalytic initiation gate: GTP dissociation precedes substrate entry, transitioning CTPS to a substrate-competent state. 2) Intermediate-dependent GTP recruitment: 4Pi-UTP formation allosterically remodels the GAT domain, licensing GTP rebinding. 3) Ammonia transfer gate: GTP directly constitutes the transient interdomain gas tunnel for ammonia delivery while stimulating the glutamine hydrolyzing. CTP synthesis completion triggers GTP dissociation, allowing glutamate release. Each catalytic cycle couples CTP production to a single GTP-binding event, demonstrating that GTP cycling operates as a catalytic-phase timer. This work provides a comprehensive model for the efficient catalysis of CTPS and establishes effector cycling as a dynamic gating mechanism in enzymes.

biochemistry↗

bioRxiv2022

Super-resolution imaging reveals dynamic skeletal cytoophidia

CTP synthase (CTPS) can form filamentous structures termed cytoophidia in cells from all three domains of life. In order to study the mesoscale structure of cytoophidia, we perform fluorescence recovery after photobleaching (FRAP) and stimulated emission depletion (STED) microscopy in human cells. By using EGFP dimeric tag, as a tool to explore the physical properties of cytoophidia, we find that cytoophidia are dynamic and reticular. The reticular structure of CTPS cytoophidia may provide space for other components such as IMPDH. In addition, we observe CTPS granules with tentacles.

biophysics↗

bioRxiv2022

Structural basis of human PRPS2 filaments

PRPP synthase (PRPS) transfers the pyrophosphate groups from ATP to ribose-5-phosphate to produce 5-phosphate ribose-1-pyrophosphate (PRPP), a key intermediate in the biosynthesis of several metabolites including nucleotides, dinucleotides and some amino acids. There are three PRPS isoforms encoded in human genome. While hPRPS1 and hPRPS2 are expressed in most tissues, hPRPS3 is exclusively expressed in testis. Although hPRPS1 and hPRPS2 share 95% sequence identity, hPRPS2 has been shown to be less sensitive to allosteric inhibition and specifically upregulated in certain cancers in the translational level. Recent studies demonstrate that PRPS can form a subcellular compartment termed the cytoophidium in multiple organisms across prokaryotes and eukaryotes. Forming the cytoophidium is considered as a distinctive mechanism involving the polymerization of the protein. In order to investigate the function and molecular mechanism of hPRPS2 polymerization, we solve the polymer structure of hPRPS2 at 3.08[A] resolution using cryo-Electron Microscopy (cryo-EM). hPRPS2 hexamers stack into polymers in the conditions with the allosteric/competitive inhibitor ADP. The binding modes of ADP at the canonical allosteric site and at the catalytic active site are clearly determined. A point mutation disrupting the inter-hexamer interaction prevents hPRPS2 polymerization and results in significantly reduced catalytic activity. Our findings suggest that the regulation of hPRPS2 polymer is distinct from E. coli PRPS polymer and provide new insights to the regulation of hPRPS2 with structural basis.

biochemistry↗

bioRxiv2022

Detecting apoptosis via DODO

The real-time detection of intracellular biological processes by coded sensors has broad application prospects. Here we develop a degron based modular reporting system: the Device of Death Operation (DODO), which can be used to detect a series of biological processes. The DODO system consists of "reporter", "sensor" and degron. After protease activation and cleavage, the degron will be released from the fluorescent protein and eventually lead to the stabilization of the fluorescent protein. By replacing different "sensors" and "reporters", a series of biological processes can be reported through different signals. The system can effectively report the existence of TEV. To prove this concept, we successfully apply the DODO system to report apoptosis. In addition, the reporter based on degron will help to design protease reporters other than caspase.

molecular biology↗

bioRxiv2022

Fat body-specific reduction of CTPS alleviates HFD-induced obesity

High fat diet (HFD)-induced obesity is a multi-factorial disease including genetic, physiological, behavioral, and environmental components. Drosophila has emerged as a useful model of metabolic diseases. Cytidine 5'-triphosphate synthase (CTPS) is a key enzyme for the de novo synthesis of CTP, governing cellular level of CTP and phospholipid synthesis. We have demonstrated that CTPS show the capacity to assemble into large filaments termed cytoophidia, which are evolutionarily conserved in bacteria, archaea and eukaryotes. Here, we show that CTPS acts in fat body to regulate body weight and starvation resistant in Drosophila. HFD-induced obesity elevates CTPS transcription and elongates cytoophidia in larval adipocytes. Fat body-specific depletion of CTPS alleviated HFD-induced obesity including body weight gain, lipid storage and TAG level. Moreover, a dominant negative form of CTPS reduces lipid accumulation and down regulates lipogenic genes. Therefore, our data not only provide a functional link between CTPS and lipid homeostasis, but also highlight the potential application of manipulating CTPS in the treatment of HFD-induced obesity.

