Biology subjects

Mai, X.

Publications and source records attributed to Mai, X..

13 recordsLinked to original sources

bioRxiv2025

Ex vivo and in vivo CRISPR/Cas9 screenings identify the roles of protein N-glycosylation in regulating T-cell activation and functions

Cytotoxic CD8+ T-cells play central roles in tumor immunotherapy. Understanding mechanisms that regulate development, differentiation, and functions of cytotoxic CD8+ T-cells leads to development of better immunotherapies. By combining primary T-cell culture and a syngeneic mouse tumor model with both genome-wide and custom CRISPR/Cas9 screenings, we systematically identified genes and pathways that regulate PD-1 expression and functions of CD8+ T-cells. Among them, inactivation of a key enzyme in glycoconjugate biosynthesis, beta 1, 4-galatosyltransferase 1 (B4GALT1), leads to significantly enhanced T-cell receptor (TCR) activation and functions of CD8+ T-cell. Interestingly, suppression of B4GALT1 enhances functions of TCR-T-cells, but has no effect on chimeric antigen receptor T (CAR-T) cells. We systematically identified the substrates of B4GALT1 on CD8+ T-cell surface by affinity purification and mass spectrometry analysis, which include protein components in both TCR and its co-receptor complexes. The galactosylation of TCR and CD8 leads to reduced interaction between TCR and CD8 that is essential for TCR activation. Artificially tethering TCR and CD8 by a TCR-CD8 fusion protein could bypass the regulation of B4GALT1 in CD8+ T-cells. Finally, the expression levels of B4GALT1 normalized to tumor infiltrated CD8+ T-cells in tumor microenvironment are significant and negatively associated with prognosis of human patients. Our results reveal the important roles of protein N-glycosylation in regulating functions of CD8+ T-cells and prove that B4GALT1 is a potential target for tumor immunotherapy.

immunology↗

bioRxiv2025

DMBT1 promotes SARS-CoV-2 infection and its SRCR-derived peptide inhibits SARS-CoV-2 infection

DMBT1 is a large scavenger receptor cysteine rich (SRCR) B protein that has been reported as a tumor suppressor gene and a co-receptor for HIV-1 infection. Here we found DMBT1 is a major mucosal protein bound to SARS-CoV-2. Overexpression of DMBT1 in 293T cells enhanced infection by SARS-CoV-2 in ACE2 dependent manner. Blocking experiments using overlapping peptide library of SRCR domain of DMBT1 showed that CQGRVEVLYRGSWGTV peptide, which contains bacteria-binding VEVLXXXXW motif, could inhibit SARS-CoV-2 infection. High concentration of the peptide can significantly inhibit the replication of SARS-CoV-2 in hamsters. Single cell sequencing analysis showed the highest expression abundance of DMBT1 at airway submucosal glands. These results demonstrate that membrane DMBT1 can promote SARS-CoV-2 infection, and serve as a candidate target for antiviral development. Author SummaryWe discovered that a protein named as DMBT1, which is found in our mucous membranes, plays a significant role in SARS-CoV-2 infection. DMBT1 is a large glycoprotein that has previously reported linked to tumor suppression and HIV-1 infection. Our single-cell sequencing analysis revealed that DMBT1 is most abundant in the airway submucosal glands. In our study, we found that when DMBT1 is overexpressed in cells, it enhances SARS-CoV-2 infection. Using a peptide library from DMBT1, we pinpointed a specific peptide, CQGRVEVLYRGSWGTV, which can inhibit SARS-CoV-2 infection. At high concentrations, it significantly reduces the viruss ability to replicate in hamsters. These findings suggest that DMBT1 on the cell membrane can promote SARS-CoV-2 infection and highlight DMBT1 as a potential target for developing antiviral treatments.

