Biology subjects

Xiao, P.

Publications and source records attributed to Xiao, P..

26 records · Page 2Linked to original sources

bioRxiv2020

Structural basis of bile acid receptor activation and Gs coupling

G protein-coupled bile acid receptor (GPBAR) is a membrane receptor that senses bile acids to regulate diverse functions through Gs activation. Here, we report the cryo-EM structures of GPBAR-Gs complexes stabilized by either high-affinity P395 or the semisynthesized bile acid derivative INT-777 at 3-[A] resolution. These structures revealed a large oval-shaped ligand pocket with several sporadic polar groups to accommodate the amphipathic cholic core of bile acids. A fingerprint of key residues recognizing diverse bile acids in the orthosteric site, a putative second bile acid binding site with allosteric properties and structural features contributing to bias property were identified through structural analysis and mutagenesis studies. Moreover, structural comparison of GPBAR with other GPCRs uncovered an atypical mode of receptor activation and G-protein- coupling, featuring a different set of key residues connecting the ligand binding pocket to the Gs coupling site, and a specific interaction motif localized in intracellular loop 3. Overall, our study not only provides unique structural features of GPBAR in bile acid recognition, allosteric effects and biased signaling, but also suggests that distinct allosteric connecting mechanisms between the ligand binding pocket and the G protein binding site exist in the GPCR superfamily.

biochemistry↗

bioRxiv2020

Single-cell Analysis of ACE2 Expression in Human Kidneys and Bladders Reveals a Potential Route of 2019-nCoV Infection

Since December 2019, a novel coronavirus named 2019 coronavirus (2019-nCoV) has emerged in Wuhan of China and spread to several countries worldwide within just one month. Apart from fever and respiratory complications, acute kidney injury has been observed in some patients with 2019-nCoV. In a short period of time, angiotensin converting enzyme II (ACE2), have been proposed to serve as the receptor for the entry of 2019-nCoV, which is the same for severe acute respiratory syndrome coronavirus (SARS). To investigate the possible cause of kidney damage in 2019-nCoV patients, we used both published kidney and bladder cell atlas data and an independent unpublished kidney single cell RNA-Seq data generated in-house to evaluate ACE2 gene expressions in all cell types in healthy kidneys and bladders. Our results showed the enriched expression of all subtypes of proximal tubule cells of kidney and low but detectable levels of expression in bladder epithelial cells. These results indicated the urinary system is a potential route for 2019-nCoV infection, along with the respiratory system and digestion system. Our findings suggested the kidney abnormalities of SARS and 2019-nCoV patients may be due to proximal tubule cells damage and subsequent systematic inflammatory response induced kidney injury. Beyond that, laboratory tests of viruses and related indicators in urine may be needed in some special patients of 2019-nCoV.

