Biology subjects

Zou, X.

Publications and source records attributed to Zou, X..

At least 73 records · Page 4Linked to original sources

bioRxiv2021

MAL suppresses OSCC tumorigenesis by maintaining epithelial cell differentiation

Oral squamous cell carcinoma (OSCC) is widely recognized as an optimal model for precise medicine guided molecular biomarkers of cancer, however, few clinical practices were applied till now. Based on the data from our own studies and published papers, it was found that the expression of MAL was significantly decreased in epithelial cancer as compared with normal tissues, and exhibited a opposite association with pathological grade. To study the molecular events related to deficiency of MAL during carcinogenesis, occurrence and development, a Mal knockout mouse model was constructed and consistently reproduced and bred. The Mal knockout mice are highly vulnerable to tumor induction by carcinogen of 4NQO, evidenced by their extremely earlier carcinogenesis, higher incidence, and more aggressive growth. Analysis of scRNA-seq data indicated that Mal knockout mice lost the ability in maintaining epithelial cell differentiation and get more prone to carcinogen with a remarkably higher incidence of epithelial malignancy. Further analyses identified putative co-functional genes of MAL, including DSG1, AQP3 and S100A8, which are key factors in maintaining epithelial cell differentiation. To conclude, the current study exhibits the clinical significance and explains the tumor suppressing function of MAL. The results also suggest the potential of MAL and its co-functional genes being biomarkers for designing the prevention and/or differentiation therapy strategies in OSCC. SignificanceMAL is found to be strongly opposite with tumor pathological grade from clinical and in vivo studies in OSCC. We propose MAL and its co-functional genes, including DSG1, AQP3 and S100A8, as key factors in maintaining epithelial cell differentiation and are valuable targets for designing prevention and differentiation therapy strategies in OSCC.

cell biology↗

bioRxiv2021

Screening and selection of 21 novel microhaplotype markers for ancestry inference in ten Chinese Subpopulations

Genetic findings suggested that ethnolinguistically diverse populations in China harbor differentiated genetic structure and complex evolutionary admixture history, which provide the genetic basis and theoretical foundation for forensic biogeographical ancestry inference (BGAI). Forensic assays for BGAI among intracontinental eastern Eurasians were previously conducted mainly based on the SNPs or InDels. Microhaplotypes, as a set of closely linked SNPs within 200 base pairs, possess the advantages of both STR and SNP and have great potential in forensic ancestry inference. However, the developed forensic assay based on the ancestry informative microhaplotypes in the BGAI remained to be comprehensively explored, especially in China with enriching genetic diversity. Here, we described a new BGAI panel based on 21 novel identified ancestry informative microhaplotypes that focused on dissected finer-scale ancestry composition of Chinese populations. We initially screened all possible microhaplotypes with high Fst values among five East Asian populations and finally employed 21 candidate microhaplotypes in two multiplex SNaPshot assays. Forensic amplification efficiency and statistically/physically phased haplotypes of the 21 microhaplotypes were validated using both SNaPshot and massively parallel sequencing (MPS) platforms. Followingly, we validated the efficiency of these microhaplotypes for BGAI in 764 individuals from ten Chinese populations. Fine-scale ancestry source and ancestry proportion estimated by the principal component analysis (PCA), multidimensional scaling (MDS), phylogenetic tree and model-based STRUCTURE among worldwide populations and East Asians showed that our customized panel could provide a higher discrimination resolution in both continental population stratification and East Asian regional substructure. East Asian populations could be further classified into linguistically/geographically different intracontinental subpopulations (Tibeto-Burman, Tai-Kadai and others). Finally, we obtained a higher estimated accuracy using training and tested datasets in the microhaplotype-based panel than traditional SNP-based panels. Generally, the above results demonstrated that this microhaplotype panel was robust and suitable for forensic BGAI in Chinese populations, which not only provided a high discriminatory power for continental populations but also discriminated East Asians into linguistically restricted subpopulations.