microbiology↗

bioRxiv2024

Mechanism of D-type cyclins recognition by the AMBRA1 E3 ligase receptor

AMBRA1 is a tumour suppressor protein that functions as a substrate receptor in the ubiquitin conjugation system and regulates the stability of D-type cyclins and cell proliferation. Here, we present the cryo-EM structure of cyclin D1 bound AMBRA1-DDB1 complex at 3.5 [A] resolution. The structure reveals a substrate interaction surface on the AMBRA1 WD40 domain that specifically binds to the C-terminal region of D-type cyclins. This interaction is dependent on the phosphorylation of Thr286 residue in the C-terminal phosphodegron site of D-type cyclins. The phosphodegron motif folds into a turn-like conformation followed by a 310 helix that promotes its assembly with AMBRA1. Additionally, we show that AMBRA1 mutants, which are defective in cyclin D1 binding, lead to cyclin D1 accumulation and DNA damage. Understanding the AMBRA1-D-type cyclins structure enhances the knowledge of the molecular mechanisms that govern the cell cycle control and may lead to new therapeutic approaches for cancers linked to abnormal cyclin D activity.

biochemistry↗

bioRxiv2024

Structural basis for membrane remodelling by the AP5:SPG11-SPG15 complex

The human spastizin (spastic paraplegia 15, SPG15) and spatacsin (spastic paraplegia 11, SPG11) complex is involved in cargo sorting from late endosomes to the Golgi, and mutations in these two proteins are linked with hereditary autosomal recessive spastic paraplegia (HSP). SPG11-SPG15 can cooperate with evolutionarily ancient fifth adaptor protein complex (AP5). We employed cryo-electron microscopy and in silico predictions to investigate the structural assemblies of SPG11-SPG15 and AP5:SPG11-SPG15 complex. The W-shaped SPG11-SPG15 intertwined in a head-to-head fashion, and the N-terminal region of SPG11 is required for AP5 complex interaction and assembly. The AP5 complex is in a super open conformation. We employed in vitro lipid binding assays and cellular localization analysis to investigate AP5:SPG11-SPG15 membrane binding properties. Here we solve a major problem in understanding AP5:SPG11-SPG15 function in autophagic lysosome reformation (ALR), using a fully reconstituted system. We reveal that the AP5:SPG11-SPG15 complex binds PI3P molecules, can sense membrane curvature and drive membrane remodelling in vitro. These studies provide key insights into the structure and function of the spastic paraplegia AP5:SPG11-SPG15 complex, which is essential for the initiation of autolysosome tubulation.

biochemistry↗

bioRxiv2024

Enhanced Recognition of a Herbal Compound Epiberberine by a DNA Quadruplex-Duplex Structure

The small molecule epiberberine (EPI) is a natural alkaloid with versatile bioactivities against several diseases, including cancer and bacterial infection. EPI can induce the formation of a unique binding pocket at the 5' side of a human telomeric G-quadruplex (HTG) sequence Q4, resulting in a nanomolar binding affinity (KD approximately 26 nM) with significant fluorescence enhancement upon binding. It is important to understand (1) how EPI binding affects HTG structural stability and (2) how enhanced EPI binding may be achieved through the engineering of the DNA binding pocket. In this work, the EPI binding-induced HTG structure stabilization effect was probed by a peptide nucleic acid (PNA) invasion assay in combination with a series of biophysical techniques. We show that the PNA invasion-based method may be useful for the characterization of compounds binding to DNA (and RNA) structures in physiological conditions without the need to vary the solution temperature or buffer components, which are typically needed for structural stability characterization. Importantly, the combination of theoretical modeling and experimental quantification allows us to successfully engineer the Q4 derivative Q4-ds-A by a simple extension of a duplex structure to Q4 at the 5' end. Q4-ds-A is a superb EPI binder with a KD of 8 nM, with the binding enhancement achieved through the preformation of a binding pocket and a reduced dissociation rate. The tight binding of Q4 and Q4-ds-A with EPI allows us to develop a novel magnetic bead-based affinity purification system to effectively extract EPI from Rhizoma coptidis (Huang Lian) extracts.

biochemistry↗

bioRxiv2023

Lactose and its derivatives are a new class of immune checkpoint inhibitors for cancer therapy