genomics↗

bioRxiv2019

The COP9 Signalosome Suppresses Cardiomyocyte Necroptosis

BackgroundLoss of cardiomyocyte (CMs) due to apoptosis and regulated necrosis contributes to heart failure. However, the molecular mechanisms governing regulated CM necrosis remain obscure. The COP9 signalosome (CSN) formed by 8 unique protein subunits (COPS1 through COPS8) functions to deneddylate Cullin-RING ligases (CRLs), thereby regulating the functioning of the CRLs. Mice with CM-restricted knockout of Cops8 (Cops8-cko) die prematurely, following reduced myocardial performance of autophagy and the ubiquitin-proteasome system (UPS) as well as massive CM necrosis. This study was aimed to determine the nature and underlying mechanisms of the CM necrosis in Cops8-cko mice. MethodsWe examined myocardial expression and activities of key proteins that reflect the status of the RIPK1-RIPK3 pathway, redox, and caspase 8 in Cops8-cko mice. Moreover, we used in vivo CM uptake of Evans blue dye (EBD) as an indicator of necrosis and performed Kaplan-Meier survival analyses to test whether treatment with a RIPK1 kinase inhibitor (necrostatin-1) or an antioxidant (N-acetyl-L-cysteine), global knockout of the RIPK3 or the Ppif gene, CM-restricted knockout of the Nrf2 gene, or cardiac HMOX1 overexpression could rescue the Cops8-cko phenotype. ResultsCompared with littermate control mice, myocardial protein levels of RIPK1, RIPK3, MLKL, the RIPK1-bound RIPK3, protein carbonyls, full-length caspase 8, Nrf2, Ser40-phosphorylated Nrf2 and BCL2, as well as histochemical staining of superoxide anions were significantly increased but the cleaved caspase 8 and the overall caspase 8 activity were markedly decreased in Cops8-cko mice, indicating that the RIPK1-RIPK3 and the Nrf2 pathways are activated and caspase 8 activation is suppressed by Cops8-cko. Continuous necrostatin-1 infusion initiated at 2 weeks of age nearly completely blocked CM necrosis at 3 weeks and markedly delayed premature death of Cops8-cko mice. RIPK3 haploinsufficiency or cardiac-specific Nrf2 heterozygous knockout discernably attenuated CM necrosis and/or delayed mouse premature death; conversely, Ppif knockout, N-acetyl-L-cysteine treatment, and cardiac overexpression of HMOX1 exacerbated CM necrosis and mouse premature death. ConclusionsCardiac Cops8/CSN malfunction causes RIPK1-RIPK3 mediated CM necroptosis in mice; sustained Nrf2 activation and reductive stress pivot cardiomyocytes to necroptosis when autophagy and the UPS are impaired; and the CSN plays an indispensable role in suppressing CM necroptosis.

cell biology↗

bioRxiv2019

Developing a DeSipher method for conformational change detection of membrane receptor complex at residue resolution

While one dimensional 1H nuclear magnetic resonance (1D 1H-NMR) spectroscopy is one of the most important and convenient method for measuring conformation change in biomacromolecules, characterization of protein dynamics in large membrane protein complexes by 1D 1H-NMR remains challenging, due to the difficulty of spectra assignment, low signal-to-noise ratio (S/N) and the need for large amount of protein. Here we report the site-specific incorporation of 4-trimethylsilyl phenylalanine (TMSiPhe) into proteins, through genetic code expansion in Escherichia coli cells, and the measurement of multiple conformational states in membrane protein complex by 1D 1H-NMR. The unique up-field 1H-NMR chemical shift of TMSiPhe, highly efficient and specific incorporation of TMSiPhe enabled facile assignment of the TMSiPhe 1H-NMR signal, and characterization of multiple conformational state in a 150 kilodalton (kD) membrane protein complex, using only 5 M of protein and 20 min spectra accumulation time. This highly efficient and convenient methods should be broadly applicable for the investigation of dynamic conformation change of protein complexes.

biochemistry↗

bioRxiv2019

PTP-MEG2 regulates quantal size and fusion pore opening through two distinct structural bases and substrates

Tyrosine phosphorylation of secretion machinery proteins is a crucial regulatory mechanism for exocytosis. However, the participation of protein tyrosine phosphatases (PTPs) in different exocytosis stages has not been defined. Here we demonstrated that PTP-MEG2 controls multiple steps of catecholamine secretion. Biochemical and crystallographic analyses revealed key residues that the interactions between govern the PTP-MEG2 and NSF-pY83 site, specify PTP-MEG2 substrate selectivity and modulate the fusion of catecholamine-containing vesicles. Unexpectedly, delineation of PTP-MEG2 mutants along with the NSF binding interface revealed that PTP-MEG2 controls the fusion pore opening through non-NSF dependent mechanisms. Utilizing bioinformatics search and biochemical and electrochemical screening approaches, we discovered that PTP-MEG2 regulates the opening and extension of the fusion pore by dephosphorylating the DYNAMIN2-pY125 and MUNC18-1-pY145 site. Further structural and biochemical analysis confirmed the interaction of PTP-MEG2 with MUNC18-1-pY145 or DYNAMIN2-pY125 through a distinct structural basis compared with that of the NSF-pY83 site. Our studies extended mechanistic insights in complex exocytosis processes. HIGHLIGHTSPTP-MEG2 regulates multiple steps of exocytosis. A crystal structure of the PTP-MEG2/phosphor-NSF-pY83 segment was obtained. Functional delineation of the PTP-MEG2/NSF interface led to the discovery of new PTP-MEG2 substrates. PTP-MEG2 regulates fusion pore opening and extension through the DYNAMIN2-pY125 site and MUNC18-1 pY145 site. The distinct structural basis of the recognition of substrates by PTP-MEG2 allows selective inhibitor design.

