Biology subjects

Che, Y.

Publications and source records attributed to Che, Y..

30 records · Page 2Linked to original sources

bioRxiv2022

Bile Acid Restrained T Cell Activation Explains Cholestasis Aggravated Hepatitis B Virus Infection

Cholestasis is a common complication of Hepatitis B Virus (HBV) infection, characterized by increased intrahepatic and plasma bile acid levels. Cholestasis was found negatively associated with hepatitis outcome, however; the exact mechanism by which cholestasis impact on anti-viral immunity and impede HBV clearance remains elusive. Here, we found that cholestatic mice are featured with dysfunctional T cell response, and bile acids inhibit the activation and metabolic reprogramming of CD4+ T cells. Mechanistically, bile acids disrupt intracellular calcium homeostasis via inhibiting mitochondria calcium uptake and elevating cytoplasmic Ca2+ concentration of CD4+ T cells, leading to STIM1 and ORAIL1 decoupling and impaired store-operated Ca2+ entry which is essential for NFAT signaling and T cell activation. Moreover, in a transgenic mouse model of HBV infection, it was confirmed that cholestasis compromised T cells activation resulting in poor viral clearance. Collectively, our results suggest that bile acids play pivotal roles in anti-HBV infection via controlling T cells activation and metabolism, and that targeting regulation of bile acids may be a therapeutic strategy for host virus defense.

immunology↗

bioRxiv2021

Reactivities of the Front Pocket N-Cap Cysteines in Human Kinases

Discovery of targeted covalent inhibitors directed at nucleophilic cysteines is attracting enormous interest. The front pocket (FP) N-cap cysteine has been the most popular site of covalent modification in kinases. Curiously, a long-standing hypothesis associates the N-cap position with cysteine hyper-reactivity; however, traditional computational methods suggest that the FP N-cap cysteines in all human kinases are predominantly unreactive at physiological pH. Here we applied a newly developed GPU-accelerated continuous constant pH molecular dynamics (CpHMD) tool to test the N-cap hypothesis and elucidate the cysteine reactivities. Simulations showed that the N-cap cysteines in BTK/BMX/TEC/ITK/TXK, JAK3, and MKK7 sample the reactive thiolate form to varying degrees at physiological pH; however, those in BLK and EGFR/ERBB2/ERBB4 which contain an Asp at the N-cap+3 position adopt the unreactive thiol form. The latter argues in favor of the base-assisted thiol-Michael addition mechanisms as suggested by the quantum mechanical calculations and experimental structure-function studies of EGFR inhibitors. Analysis revealed that the reactive N-cap cysteines are stabilized by hydrogen bond as well as electrostatic interactions, and in their absence a N-cap cysteine is unreactive due to desolvation. To test a corollary of the N-cap hypothesis, we also examined the reactivities of the FP N-cap+2 cysteines in JNK1/JNK2/JNK3 and CASK. Additionally, our simulations predicted the reactive cysteine and lysine locations in all 15 kinases. Our findings offer a systematic understanding of cysteine reactivities in kinases and demonstrate the predictive power and physical insights CpHMD can provide to guide the rational design of targeted covalent inhibitors.

biophysics↗

bioRxiv2021

Coral symbionts exhibit a polycistronic flavodiiron gene leading to functional proteins in photosynthesis

Photosynthesis in cyanobacteria, green algae, and basal land plants is protected against excess reducing pressure on the photosynthetic chain by flavodiiron proteins (FLV) that dissipate photosynthetic electrons by reducing O2. In these organisms, the genes encoding FLV are always conserved in the form of a pair of two-type isozymes (FLVA and FLVB) that are believed to function in O2 photo-reduction as a heterodimer. While coral symbionts (dinoflagellates of the family Symbiodiniaceae) are the only algae to harbor FLV in photosynthetic red plastid lineage, only one gene is found in transcriptomes and its role and activity remain unknown. Here, we characterized the FLV genes in Symbiodiniaceae and found that its coding region is composed of tandemly repeated FLV sequences. By measuring the O2-dependent electron flow and P700 oxidation, we suggest that this atypical FLV is active in vivo. Based on the amino-acid sequence alignment and the phylogenetic analysis, we conclude that in coral symbionts, the gene pair for FLVA and FLVB have been fused to construct one coding region for a hybrid enzyme, which presumably occurred when or after both genes were inherited from basal green algae to the dinoflagellate. Immunodetection suggested the FLV polypeptide to be cleaved by a post-translational mechanism, adding it to the rare cases of polycistronic genes in eukaryotes. Our results demonstrate that FLV are active in coral symbionts with genomic arrangement that is unique to these species. The implication of these unique features on their symbiotic living environment is discussed.