Understanding the mechanisms by which the immune system surveils cancer is the key to developing better tumor immunotherapy strategies. By CRISPR/Cas9 screenings, we identified that inactivation of beta-1,4-galactosyltransferase-1 (B4GALT1), a key enzyme in glycoconjugate biosynthesis, leads to enhanced T-cell receptor (TCR) activation and functions of CD8+ T-cells. Via proximity-dependent-intercellular-protein-spreading (PDICPS), cancer cells transfer surface-bound galectin-1 (Gal-1) proteins, which recognize and bind galactosylated membrane proteins, to CD8+ T-cells, thereby suppressing T-cell-mediated cytolysis. B4GALT1-deficiency leads to reduced cell-surface galactosylation and Gal-1 binding of CD8+ T-cells. Proteomic analysis revealed reduced binding of Gal-1 with TCR and its coreceptor CD8 on B4GALT1-deficient CD8+ T-cells, leading to enhanced TCR-CD8 colocalization and T-cell activation. Lactose, a structure-mimicking competitive inhibitor of N-glycan galactosylation, enhances the functions of CD8+ T-cells and tumor immunosurveillance. Results from various preclinical tumor models demonstrate that lactose and its derivatives are a new class of immune checkpoint inhibitors for tumor immunotherapy.

immunology↗

bioRxiv2023

A muscle hypertrophy-derived myokine reprograms the stromal vascular fractions differentiation towards thermogenic adipocytes in subcutaneous adipose tissue

Skeletal muscle plays a significant role in both local and systemic energy metabolism; however, the cross-talk between skeletal muscle and adipose tissue remains largely unexplored. In this study, we identify the HIF2-Bambi-Thbs4 axis as a critical driver of muscle hypertrophy and metabolic improvement. Deletion of Bambi induces muscle hypertrophy and oxidative switching, thereby enhancing local and systemic metabolism under conditions of rodent chow and high-fat diets (HFD). Other than the metabolic regulation of skeletal muscle hypertrophy, we hypothesize that a portion of this improvement may be attributed to the metabolic reprogramming of the stromal vascular fraction (SVFs) of iWAT, promoting the development of beige adipocytes. Leveraging multi-omics approaches, we establish Thbs4 as a newly identified, muscle hypertrophy-derived myokine that serves as an essential regulator of this metabolic reprogramming. Thbs4 is upregulated in skeletal muscle during hypertrophy and aerobic exercise, localizes to the iWAT membrane, and facilitates the transformation of white adipocytes into beige adipocytes under conditions such as cold exposure, muscle hypertrophy, or aerobic exercise. Moreover, overexpression of Thbs4 elevates its plasma levels and leads to the transformation of white to beige adipocytes, therefore offering long-term protection against HFD-induced metabolic disorders. Conversely, Thbs4 knockout (Thbs4-KO) disrupts the metabolic reprogramming of SVFs and exacerbates metabolic syndromes induced by an HFD, mirroring the observed decline in Thbs4 levels in aged mice. Therefore, our findings underscore Thbs4s potential as both a long-term metabolic protective factor and a therapeutic target for the treatment of metabolic disorders. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=197 HEIGHT=200 SRC="FIGDIR/small/558627v1_ufig1.gif" ALT="Figure 1"> View larger version (63K): org.highwire.dtl.DTLVardef@1178e6org.highwire.dtl.DTLVardef@1184d4eorg.highwire.dtl.DTLVardef@7b592aorg.highwire.dtl.DTLVardef@1b0b03_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIBambi, a direct target gene of HIF2 in skeletal muscle, when deleted, triggers muscle hypertrophy and the transformation of white into beige adipocytes, improving systemic energy metabolism. C_LIO_LIThe metabolic reprogramming of stromal vascular fractions (SVFs) contributes to the observed improvements in energy metabolism and adipocyte hyperplasia. C_LIO_LIThbs4, a novel myokine produced in skeletal muscle during hypertrophy and aerobic exercise, plays a key role in the transformation of white into beige adipocytes. C_LIO_LIOverexpression of Thbs4 provides enduring protection against high-fat diet (HFD)-induced metabolic disorders, demonstrating its potential as a therapeutic target. C_LIO_LIThbs4 deletion abrogates the beneficial metabolic reprogramming of SVFs and exacerbates HFD-induced metabolic syndromes, further underlining its crucial role in metabolic regulation. C_LI