genetics↗

bioRxiv2021

Spatially-resolved transcriptomics analyses of invasive fronts in solid tumors

Solid tumors are complex ecosystems, and heterogeneity is the major challenge for overcoming tumor relapse and metastasis. Uncovering the spatial heterogeneity of cell types and functional states in tumors is essential for developing effective treatment, especially in invasive fronts of tumor, the most active region for tumor cells infiltration and invasion. We firstly used SpaTial Enhanced REsolution Omics-sequencing (Stereo-seq) with a nanoscale resolution to characterize the tumor microenvironment of intrahepatic cholangiocarcinoma (ICC). Enrichment of distinctive immune cells, suppressive immune microenvironment and metabolic reprogramming of tumor cells were identified in the 500{micro}m-wide zone centered bilaterally on the tumor boundary, namely invasive fronts of tumor. Furthermore, we found the damaged states of hepatocytes with overexpression of Serum Amyloid A (SAA) in invasive fronts, recruiting macrophages for facilitating further tumor invasion, and thus resulting in a worse prognosis. We also confirmed these findings in hepatocellular carcinoma and other liver metastatic cancers. Our work highlights the remarkable potential of high-resolution-spatially resolved transcriptomic approaches to provide meaningful biological insights for comprehensively dissecting the tumor ecosystem and guiding the development of novel therapeutic strategies for solid tumors.

cancer biology↗

bioRxiv2021

Demographic model and biological adaptation inferred from the genome-wide SNP data reveal tripartite origins of southernmost Chinese Huis

The culturally unique Sanya Hui (SYH) people are regarded as the descendants of ancient Cham people in Central Vietnam (CV) and exhibit a scenario of complex migration and admixture history, who were likely to first migrate from Central and South Asia (CSA) to CV and then to South Hainan and finally assimilated with indigenous populations and resided in the tropical island environments since then. A long-standing hypothesis posits that SYH derives from different genetic and cultural origins, which hypothesizes that SYH people are different from the genetically attested admixture history of northern Hui people possessing major Han-related ancestry and minor western Eurasian ancestry. However, the effect of the cultural admixture from CSA and East Asia (EA) on the genetic admixture of SYH people remains unclear. Here, we reported the first batch of genome-wide SNP data from 94 SYH people from Hainan and comprehensively characterized their genetic structure, origin, and admixture history. Our results found that SYH people were genetically different from the northern Chinese Hui people and harbored a close genomic affinity with indigenous Vietnamese but a distinct relationship with Cham, which confirmed the hypothesis of documented recent historical migration from CV and assimilation with Hainan indigenous people. The fitted admixture models and reconstructed demographic frameworks revealed an additional influx of CSA and EA ancestries during the historical period, consisting of the frequent cultural communication along the Southern Maritime Silk Road and extensive interaction with EA. Analyses focused on natural-selected signatures of SYH people revealed a similar pattern with mainland East Asians, which further confirmed the possibility of admixture-induced biological adaptation of island environments. Generally, three genetically attested ancestries from CV, EA, and CSA in modern SYH people supported their tripartite model of genomic origins.

genetics↗

bioRxiv2021

Genomic insights into the population history and biological adaptation of Southwestern Chinese Hmong-Mien people

Hmong-Mien-speaking (HM) populations, widely distributed in South China, North of Thailand, Laos and Vietnam, have experienced different settlement environments, dietary habits and pathogen exposure. However, their specific biological adaptation also remained largely uncharacterized, which is important in the population evolutionary genetics and Trans-Omics for regional Precision Medicine. Besides, the origin and genetic diversity of HM people and their phylogenetic relationship with surrounding modern and ancient populations are unknown. Here, we reported genome-wide SNPs in 52 representative Miao people and combined them with 144 HM people from 13 geographically representative populations to characterize the full genetic admixture and adaptive landscape of HM speakers. We found that obvious genetic substructures existed in geographically different HM populations and also identified one new ancestral lineage specifically exited in HM people, which spatially distributed from Sichuan and Guizhou in the North to Thailand in the South and temporally dated to at least 500 years. The sharing patterns of the newly-identified homogeneous ancestry component combined the estimated admixture times via the decay of Linkage Disequilibrium and haplotype sharing in GLOBETROTTER suggested that the modern HM-speaking populations originated from Southwest China and migrated southward recently, which is consistent with the reconstructed phenomena of linguistic and archeological documents. Additionally, we identified specific adaptive signatures associated with several important human nervous system biological functions. Our pilot work emphasized the importance of anthropologically-informed sampling and deeply genetic structure reconstruction via whole-genome sequencing in the next step in the deep Chinese population genomic diversity project (CPGDP), especially in the ethnolinguistic regions.

genetics↗

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

Structural basis for the therapeutic advantage of dual and triple agonists at the human GIP, GLP-1 or GCG receptors