plant biology↗

bioRxiv2020

BNT162b vaccines are immunogenic and protect non-human primates against SARS-CoV-2

A safe and effective vaccine against COVID-19 is urgently needed in quantities sufficient to immunise large populations. We report the preclinical development of two BNT162b vaccine candidates, which contain lipid-nanoparticle (LNP) formulated nucleoside-modified mRNA encoding SARS-CoV-2 spike glycoprotein-derived immunogens. BNT162b1 encodes a soluble, secreted, trimerised receptor-binding domain (RBD-foldon). BNT162b2 encodes the full-length transmembrane spike glycoprotein, locked in its prefusion conformation (P2 S). The flexibly tethered RBDs of the RBD-foldon bind ACE2 with high avidity. Approximately 20% of the P 2S trimers are in the two-RBD down, one-RBD up state. In mice, one intramuscular dose of either candidate elicits a dose-dependent antibody response with high virus-entry inhibition titres and strong TH1 CD4+ and IFN{gamma}+ CD8+ T-cell responses. Prime/boost vaccination of rhesus macaques with BNT162b candidates elicits SARS-CoV-2 neutralising geometric mean titres 8.2 to 18.2 times that of a SARS-CoV-2 convalescent human serum panel. The vaccine candidates protect macaques from SARS-CoV-2 challenge, with BNT162b2 protecting the lower respiratory tract from the presence of viral RNA and with no evidence of disease enhancement. Both candidates are being evaluated in phase 1 trials in Germany and the United States. BNT162b2 is being evaluated in an ongoing global, pivotal Phase 2/3 trial (NCT04380701, NCT04368728).

immunology↗

bioRxiv2020

Mitochondria fragment and reassemble to initiate the formation and development of the nucleus

The function of nuclear-localized mitochondria remains unknown. We found that mitochondria assembled dense particles to fragment and disperse into the particles, which reassembled to initiate nuclear formation and development. Individually-formed nuclei in one single cell were joined together by mitochondrial fragmentation concurrently partitioning cytoplasm to form an intranuclear inclusion (INC), whose creation was not related to herniation or invagination of the cytoplasm. Along with the nuclear transition of a mitochondrion and its neighboring counterparts, the organelle included itself in the nucleus to become nuclear mitochondrion through peripherally assembling of dense particles. New medium reversed the nuclear formation of the organelles to recovery and re-establishment via the return of the particles, which consisted of dense microvesicles (MIVs).

cell biology↗

bioRxiv2020

A prefusion SARS-CoV-2 spike RNA vaccine is highly immunogenic and prevents lung infection in non-human primates

To contain the coronavirus disease 2019 (COVID-19) pandemic, a safe and effective vaccine against the new severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is urgently needed in quantities sufficient to immunise large populations. In this study, we report the design, preclinical development, immunogenicity and anti-viral protective effect in rhesus macaques of the BNT162b2 vaccine candidate. BNT162b2 contains an LNP-formulated nucleoside-modified mRNA that encodes the spike glycoprotein captured in its prefusion conformation. After expression of the BNT162b2 coding sequence in cells, approximately 20% of the spike molecules are in the one-RBD up, two-RBD down state. Immunisation of mice with a single dose of BNT162b2 induced dose level-dependent increases in pseudovirus neutralisation titers. Prime-boost vaccination of rhesus macaques elicited authentic SARS-CoV-2 neutralising geometric mean titers 10.2 to 18.0 times that of a SARS-CoV-2 convalescent human serum panel. BNT162b2 generated strong TH1 type CD4+ and IFN{gamma}+ CD8+ T-cell responses in mice and rhesus macaques. The BNT162b2 vaccine candidate fully protected the lungs of immunised rhesus macaques from infectious SARS-CoV-2 challenge. BNT162b2 is currently being evaluated in a global, pivotal Phase 2/3 trial (NCT04368728).

