Biology subjects

Mao, C.

Publications and source records attributed to Mao, C..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Characterization of human XPD helicase activity with Single Molecule Magnetic Tweezers

XPD helicase is a DNA unwinding enzyme involved in multiple cellular processes. As part of TFIIH, XPD opens a repair bubble in DNA for access by proteins in the nucleotide excision repair pathway. XPD uses the energy from ATP hydrolysis to translocate in the 5 to 3 direction on one strand of duplex DNA, displacing the opposite strand in the process. We used magnetic tweezers assays to measure the double-stranded DNA (dsDNA) unwinding and single-stranded DNA (ssDNA) translocation activities of human XPD by itself. In our experimental setup, hXPD exhibits low unwinding processivity of ~14 bp and slow overall unwinding rate of ~0.3 bp/s. Individual unwinding and translocation events were composed of fast and slow runs and pauses. Analysis of these events gave similar mean run sizes and rates for unwinding and translocation, suggesting that unwinding is a reflection of translocation. The analysis also revealed that hXPD spent similar time stalling and unwinding. hXPD translocated on ssDNA at a similar overall rate as that of unwinding, pointing to an active helicase. However, we observed modest effects of DNA sequence on stalling and unwinding initiation position. Considering the slow unwinding rate, high probability of base pair separation at the ssDNA/dsDNA fork, and the observed DNA sequence dependences, we propose that hXPD is most likely a partially active helicase. Our results provide detailed information on the basal activity of hXPD which enhances our mechanistic understanding of hXPD activity. SIGNIFICANCEHuman XPD helicase is a major component of the general transcription factor TFIIH. TFIIH is essential in both transcription and nucleotide excision repair. Mutations in hXPD are associated with cancers and autosomal recessive disorders. Here we directly measured the dsDNA unwinding and ssDNA translocation of human XPD helicase by itself. Our measurements provide detailed information on the basal activity of human XPD, which enhance our mechanistic understanding of the activity of XPD in the cell, provide a basis for better understanding of the clinical phenotypes, and aid in drug design targeting hXPD related diseases.

biophysics↗

bioRxiv2023

Protein Sequence Design by Entropy-based Iterative Refinement

Inverse Protein Folding (IPF) is an important task of protein design, which aims to design sequences compatible with a given backbone structure. Despite the prosperous development of algorithms for this task, existing methods tend to leverage limited and noisy residue environment when generating sequences. In this paper, we develop an iterative sequence refinement pipeline, which can refine the sequence generated by existing sequence design models. It selects and retains reliable predictions based on the models confidence in predicted distributions, and decodes the residue type based on a partially visible environment. The proposed scheme can consistently improve the performance of a number of IPF models on several sequence design benchmarks, and increase sequence recovery of the SOTA model by up to 10%. We finally show that the proposed model can be applied to redesign Transposon-associated transposase B. 8 variants exhibit improved gene editing activity among the 20 variants we proposed. Our code and a demo of the refinement pipeline are provided in the online colab.

bioinformatics↗

bioRxiv2022

Structural Genomics of the Human Dopamine Receptor System

The dopamine system, including five dopamine receptors (D1R to D5R), plays essential roles in the central nervous system (CNS) and ligands that activate dopamine receptors have been used to treat many neuropsychiatric disorders, including Parkinsons Disease (PD) and schizophrenia. Here, we report five cryo-EM structures of all subtypes of human dopamine receptors in complex with G-protein and bound to the pan agonist, Rotigotine, which is used to treat PD and restless legs syndrome. The structures reveal the basis of Rotigotine binding modes to different dopamine receptors. Structural analysis together with functional assays illuminate determinants of ligand polypharmacology and selectivity. The structures also uncover the mechanisms of the dopamine receptor activation, unique structural features among the five receptor subtypes, and the basis of G-protein coupling specificity. Our works provide a comprehensive set of structural templates for the rational design of specific ligands to treat CNS diseases targeting the dopaminergic system.

biochemistry↗

bioRxiv2022

Universal mechanism of hormone and allosteric agonist mediated activation of glycoprotein hormone receptors as revealed by structures of follicle stimulating hormone receptor