Glucose homeostasis, regulated by glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1) and glucagon (GCG) is critical to human health. Several multi-targeting agonists at GIPR, GLP-1R or GCGR, developed to maximize metabolic benefits with reduced side-effects, are in clinical trials to treat type 2 diabetes and obesity. To elucidate the molecular mechanisms by which tirzepatide, a GIPR/GLP-1R dualagonist, and peptide 20, a GIPR/GLP-1R/GCGR triagonist, manifest their superior efficacies over monoagonist such as semaglutide, we determined cryo-electron microscopy structures of tirzepatide-bound GIPR and GLP-1R as well as peptide 20-bound GIPR, GLP-1R and GCGR The structures reveal both common and unique features for the dual and triple agonism by illustrating key interactions of clinical relevance at the atomic level. Retention of glucagon function is required to achieve such an advantage over GLP-1 monotherapy. Our findings provide valuable insights into the structural basis of functional versatility and therapeutic supremacy of tirzepatide and peptide 20.

molecular biology↗

bioRxiv2021

The northern gene flow into southeastern East Asians inferred from genome-wide array genotyping

The population history of Southeast China remains poorly understood due to the sparse sampling of present-day populations and far less modeling with ancient genomic data. We here newly reported genome-wide genotyping data from 207 present-day Han Chinese and Hmong-Mien-speaking She people from Fujian and Taiwan, southeast China. We co-analyzed with 66 early-Neolithic to Iron-Age ancient Fujian and Taiwan individuals obtained from literature to explore the genetic continuity and admixture based on the genetic variations of high-resolution time transect. We found the genetic differentiation between northern and southern East Asians defined by a north-south East Asian genetic cline and the studied southern East Asians were clustered in the southern end of this cline. We also found that southeastern coastal continental modern East Asians harbored the genetic differentiation with other southern Tai-Kadai, Hmong-Mien, Austronesian and Austroasiatic speakers, as well as geographically close Neolithic-to-Iron Age populations, but relatedly close to post-Neolithic Yellow River ancients, which suggested the influence of southward gene flow on the modern southern coastal gene pool. Besides, we also identified one new Hmong-Mien genetic cline in East Asia with the coastal Fujian She localizing at the intersection position between Hmong-Mien and Han clines in the principal component analysis. She people show stronger genetic affinity with southern East Asian indigenous populations with the main ancestry deriving from Hanben-related populations. The southeastern Han Chinese could be modeled with the primary ancestry deriving from the group related to the Yellow River Basin millet farmers and the remaining from groups related to southeastern ancient indigenous rice farmers, which was consistent with the northern China origin of modern southeastern Han Chinese and in line with the historically and archaeologically attested southward migrations of Han people and their ancestors. Interestingly, f4-statistics and three-way admixture model results showed both coastal ancient sources related to Austronesian speakers and inland ancient sources related to Austroasiatic speakers complexed the modern observed fine-scale genetic structure here. Our estimated north-south admixture time ranges based on the decay of the linkage disequilibrium spanned from the Bronze age to historic periods, suggesting the recent large-scale population migrations and subsequent admixture participated in the formation of modern Han in Southeast Asia.

genetics↗

bioRxiv2021

Convergence of MCR-8.2 and chromosome-mediated resistance to colistin and tigecycline in an NDM-5-producing ST656 Klebsiella pneumoniae from a lung transplant patient

For infection caused by NDM-5-producing Klebsiella pneumoniae, tigecycline and colistin are the last treatment options. In this study, we characterized the first NDM-5 and MCR-8.2 co-harboring K. pneumoniae clinical isolate, combining with chromosomal gene-mediated resistance to colistin and tigecycline. The K. pneumoniae KP32558 was isolated from the bronchoalveolar lavage fluid from a lung transplant male patient. Whole genome sequencing was carried out using Illumina HiSeq sequencing platform as well as nanopore sequencing method. The K. pneumoniae KP32558 was identified as a pan-drug resistant bacteria, belonged to ST656, and harbored plasmid-encoded blaNDM-5 and mcr-8.2 genes. The blaNDM-5 gene was located on an IncX3 type plasmid, which was successfully transferred to Escherichia coli strain J53 without visible fitness cost. The mcr-8.2 gene was located on a conjugative plasmid pKP32558-2-mcr8. It had two replicons, IncFII(K) and IncQ1, harbored previously by another two mcr-8.2-carrying plasmids pMCR8_020135 and pMCR8_095845. These three plasmids were clustered into the same clade and derived from K. pneumoniae isolates of the same clonal complex, indicating that pKP32558-2-mcr8 may come from pMCR8_020135 and pMCR8_095845 related ancestor. The MIC of KP32558 for colistin was 256 mg/L, the 6 amino acid substitutions in the two-component system may involve in the high-level colistin resistance. The truncation in acrR gene, related to tigecycline resistance, was also identified. K. pneumoniae has evolved a variety of complex resistance mechanisms to the last-resort antimicrobials, close surveillance is urgently needed to monitor the prevalence of this clone.

