Biology subjects

Hu, H.-H.

Publications and source records attributed to Hu, H.-H..

5 recordsLinked to original sources

bioRxiv2025

VitisC: Visualizing interested target via integrated scaffold of cytoophidium

Cryo-electron microscopy (cryo-EM) single-particle analysis has become a widely used technique for high-resolution structural determination of biological macromolecules and complexes. However, the determination of the structure of small molecule proteins remains a limitation of this technology. To address this issue, here we develop a novel approach termed Visualizing interested target via integrated scaffold of cytoophidium (VitisC). We use a filamentous structure formed by Escherichia coli CTP synthase (CTPS) as the scaffold, termed the scaffold of cytoophidium. Through artificial design and modification, small proteins can be attached to this symmetrical scaffold, which is very suitable for cryo-EM imaging. The formation conditions for stable filament structures of the fusion protein are optimized in vitro, and the three-dimensional structure of the fusion protein is reconstructed using cryo-EM single-particle analysis, achieving an overall resolution of 3.39 [A]. Therefore, cryo-EM is successfully applied to visualize small proteins. VitisC not only demonstrates the feasibility of employing cytoophidia as scaffolds, but also provides an approach for high-resolution structural analysis of small proteins using Cryo-EM.

biochemistry↗

bioRxiv2024

AMPK acts to remove immune barriers to CD8+ T cell-mediated immunity against hepatocellular carcinoma

Dysregulated metabolism in tumor tissues, and para-tumor tissues alike, can lead to immunosuppression, which may underlie cancer development. However, metabolic intervention as a therapeutic strategy has been of no avail. In this study, we explored the anti-cancer therapeutic effect of aldometanib that specifically targets lysosome-associated aldolase to mimic glucose starvation and thereby activates the lysosomal AMP-activated protein kinase (AMPK), a master regulator of metabolic homeostasis. We show that aldometanib inhibits the growth of HCC in an AMPK-dependent manner, allowing hepatoma-bearing mice to survive to mature ages, although aldometanib does not possess cytotoxicity towards HCC or normal cells. Intriguingly, aldometanib exerts anti-cancer effects only in immune-competent host mice, but not in immune-defective mice. We have further found that the HCC tissues in aldometanib-treated mice are massively infiltrated with CD8+ T cells, which is not seen in mice with liver-specific knockout of AMPK. Our findings thus suggest that the metabolic regulator AMPK rebalances the tumor microenvironment to allow the cytotoxic immune cells inside the body to eliminate cancer cells and effectively contain the tumor tissues. The finding that metabolic intervention can make cancer a life-long manageable disease, may potentially usher in a new era of cancer therapy.

immunology↗

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↗

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↗