cell biology↗

bioRxiv2019

Extensive adaptive immune response of AAVs and Cas proteins in non-human primates

The CRISPR-mediated Cas system is the most widely used tool in gene editing and gene therapy for its convenience and efficiency. Delivery of the CRISPR system by adeno-associated viruses (AAVs) is currently the most promising approach to gene therapy. However, pre-existing adaptive immune responses against CRISPR nuclease (PAIR-C) and AAVs has been found in human serum, indicating that immune response is a problem that cannot be ignored, especially for in vivo gene correction. Non-human primates (NHPs) share many genetic and physiological traits with human, and are considered as the bridge for translational medicine. However, whether NHPs have same PAIR-C status with human is still unknown. Here, macaques (rhesus and cynomolgus), including normal housed and CRISPR-SpCas9 or TALENs edited individuals, were used to detect PAIR-C which covered SaCas9, SpCas9, AsCas12a and LbCas12a. Dogs and mice were also detected to expand the range of species. In addition, pre-existing adaptive antibodies to AAV8 and AAV9 were performed against macaques of different ages. The results showed that adaptive immunity was pre-existing in the macaques regardless of Cas proteins and AAVs. These findings indicate that the pre-existing adaptive immune of AAV-delivered CRISPR construction and correction system should be concerned for in vivo experiments.

immunology↗

bioRxiv2017

Acute and chronic toxicity assessment of benzylpenicillin G residue in cooked meat

The current level of penicillin use and its persisting residues in livestock is potentially concerning; the toxicity of penicillin residues in heat-treated animal food products (HAFP) is yet to be elucidated. In this study, the acute and chronic toxicity of benzylpenicillin G (BPG) residues in HAFP was investigated in a mouse model. The calculated LD50 of BPG heated to cooking temperature (BPHCT) was 933.04 mg kg-1 [b.w.] intraperitoneally corresponding to 3.75 times lower than its prototype. Mice fed on the experimental diet containing heat-treated beef with high BPG levels for 6 months displayed a reduction in body weight and altered serum values indicating for liver and renal function. Further, the organ ratios of intestinal and spleen were increased. Histopathological changes were observed in the liver, lung and parenchyma testis tissue. BPHCT residue induced sperm aberration and micronucleated polychromatic erythrocytes formation. Present results indicate that prolonged exposure of BPHCT at higher levels of residue might have an impact on public health. Importantly the toxic concentrations of BPHCT are relatively high compared with levels that would result from the degradation of antibiotic residues in meat from animals that have received a therapeutic dose of BPG.

pharmacology and toxicology↗

bioRxiv2017

Metabolomic profiling reveals effects of marein on energy metabolism in HepG2 cells

Previous studies have suggested that Coreopsis tinctoria improves insulin resistance in rats fed with high-fat diet. But little is known about the antidiabetic effects of marein which is the main component of C. tinctoria. This study investigated the effects of ethyl acetate extract of C. tinctoria (AC) on insulin resistance (IR) in rats fed a high-fat diet. High glucose and fat conditions cause a significant increase in blood glucose, insulin, serum TC,TG and LDL-C, leading to an abnormal IR in rats. However, treatment with AC protects against HFD-induced IR by improving fasting serum glucose and lipid homeostasis. High glucose conditions cause a significant decrease in glycogen synthesis and increases PEPCK and G6Pase protein levels and Krebs-cycle-related enzymes levels, leading to an abnormal metabolic state in HepG2 Cells. However, treatment with Marein improves IR by increasing glucose uptake and glycogen synthesis and by downregulating PEPCK and G6Pase protein levels. The statistical analysis of HPLC/MS data demonstrates that Marein restores the normal metabolic state. The results show that AC ameliorates IR in rats and Marein has the potential effect in improving IR by ameliorating glucose metabolic disorders.\n\nAbbreviations

pharmacology and toxicology↗