Follicle stimulating hormone (FSH) is an essential glycoprotein hormone for human reproduction, which functions are mediated by a G protein-coupled receptor, FSHR. Aberrant FSH-FSHR signaling causes infertility and ovarian hyperstimulation syndrome. Here we report cryo-EM structures of FSHR in both inactive and active states, with the active structure bound to FSH and an allosteric agonist compound 21f. The structures of FSHR are similar to other glycoprotein hormone receptors, highlighting a conserved activation mechanism of hormone-induced receptor activation. Compound 21f formed extensive interactions with the TMD to directly activate FSHR. Importantly, the unique residue H6157.42 in FSHR plays an essential role in determining FSHR selectivity for various allosteric agonists. Together, our structures provide a molecular basis of FSH and small allosteric agonist-mediated FSHR activation, which could inspire the design of FSHR-targeted drugs for the treatment of infertility and controlled ovarian stimulation for in vitro fertilization.

cell biology↗

bioRxiv2022

Mechanical Deformation Behaviors and Structural Properties of Ligated DNA Crystals

DNA self-assembly has emerged as a powerful strategy for constructing complex nanostructures. While the mechanics of individual DNA strands have been studied extensively, the deformation behaviors and structural properties of self-assembled architectures are not well understood. This is partly due to the small dimensions and limited experimental methods available. DNA crystals are macroscopic crystalline structures assembled from nanoscale motifs via sticky-end association. The large DNA constructs may thus be an ideal platform to study structural mechanics. Here we have investigated the fundamental mechanical properties and behaviors of ligated DNA crystals made of tensegrity triangular motifs. We performed coarse-grained molecular dynamics simulations and confirmed the results with nanoindentation experiments using atomic force microscopy. We observed various deformation modes including un-tension, linear elasticity, duplex dissociation, and single-stranded component stretch. We found that the mechanical properties of a DNA architecture are correlated with those of its components, however the structure shows complex behaviors which may not be predicted by components alone.

biophysics↗

bioRxiv2022

Lipidomic change in Aedes aegypti female following blood feeding reveals similar changes in functionally related lipids

We conducted a lipidomic analysis of the whole body of female Aedes aegypti mosquitoes at different time points over the course of feeding and reproduction. There were temporal biphasic increases of more than 80% of lipids identified at the time of feeding and from 16 h to 30 h post blood meal. During these two increases, the abundance of many lipids dropped while body weight remained stable, probably reflecting blood lipid digestion and the synthesis of vitellogenin in this period. A concerted temporal pattern was particularly strong at the second peak for membrane and signalling lipids such as PE, PI, CL, HexCer and LPA. Lyso-glycerophospholipids showed three distinct change patterns which are functionally related: LPE and LPC, which are membrane lipids, showed little change; LPA, a signalling lipid, showed a dramatic increase from 16 to 30 h PBM; LPI, a bioactive lipid, and both LPG and LPS which are bacterial membrane lipids, showed one significant increase from the time of feeding to 16 hours post blood meal. The result of our study on the anautogenous insect Ae. aegypti point to specific lipids likely to be important in the reproductive process with a role in the formation and growth of ovarian follicles.

biochemistry↗

bioRxiv2022

Anaerobic degradation of acid red 73 by obligately aerobic Aspergillus tabacinus LZ-M though a self-redox mechanism

Fungi are potential biological resources for refractory organics degradation, but their anaerobic degradation of azo dyes are rarely reported. In this study, a fungus Aspergillus tabacinus LZ-M was isolated grown aerobically and degraded acid red 73 (AR73) with a decolorization rate of 90.28% in 5 days at 400 mg/L of concentration anaerobically. Metabolic pathway showed that AR73 was reduced into 2-hydroxynaphthalene and aniline then mineralized into CO2. The anaerobic self-redox process revealed electrons generated in carbon oxidation and transferred to -C-N= and -N=N, resulting in complete mineralization of AR73 in strain LZ-M. Data of transcriptome analysis showed that the benzene compounds produced from AR73 by declorizing reductase entered the catechol pathway and glycolysis process to mineralize. Enzymes involved in aromatics degradation, glycolysis processes, cytochrome C and quinone oxidoreductases were up-regulate, but the key reductase responsible to cleave AR73 to phenylhydrazine was not found. A novel enzyme Ord95 containing a glutamate S-transferase domain was identified in the unknown genes as a reductase which cleaving -C-N= in AR73 using NADH as electron donor, and three arginines key active sites. These observations reveal a new degradation mechanism of AR73 in strain LZ-M which would be potential candidate for treatment of azo dyes wastewater. Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

microbiology↗

bioRxiv2022

Unique molecular signatures sustained in circulating monocytes and regulatory T cells in Convalescent COVID-19 patients