physiology↗

bioRxiv2023

Structural basis for lipid transfer by the ATG2A-ATG9A complex

Autophagy is characterized by the formation of double-membrane vesicles called autophagosomes. ATG2A and ATG9A play an essential role in autophagy by mediating lipid transfer and re-equilibration between membranes for autophagosome formation. Here we report the cryo-EM structures of human ATG2A-WIPI4 complex at 3.2 [A], and ATG2A-WIPI4-ATG9A complex at 7 [A] resolution. The ATG2A structure is characterized by a central hydrophobic cavity formed by a network of {beta}-strands that facilitates lipid transfer, and highly flexible N- and C-terminal domains. Molecular dynamics simulations of the ATG2A N-terminal domain revealed the mechanism of lipid-extraction from the donor membranes while the ATG2A-ATG9A complex structure provides insights into the later stages of the lipid transfer reaction. ATG9A-ATG2A structural analysis revealed a 1:1 stoichiometry, directly aligning the ATG9A lateral pore with ATG2A lipid transfer cavity, hence allowing for a direct transfer of lipids from ATG2A. The ATG9A trimer can interact with both N- and C-terminal tip of rod-shaped ATG2A. Cryo-electron tomography of ATG2A-liposome binding states shows that ATG2A tethers lipid vesicles at different orientations. In summary, this study provides a molecular basis for the growth of the phagophore membrane, and lends structural insights into spatially coupled lipid transport and re-equilibration during autophagosome formation.

biochemistry↗

bioRxiv2023

Dissecting peri-implantation development using cultured human embryos and embryo-like assembloids

Studies of cultured embryos have provided insights into human peri-implantation development. However, detailed knowledge of peri-implantation lineage developments as well as underlying mechanisms remain obscure. Using 3D-cultured human embryos, herein we report a complete cell atlas of the early post-implantation lineages and decipher cellular composition and gene signatures of the epiblast and hypoblast derivatives. In addition, we develop an embryo-like assembloid (E-assembloid) by assembling naive hESCs and extraembryonic cells. Using human embryos and E-assembloids, we reveal that WNT, BMP and Nodal signaling synergistically, but functionally differently, orchestrate human peri-implantation lineage development. Specially, we dissect mechanisms underlying extraembryonic mesoderm and extraembryonic endoderm specifications. Finally, an improved E-assembloid is developed to recapitulate the epiblast and hypoblast developments and tissue architectures in the pre-gastrulation human embryo. Our findings provide insights of the human peri-implantation development, and the E-assembloid offers a useful model to disentangle cellular behaviors and signaling interactions that drive human embryogenesis.

developmental biology↗

bioRxiv2023

Acute stress reshapes third-party punishment and help decisions: Behavioral evidence and neurocomputational mechanisms

People tend to intervene in others injustices by either punishing the transgressor or helping the victim. Injustice events often occur under stressful circumstances. However, how acute stress affects a third partys intervention in injustice events remains open. Here, we show a stress-induced shift in third parties willingness to engage in help instead of punishment by acting on emotional salience and central-executive and theory-of-mind networks. Acute stress decreased the third partys willingness to punish the violator and the severity of the punishment and increased their willingness to help the victim. Computational modeling revealed a shift in intervention severity bias from punishment toward help under stress. This finding is consistent with the increased dorsolateral prefrontal engagement observed with higher amygdala activity and greater connectivity with the ventromedial prefrontal cortex. A brain connectivity theory-of-mind network predicted stress-induced severity bias in punishment. Our findings suggest a neurocomputational mechanism of how acute stress reshapes third parties decisions by reallocating neural resources in emotional, executive and mentalizing networks to inhibit punishment bias and decrease punishment severity.

neuroscience↗

bioRxiv2022

Structural basis for substrate recruitment by AMBRA1 E3 ligase receptor

AMBRA1 is a tumor suppressor protein that functions as a substrate receptor of the ubiquitin conjugation system as part of autophagy and cell-cycle regulatory network. The highly intrinsic disorder of AMBRA1 has so far precluded its structural determination. To solve this problem, we analyzed the domain organization and dynamics of AMBRA1 using hydrogen deuterium exchange mass spectrometry (HDX-MS). High deuterium uptake indicates that AMBRA1 is a dynamic and largely unstructured protein, and can be stabilized upon interaction with DDB1, the adaptor of the Cullin4A/B E3 ligase complex. Here we present the cryo-EM structure of AMBRA1 in complex with DDB1 at 3 [A] resolution. The structure shows that parts of N- and C-terminal structural regions in AMBRA1 fold together into the highly dynamic WD40 domain, and reveals how DDB1 engages with AMBRA1 to create a binding scaffold for substrate recruitment. AMBRA1 uses its N-terminal helix-loop-helix and WD40 domain to bind the double-propeller fold of DDB1, whereas different regions target the specific cellular substrates for ubiquitination. We also demonstrate that DDB1 binding-defective AMBRA1 mutants prevent ubiquitination of the substrate Cyclin D1 in vitro and decreased number of autophagosomes in the cells. Together, these results provide structural insights into AMBRA1-ubiquitin ligase complex and suggests a mechanism by which the AMBRA1 acts as a hub involved in various physiological processes.