cell biology↗

bioRxiv2022

Allosteric inhibition of PRPS is moderated by filamentous polymerization

Phosphoribosyl pyrophosphate (PRPP) is an important intermediate for the biosynthesis of purine and pyrimidine nucleotides, histidine, tryptophan, and cofactors NAD and NADP. Abnormal regulation of PRPP synthase (PRPS) has been associated with human disorders including Arts syndrome, retinal dystrophy and gouty arthritis. Recent studies have revealed that PRPS can form filamentous cytoophidia in prokaryotes and eukaryotes. Here we resolve two distinct filament structures of E. coli PRPS at near-atomic resolution under Cryo-EM. Formation of two types of filaments is controlled by the binding of different ligands. While the type A filament attenuates the allosteric inhibition of PRPS by ADP, the type B filament enhances the inhibition. In addition, a novel conformation of the regulatory flexible loop of PRPS was found occupying the ATP binding site. AMP/ADP bound at a noncanonical allosteric site interacts with the regulatory flexible loop and facilitates the binding of ATP. Our findings not only reveal molecular mechanisms of the regulation of PRPS with structural basis, but also suggest a distinctive bidirectional regulatory system for PRPP production via PRPS polymerization.

biochemistry↗

bioRxiv2022

Molecular Crowding Facilitates Bundling of IMPDH Polymers and Cytoophidium Formation

The cytoophidium is a unique type of membraneless compartment comprising of filamentous protein polymers. Inosine monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting step of de novo GTP biosynthesis and plays critical roles in active cell metabolism. However, the molecular regulation of cytoophidium formation is poorly understood. Here we show that human IMPDH2 polymers bundle up to form cytoophidium-like aggregates in vitro when macromolecular crowders are present. The self-association of IMPDH polymers is suggested to rely on electrostatic interactions. In cells, the increase of molecular crowding with hyperosmotic medium induces cytoophidia, while the decrease of that by the inhibition of RNA synthesis perturbs cytoophidium assembly. In addition to IMPDH, CTPS and PRPS cytoophidium could be also induced by hyperosmolality, suggesting a universal phenomenon of cytoophidium-forming proteins. Finally, our results indicate that the cytoophidium can prolong the half-life of IMPDH, which is proposed to be one of conserved functions of this subcellular compartment.

cell biology↗

bioRxiv2021

Ubiquitination regulates cytoophidium assembly in Schizosaccharomyces pombe

CTP synthase (CTPS), a metabolic enzyme responsible for the de novo synthesis of CTP, can form filamentous structures termed cytoophidia, which are evolutionarily conserved from bacteria to humans. Here we used Schizosaccharomyces pombe to study the cytoophidium assembly regulation by ubiquitination. We tested the CTP synthases capacity to be epigenetically modified by ubiquitin or be affected by the ubiquitination state of the cell, showed that CTPS is immunoprecipitated with ubiquitin, and that ubiquitination is important for the maintenance of the CTPS filamentous structure in fission yeast. We have identified proteins which are in complex with CTPS, including specific ubiquitination regulators which significantly affect CTPS filamentation, and mapped probable ubiquitination targets on CTPS. Furthermore, we discovered that a cohort of deubiquitinating enzymes is significant for the regulation of cytoophidium morphology. Our study provides a framework for the analysis of the effects that ubiquitination and deubiquitination have on the formation of CTPS filaments.

cell biology↗

bioRxiv2021

Coupling adipose tissue architecture and metabolism via cytoophidia

Tissue architecture determines its unique physiology and function. How these properties are intertwined has remained unclear. Here, we show that the metabolic enzyme CTP synthase (CTPS) form filamentous structures termed cytoophidia along the adipocyte cortex in Drosophila adipose tissue. Interestingly, loss of cytoophidia, whether due to reduced CTPS expression or a point mutation that specifically abrogates its polymerization ability, leads to downregulated Collagen-Integrin signaling, weakened adipocyte adhesion, and defective adipose architecture. Strikingly, CTPS specifically binds with Integrin subunit 2, which influences Integrin function and Collagen IV deposition. cytoophidia promote Collagen IV mRNA expression and thus its extracellular deposition to strengthen adipocyte adhesion. Remarkably, Collagen IV-Integrin signaling reciprocally regulates cytoophidium formation at a post-translational level. Together, we demonstrate that a positive feedback signaling loop containing both cytoophidia and Integrin adhesion complex couples tissue architecture and metabolism in the fly adipose.