immunology↗

bioRxiv2020

A valid protective immune response elicited in rhesus macaques by an inactivated vaccine is capable of defending against SARS-CoV-2 infection

With the relatively serious global epidemic outbreak of SARS-CoV-2 infection, public concerns focus on not only clinical therapeutic measures and public quarantine for this disease but also the development of vaccines. The technical design of our SARS-CoV-2 inactivated vaccine provides a viral antigen that enables the exposure of more than one structural protein based upon the antibody composition of COVID-19 patients convalescent serum. This design led to valid immunity with increasing neutralizing antibody titers and a CTL response detected post-immunization of this vaccine by two injections in rhesus macaques. Further, this elicited immunoprotection in macaques enables not only to restrain completely viral replication in tissues of immunized animals, compared to the adjuvant control and those immunized by an RBD peptide vaccine, but also to significantly alleviate inflammatory lesion in lung tissues in histo-pathologic detection, compared to the adjuvant control with developed interstitial pneumonia. The data obtained from these macaques immunized with the inactivated vaccine or RBD peptide vaccine suggest that immunity with a clinically protective effect against SARS-CoV-2 infection should include not only specific neutralizing antibodies but also specific CTL responses against at least the S and N antigens.

immunology↗

bioRxiv2019

Evolution from total variation to nonlinear sparsifying transform for sparse-view CT image reconstruction

Sparse-view CT has been widely studied as an effective strategy for reducing radiation dose to patients. However, the conventional image reconstruction algorithms, such as filtered back-projection method and classical algebraic reconstruction techniques, can no longer be competent in the image reconstruction task of sparse-view CT. A new principle, called compressed sensing (CS), has been therefore developed to provide an effective solution for the ill-posed inverse problem of sparse-view CT image reconstruction. Total variation (TV) minimization, which is most extensively studied among the existing CS techniques, has been recognized as a powerful tool for dealing with this difficult problem in image reconstruction community. However, in recent years, the drawbacks of TV are being increasingly reported, which are appearance of patchy artifacts, depict of incorrect object boundaries, and loss in image textures or smooth intensity changes. These degradations appear especially in reconstructing actual CT images with complicated intensity changes. In order to address these drawbacks, a series of advanced algorithms using nonlinear sparsifying transform (NLST) have been proposed very recently. The NLST-based CS is based on a different framework from the TV, and it achieves an improvement in image quality. Since it is a relatively newly proposed idea, within the scope of our knowledge, there exist few literatures that discusses comprehensively how the image quality improvement occurs in comparison with the conventional TV method. In this study, we investigated the image quality differences between the conventional TV minimization and the NLST-based CS, as well as image quality differences among different kinds of NLST-based CS algorithms in the sparse-view CT image reconstruction. More specifically, image reconstructions of actual CT images of different body parts were carried out to demonstrate the image quality differences.

bioinformatics↗

bioRxiv2019

Machine Learning for Prioritization of Thermostabilizing Mutations for G-protein Coupled Receptors

Although the three-dimensional structures of G-protein-coupled receptors (GPCRs), the largest superfamily of drug targets, have enabled structure-based drug design, there are no structures available for 87% of GPCRs. This is due to the stiff challenge in purifying the inherently flexible GPCRs. Identifying thermostabilized mutant GPCRs via systematic alanine scanning mutations has been a successful strategy in stabilizing GPCRs, but it remains a daunting task for each GPCR. We developed a computational method that combines sequence, structure and dynamics based molecular properties of GPCRs that recapitulate GPCR stability, with four different machine learning methods to predict thermostable mutations ahead of experiments. This method has been trained on thermostability data for 1231 mutants, the largest publicly available dataset. A blind prediction for thermostable mutations of the Complement factor C5a Receptor retrieved 36% of the thermostable mutants in the top 50 prioritized mutants compared to 3% in the first 50 attempts using systematic alanine scanning.\n\nStatement Of SignifiganceG-protein-coupled receptors (GPCRs), the largest superfamily of membrane proteins play a vital role in cellular physiology and are targets to blockbuster drugs. Hence it is imperative to solve the three dimensional structures of GPCRs in various conformational states with different types of ligands bound. To reduce the experimental burden in identifying thermostable GPCR mutants, we report a computational framework using machine learning algorithms trained on thermostability data for 1231 mutants and features calculated from analysis of GPCR sequences, structure and dynamics to predict thermostable mutations ahead of experiments. This work represents a significant advancement in the development, validation and testing of a computational framework that can be extended to other class A GPCRs and helical membrane proteins.