Over two years into the COVID-19 pandemic, the human immune response to SARS-CoV-2 during the active disease phase has been extensively studied. However, the long-term impact after recovery, which is critical to advance our understanding SARS-CoV-2 and COVID-19-associated long-term complications, remains largely unknown. Herein, we characterized multi-omic single-cell profiles of circulating immune cells in the peripheral blood of 100 patients, including covenlesent COVID-19 and sero-negative controls. The reduced frequencies of both short-lived monocytes and long-lived regulatory T (Treg) cells are significantly associated with the patients recovered from severe COVID-19. Consistently, sc-RNA seq analysis reveals seven heterogeneous clusters of monocytes (M0-M6) and ten Treg clusters (T0-T9) featuring distinct molecular signatures and associated with COVID-19 severity. Asymptomatic patients contain the most abundant clusters of monocyte and Treg expressing high CD74 or IFN-responsive genes. In contrast, the patients recovered from a severe disease have shown two dominant inflammatory monocyte clusters with S100 family genes: S100A8 & A9 with high HLA-I whereas S100A4 & A6 with high HLA-II genes, a specific non-classical monocyte cluster with distinct IFITM family genes, and a unique TGF-{beta} high Treg Cluster. The outpatients and seronegative controls share most of the monocyte and Treg clusters patterns with high expression of HLA genes. Surprisingly, while presumably short-ived monocytes appear to have sustained alterations over 4 months, the decreased frequencies of long-lived Tregs (high HLA-DRA and S100A6) in the outpatients restore over the tested convalescent time (>= 4 months). Collectively, our study identifies sustained and dynamically altered monocytes and Treg clusters with distinct molecular signatures after recovery, associated with COVID-19 severity.

immunology↗

bioRxiv2021

Transcription factor induction of vascular blood stem cell niches in vivo

The hematopoietic niche is a supportive microenvironment comprised of distinct cell types, including specialized vascular endothelial cells that directly interact with hematopoietic stem and progenitor cells (HSPCs). The molecular factors that specify niche endothelial cells and orchestrate HSPC homeostasis remain largely unknown. Using multi-dimensional gene expression and chromatin accessibility analyses, we define a conserved gene expression signature and cis-regulatory landscape unique to sinusoidal endothelial cells in the HSPC niche. Using enhancer mutagenesis and transcription factor overexpression, we elucidate a transcriptional code involving members of the Ets, Sox and Nuclear Hormone Receptor families that is sufficient to induce ectopic niche endothelial cells that associate with mesenchymal stromal cells and support the recruitment, maintenance and division of HSPCs in vivo. These studies set forth an approach for generating synthetic HSPC niches, in vitro or in vivo, and for effective therapies to modulate the endogenous niche.

developmental biology↗

bioRxiv2021

Bicolor angelfish (Centropyge bicolor) provides the first chromosome-level genome of the Pomacanthidae family

The Bicolor Angelfish, Centropyge bicolor, is a tropical coral reef fish. It is named for its striking two-color body. However, a lack of high-quality genomic data means little is known about the genome of this species. Here, we present a chromosome-level C. bicolor genome constructed using Hi-C data. The assembled genome is 650 Mbp in size, with a scaffold N50 value of 4.4 Mbp, and a contig N50 value of 114 Kbp. Protein-coding genes numbering 21,774 were annotated. Our analysis will help others to choose the most appropriate de novo genome sequencing strategy based on resources and target applications. To the best of our knowledge, this is the first chromosome-level genome for the Pomacanthidae family, which might contribute to further studies exploring coral reef fish evolution, diversity and conservation.

genomics↗

bioRxiv2021

Constitutive signal bias mediated by the human GHRHR splice variant 1

Alternative splicing of G protein-coupled receptors has been observed, but their functions are largely unknown. Here, we report that a splice variant (SV1) of the human growth hormone releasing hormone receptor (GHRHR) is capable of transducing biased signal. Differing only at the receptor N terminus, GHRHR predominantly activates Gs while SV1 selectively couples to {beta}-arrestins. Based on the cryo-electron microscopy structures of SV1 in the apo state or in complex with the Gs protein, molecular dynamics simulations reveal that the N termini of GHRHR and SV1 differentiate the downstream signaling pathways, Gs vs. {beta}-arrestins. Suggested by mutagenesis and functional studies, it appears that GHRH-elicited signal bias towards {beta}-arrestin recruitment is constitutively mediated by SV1. The level of SV1 expression in prostate cancer cells is also positively correlated with ERK1/2 phosphorylation but negatively correlated with cAMP response. Our findings imply that constitutive signal bias may be a mechanism that ensures cancer cell proliferation. Significance StatementThe mechanism of functional changes induced by alternative splicing of GHRHR is largely unknown. Here, we demonstrate that GHRH-elicited signal bias towards {beta}-arrestin recruitment is constitutively mediated by SV1. The cryo-electron microscopy structures of SV1 and molecular dynamics simulations reveal the different functionalities between GHRHR and SV1 at the near-atomic level, i.e., the N termini of GHRHR and SV1 differentiate the downstream signaling pathways, Gs vs. {beta}-arrestins. Our findings provide valuable insights into functional diversity of class B1 GPCRs which may aid in the design of better therapeutic agents against certain cancers.