developmental biology↗

bioRxiv2021

CTP synthase does not form cytoophidia in Drosophila interfollicular stalks

CTP synthase (CTPS) catalyzes the final step of de novo synthesis of the nucleotide CTP. In 2010, CTPS has been found to form filamentous structures termed cytoophidia in Drosophila follicle cells and germline cells. Subsequently, cytoophidia have been reported in many species across three domains of life: bacteria, eukaryotes and archaea. Forming cytoophidia appears to be a highly conserved and ancient property of CTPS. To our surprise, here we find that polar cells and stalk cells, two specialized types of cells composing Drosophila interfollicular stalks, do not possess obvious cytoophidia. Moreover, we show that Myc level is low in these two types of cells, supporting the idea that Myc regulates cytoophidium assembly. Treatment with a glutamine analog, 6-diazo-5-oxo-l-norleucine (DON), increases cytoophidium assembly in main follicle cells, but not in polar cells or stalk cells. Our findings provide an interesting paradigm for the in vivo study of cytoophidium assembly and disassembly among different populations of follicle cells.

developmental biology↗

bioRxiv2021

Structural basis of dynamic P5CS filaments

The bifunctional enzyme {Delta}1-pyrroline-5-carboxylate synthase (P5CS) is central to the synthesis of proline and ornithine. Pathogenic mutations in P5CS gene (ALDH18A1) lead to neurocutaneous syndrome and skin relaxation connective tissue disease in humans, and P5CS deficiency seriously damages the ability to resist adversity in plants, which has an essential role in agriculture and human health. Recently, P5CS has been demonstrated forming the cytoophidium in vivo and filaments in vitro. However, the underlying mechanism for the function of P5CS filamentation and catalyze the synthesis of P5C is hardly accessible without structural basis. Here, we have succeeded in determining the full-length structures of Drosophila P5CS filament in three states at resolution from 3.1 to 4.3 [A] under cryo-electron microscopy, we observed the distinct ligand-binding states and conformational changes for GK and GPR domain separately. These structures show the distinctive spiral filament is assembled by P5CS tetramers and stabilized by multiple interfaces. Point mutations that deplete such interactions disturb P5CS filamentation and greatly reduce the activity. Our findings reveal a previously undescribed mechanism that filamentation is crucial for the coordination between GK and GPR domains, and provide insights into structural basis for catalysis function of P5CS filament.

biochemistry↗

bioRxiv2019

The proline synthesis enzyme P5CS forms cytoophidia in Drosophila

Compartmentation of enzymes via filamentation has arisen as a mechanism for the regulation of metabolism. In 2010, three groups independently reported that CTP synthase (CTPS) can assemble into a filamentous structure termed the cytoophidium. In searching for CTPS-interacting proteins, here we perform a yeast two-hybrid screening of Drosophila proteins and identify a putative CTPS-interacting protein, {Delta}1-pyrroline-5-carboxylate synthase (P5CS). Using Drosophila follicle cell as the in vivo model, we confirm that P5CS forms cytoophidia, which are associated with CTPS cytoophidia. Overexpression of P5CS increases the length of CTPS cytoophidia. Conversely, filamentation of CTPS affects the morphology of P5CS cytoophidia. Finally, in vitro analyses confirm the filament-forming property of P5CS. Our work links CTPS with P5CS, two enzymes involved in the rate-limiting steps in pyrimidine and proline biosynthesis, respectively.

cell biology↗

bioRxiv2019

TurboID-mediated proximity labelling of cytoophidium proteome in Drosophila

Proximity-based biotinylation combined with mass spectrometry has emerged as a powerful approach to study protein interaction networks and protein subcellular compartmentation. However, low kinetics and the requirement of toxic chemicals limit the broad utilisation of current proximity labelling methods in living organisms. TurboID, the newly engineered promiscuous ligase, has been reported to label bait proteins effectively in various species. Here, we systematically demonstrated the application of TurboID-mediated biotinylation in a wide range of developmental stages and tissues, and we also verified the feasibility of TurboID-mediated labelling in desired cells via cell-type-specific GAL4 driver in Drosophila. Furthermore, using TurboID-mediated biotinylation coupled with mass spectrometry, we characterized the proximate proteome of the cytoophidium, a newly identified filamentous structure containing the metabolic enzyme CTP synthase (CTPS) in Drosophila. Our study demonstrates a referable tool and resource for research in subcellular compartments of metabolic enzymes in vivo.

cell biology↗