biochemistry↗

bioRxiv2022

Parallel lipid-carried epitope pillars enhance stimulation of chimeric antigen receptor T cells

Chimeric antigen receptor T cells are genetically engineered to express a specific T cell receptor of interest, such as to target a cancer-specific antigen. Signaling events in chimeric antigen receptor T cells are essential for their proliferation, survival, and function. To achieve optimal antitumor efficacy, signaling motifs as well as the structure of the transmembrane domain of the chimeric antigen receptor have to be carefully designed. However, it remains unclear whether the arrangement, and therefore the movement and elasticity, of tumor antigens influence their stimulation of chimeric antigen receptor T cells. Here we show that MUC1 molecules tethered to a glycosylphosphatidylinositol anchor drastically increased its stimulation of chimeric antigen receptor T cells. In particular, MUC1 molecules containing one tandem repeat sequence showed significantly greater stimulatory activity than five tandem repeat sequences. Thus glycosylphosphatidylinositol-anchored, tumor antigen epitope pillars in parallel are attractive targets for the design of chimeric antigen receptor T cells. These novel findings, which we propose as the parallel lipid-carried epitope model, emphasize the importance of epitope arrangement in selecting highly effective chimeric antigen receptor T cells, potentially revolutionizing the applicability of this therapy. Furthermore, our data have implications for the necessity of methodologies to measure the elasticity, movement, and density of antigen pillars in parallel, as key tools to guide future chimeric antigen receptor T cell therapy.

immunology↗

bioRxiv2020

Transferrin receptor (Tfr1) ablation in satellite cells impacts skeletal muscle regeneration through the activation of ferroptosis

Satellite cells (SCs) are critical to the postnatal development and skeletal muscle regeneration. Inactivation of SCs is linked with the skeletal muscle loss. Leveraging on the RNAseq screening, transferrin receptor (Tfr1) is identified to be associated with muscle/SC ageing and the declined regeneration potential. Muscle-specific deletion of Tfr1 results in the growth retardation, metabolic disorder and lethality, shedding light on the importance of Tfr1 in skeletal muscle physiology. Here, our investigation reported that conditional SC-ablation of Tfr1 leads to the SCs inactivation and skeletal muscle regeneration defects, followed by the labile iron accumulation, de novo lipogenesis via fibroadipogenic progenitors (FAPs) and Gpx4/Nrf2-mediated ROS-scavenger defects. These abnormal phenomena, such as Hmox1-mediated myoglobin degradation, Tfr1-Slc39a14 functional switch and the activation of unsaturated fatty acid biosynthesis pathway are orchestrated with the occurrence of ferroptosis in skeletal muscle. Ferroptosis may further prevent SC proliferation and skeletal muscle regeneration. Ferrostatin-1, a ferroptosis inhibitor could not rescue Tfr1-ablation induced ferroptosis. However, intramuscular administration of lentivirus expressing Tfr1 could partially reduce labile iron accumulation, decrease de novo lipogenesis and promote skeletal muscle regeneration. Most importantly, Tfr1/Slc39a14 functional switch, labile iron accumulation and fatty acid biosynthesis are recapitulated in aged skeletal muscle of rodents, indicating that ferroptosis occurs in the skeletal muscles of aged rodents. The present study also bridges the gap between pathogenesis of iron and functional defects in the skeletal muscle, providing mechanistic information to develop anti-aging strategies. One Sentence SummaryConditional ablation of Tfr1 in satellite cells (SCs) results in the SC inactivation, skeletal muscle regeneration defects, labile iron accumulation, and unsaturated fatty acid biosynthesis, leading to the activation of ferroptosis, which is recapitulated in skeletal muscles of aged rodents to be a new cell death form identified in skeletal muscle and sheds light on the development of novel anti-ageing strategies.

cell biology↗