molecular biology↗

bioRxiv2021

Structures of full-length glycoprotein hormone receptor signaling complexes

Luteinizing hormone (LH) and chorionic gonadotropin (CG) are members of the glycoprotein hormone family essential to human reproduction and are important therapeutic drugs. They activate the same G protein-coupled receptor, LHCGR, by binding to the large extracellular domain (ECD). Here we report four cryo-EM structures of LHCGR, two wildtype receptor structures in the inactive and active states, and two constitutively active mutated receptor structures. The active structures are bound to CG and Gs heterotrimer, with one of the structure also containing the allosteric agonist, Org43553. The structures reveal a distinct push and pull" mechanism of receptor activation, in which the ECD is pushed by the bound hormone and pulled by the extended hinge loop next to the transmembrane domain (TMD). A highly conserved 10-residue fragment (P10) from the hinge C-terminal loop at the ECD-TMD interface functions as a tethered agonist to induce conformational changes in TMD and G-protein coupling. Org43553 binds to a TMD pocket and interacts directly with P10 that further stabilizes the receptor in the active conformation. Together, these structures provide a common model for understanding glycoprotein hormone signal transduction and dysfunction, and inspire the search for clinically suitable small molecular compounds to treat endocrine diseases.

molecular biology↗

bioRxiv2021

Based on the identification of tumor antigens and immune subtypes in renal clear cell carcinoma for mRNA vaccine development

BackgroundCancer vaccine based on mRNA is considered as a promising strategy and has become a new hot spot in cancer immunotherapy. However, its application to KIRC is not clear. A growing body of research has shown that immunotyping can reflect the comprehensive immune status and immune microenvironment of tumor, which is closely related to treatment response and vaccination potential. The aim of this study was to identify the potential antigens of KIRC for the development of anti-KIRC mRNA vaccines, and to further differentiate the immune subtypes of KIRC to construct an immune landscape for the selection of appropriate patients for vaccination. MethodsGene expression profiles and corresponding clinical information of 265 KIRC patients and RNA-seq data of 539 KIRC patients were retrieved from were collected from GEO and TCGA. cBioPortal was used to visualize and compare genetic alterations, while GEPIA2 was used to calculate the prognostic index of selected antigens. The relationship between the infiltration of antigen presenting cells and the expression of the identified antigen was visualized with TIMER, and consensus clustering analysis was used to determine the immune subtypes. Finally, the immune landscape of KIRC is visualized through the dimensionality reduction analysis based on graph learning. ResultsTwo tumor antigens associated with prognostic and antigen-presenting infiltrating cells were identified in KIRC, including LRP2, and DOCK8. KIRC patients were classified into six immune subtypes based on different molecular, cellular, and clinical characteristics. Patients with IS5 and IS6 tumors had an immune "hot" and immunosuppressive phenotype, which was associated with better survival compared to other subtypes, whereas patients with IS1-4 tumors had an immune "cold" phenotype, which was associated with a higher tumor mutation burden. In addition, the expression of immune checkpoints and immunogenic cell death modulators differed significantly in different immunosubtypes of tumors. Finally, the immune landscape of KIRC shows a high degree of heterogeneity across patients. ConclusionsLRP2 and FEM2 are potential KIRC antigens for mRNA vaccine development, and patients with immune subtypes IS1-4 are suitable for vaccination.

bioinformatics↗

bioRxiv2021

Structural insights into ligand recognition and activation of the melanocortin-4 receptor

Melanocortin-4 receptor (MC4R) plays a central role in the regulation of energy homeostasis. Its high sequence similarity to other MC receptor family members, low agonist selectivity and the lack of structural information concerning receptor activation have hampered the development of MC4R-seletive therapeutics to treat obesity. Here, we report four high-resolution structures of full-length MC4R in complex with the heterotrimeric Gs protein stimulated by the endogenous peptide -MSH, FDA-approved drugs afamelanotide (Scenesse) and bremelanotide (Vyleesi), and selective small-molecule ligand THIQ, respectively. Together with pharmacological studies, our results reveal the conserved binding mode of peptidic agonists, the distinctive molecular details of small-molecule agonist recognition underlying receptor subtype selectivity, and distinct activation mechanism for MC4R, thereby offering new insights into G protein coupling. Our work may facilitate the discovery of selective therapeutic agents targeting MC4R.