biophysics↗

bioRxiv2019

Structural basis for the transport mechanism of the human glutamine transporter SLC1A5 (ASCT2)

Alanine-serine-cysteine transporter 2 (ASCT2, SLC1A5) is the primary transporter of glutamine in cancer cells and regulates the mTORC1 signaling pathway. The SLC1A5 function involves finely tuned orchestration of two domain movements that include the substrate-binding transport domain and the scaffold domain. Here, we present cryo-EM structures of human SLC1A5 and its complex with the substrate, L-glutamine in an outward-facing conformation. These structures reveal insights into the conformation of the critical ECL2a loop which connects the two domains, thus allowing rigid body movement of the transport domain throughout the transport cycle. Furthermore, the structures provide new insights into substrate recognition, which involves conformational changes in the HP2 loop. A putative cholesterol binding site was observed near the domain interface in the outward-facing state. Comparison with the previously determined inward-facing structure of SCL1A5 provides a basis for a more integrated understanding of substrate recognition and transport mechanism in the SLC1 family. Our structures are likely to aid the development of potent and selective SLC1A5 inhibitors for the treatment of cancer and autoimmune disorders.

biophysics↗

bioRxiv2017

Embryonic lethality in mice lacking Trim59 due to impaired gastrulation development

TRIM family members have been implicated in a variety of biological processes such as differentiation and development. We here found that Trim59 plays a critical role in early embryo development from blastocyst stage to gastrula. There existed delayed development and empty yolk sacs from embryonic day (E) 8.5 in Trim59 -/- embryos. No viable Trim59 -/- embryos were observed beyond E9.5. Trim59 deficiency affected primary germ layer formation at the beginning of gastrulation. In Trim59 -/- embryos at E6.5 and E7.5, the expression of primary germ layer formation associated genes including Brachyury, lefty2, Cer1, Otx2, Wnt3 and BMP4 was reduced. Homozygous mutant embryonic epiblast was contracted and the mesoderm was absent. Trim59 could interact with actin and myosin associated proteins. Trim59 deficiency disturbed F-actin polymerization during inner cell mass differentiation. Trim59 mediated polymerization of F-actin was via WASH K63-linked ubiquitination. Thus, Trim59 may be a critical regulator for early embryo development from blastocyst stage to gastrula through modulating F-actin assembly.

developmental biology↗

bioRxiv2017

Deficiency of Voltage-gated Proton Channel Hv1 Leads to hypoinsulinaemia, hyperglycemia and glucose intolerance in mice

Here, we demonstrate that the voltage-gated proton channel Hv1 represents a regulatory mechanism for insulin secretion of pancreatic islet {beta} cell. In vivo, Hv1-de[fi]cient mice display hyperglycemia and glucose intolerance due to reduced insulin secretion, but normal peripheral insulin sensitivity. In vitro, islets of Hv1-de[fi]cient and heterozygous mice, INS-1 (832/13) cells with siRNA-mediated knockdown of Hv1 exhibit a marked defect in glucose- and K+-induced insulin secretion. Hv1 de[fi]ciency decreases both insulin and proinsulin contents, and limits glucose-induced Ca2+ entry and membrane depolarization. Furthermore, loss of Hv1 increases insulin-containing granular pH and decreases cytosolic pH. In addition, histologic studies show a decrease in {beta} cell mass in islets of Hv1-deficient mice. Collectively, our results indicate that Hv1 supports insulin secretion in the {beta} cell by calcium entry, membrane depolarization and intracellular pH regulation.

cell biology↗