molecular biology↗

bioRxiv2020

Circulating ACE2-expressing Exosomes Block SARS-CoV-2 Virus Infection as an Innate Antiviral Mechanism

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the coronavirus disease 2019 (COVID-19) with innate and adaptive immune response triggered in such patients by viral antigens. Both convalescent plasma and engineered high affinity human monoclonal antibodies have shown therapeutic potential to treat COVID-19. Whether additional antiviral soluble factors exist in peripheral blood remain understudied. Herein, we detected circulating exosomes that express the SARS-CoV-2 viral entry receptor angiotensin-converting enzyme 2 (ACE2) in plasma of both healthy donors and convalescent COVID-19 patients. We demonstrated that exosomal ACE2 competes with cellular ACE2 for neutralization of SARS-CoV-2 infection. ACE2-expressing (ACE2+) exosomes blocked the binding of the viral spike (S) protein RBD to ACE2+ cells in a dose dependent manner, which was 400- to 700-fold more potent than that of vesicle-free recombinant human ACE2 extracellular domain protein (rhACE2). As a consequence, exosomal ACE2 prevented SARS-CoV-2 pseudotype virus tethering and infection of human host cells at a 50-150 fold higher efficacy than rhACE2. A similar antiviral activity of exosomal ACE2 was further demonstrated to block wild-type live SARS-CoV-2 infection. Of note, depletion of ACE2+ exosomes from COVID-19 patient plasma impaired the ability to block SARS-CoV-2 RBD binding to host cells. Our data demonstrate that ACE2+ exosomes can serve as a decoy therapeutic and a possible innate antiviral mechanism to block SARS-CoV-2 infection.

immunology↗

bioRxiv2020

Gait-symmetry-based human-in-the-loop optimization for unilateral transtibial amputees with robotic prostheses

Gait asymmetry due to the loss of unilateral limb increases the risk of injury or progressive joint degeneration. The development of wearable robotic devices paves a way to improve gait symmetry of unilateral amputees. Moreover, the state-of-the-art studies on human-in-the-loop optimization strategies through decreasing the metabolic cost as the optimization task, have met several challenges, e.g. too long period of optimization and the optimization feasibility for unilateral amputees who have the deficit of gait symmetry. Here, in this paper, we proposed gait-symmetry-based human-in-the-loop optimization method to decrease the risk of injury or progressive joint degeneration for unilateral transtibial amputees. The experimental results (N = 3 unilateral transtibial subjects) demonstrate that only average 9.0{+/-}4.1min of convergence was taken. Compared to gait symmetry while wearing prosthetics, after optimization, the gait symmetry indicator value of the subjects wearing the robotic prostheses was improved by 21.0% and meanwhile the net metabolic energy consumption value was reduced by 9.2%. Also, this paper explores the rationality of gait indicators and what kind of gait indicators are the optimization target. These results suggest that gait-symmetry-based human-in-the-loop strategy could pave a practical way to improve gait symmetry by accompanying the reduction of metabolic cost, and thus to decrease the risk of joint injury for the unilateral amputees.

bioengineering↗

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

VST Family Proteins are Regulators of Root System Architecture in Rice and Arabidopsis

Root System Architecture (RSA) is a key factor in the efficiency of nutrient capture and water uptake in plants. Understanding the genetic control of RSA will be useful in minimizing fertilizer and water usage in agricultural cropping systems. Using a hydroponic screen and a gel-based imaging system we identified a rice gene, OsVST1, which plays a key role in controlling RSA. This gene encodes a homolog of the Arabidopsis VAP-RELATED SUPPRESSORS OF TMM (VSTs), a class of proteins that promote signaling in stomata by mediating plasma membrane-endoplasmic reticulum contacts. OsVST1 mutants have shorter primary roots, decreased root meristem size, and a more compact root system architecture. We show that the Arabidopsis VST triple mutants have similar phenotypes, with reduced primary root growth and smaller root meristems. Expression of OsVST1 largely complements the short root length and reduced plant height in the Arabidopsis triple mutant, supporting conservation of function between rice and Arabidopsis VST proteins. In a field trial, mutations in OsVST1 do not adversely affect grain yield, suggesting that modulation of this gene could be used as a way to optimize RSA without an inherent yield penalty.

plant biology↗