microbiology↗

bioRxiv2021

Genomic insights into the differentiated population admixture structure and demographic history of North East Asians

North China and South Siberia, mainly populated by Altaic-speaking populations, possess extensive ethnolinguistic diversity and serve as the crossroad for the initial peopling of America and western-eastern trans-continental communication. Yet, the complex scenarios of genetic origin, population structure, and admixture history of North-East Asia remain to be fully characterized, especially for Mongolic people in China with a genome-wide perspective. Thus, we genotyped genome-wide SNPs for 510 individuals from 38 Chinese Mongolic, Tungusic, and Sinitic populations to explore the sharing alleles and haplotypes within the studied groups and following merged it with 3508 modern and ancient Eurasian individuals to reconstruct the deep evolutionary and natural selection history of northern East Asians. We identified significant substructures within Altaic-speaking populations with the primary common ancestry linked to the Neolithic northern East Asians: Western Turkic people harbored more western Eurasian ancestry; Northern Mongolic people in Siberia and eastern Tungusic people in Amur River Basin (ARB) possessed dominant Neolithic Mongolian Plateau (MP) or ARB ancestry; Southern Mongolic people in China owned obvious genetic impact from Neolithic Yellow River Basin (YRB) farmers. Additionally, we found the differentiated admixture history between western and eastern Mongolians and geographically close Northeast Hans: the former received a genetic impact from western Eurasians and the latter retained the dominant YRB and ARB Neolithic ancestry. Moreover, we demonstrated that Kalmyk people from the northern Caucasus Mountain possessed a strong genetic affinity with Neolithic MP people, supporting the hypothesis of their eastern Eurasian origin and long-distance migration history. We also illuminated that historic pastoral empires in the MP contributed considerably to the gene pool of northern Mongolic people but rarely to southern ones. We finally found natural signatures in Mongolians associated with alcohol metabolism. Generally, our results not only illuminated that complex population migration and admixture of Neolithic ancestral sources from the MP or ARB played an important role in the spread of Altaic-speaking populations and Proto-Altaic language, which partly supported the Northeast Asia-origin hypothesis, but also demonstrated that the observed multi-sources of genetic diversity contributed significantly to the modern existing extensive ethnolinguistic diversity in North-East Asia.

genomics↗

bioRxiv2021

Essential role of translation factor eIF5a in cytokine production and cell cycle regulation in primary CD8 T lymphocytes

Translational control adjusts protein production rapidly and facilitates local cellular responses to environmental conditions. Translation can be regulated through sequestration of mRNAs by regulatory proteins or RNAs, but also by the availability of ribosomes and translation factors which enable initiation and elongation of nascent polypeptides. Traditionally initiation of mRNA translation has been considered to be a major translational control point, however, control of peptide elongation can also play a role. Here we show that post-translational modification of the elongation factor, eIF5a, controls translation of subsets of proteins in naive T-cells upon activation. Sequencing of nascent polypeptides indicated that functional eIF5a was required for the production of proteins which regulate T-cell proliferation and effector function. Control of translation in multiple immune cell lineages is required to co-ordinate immune responses and these data illustrate that translational elongation can contribute to post-transcriptional regulons important for the control of inflammation.

molecular biology↗

bioRxiv2021

Genomic history and forensic characteristics of Sherpa highlanders on the Tibetan Plateau inferred from high-resolution genome-wide InDels and SNPs

Sherpa people, one of the high-altitude hypoxic adaptive populations, mainly reside in Nepal and the southern Tibet Autonomous Region. The genetic origin and detailed evolutionary profiles of Sherpas remain to be further explored and comprehensively characterized. Here we analyzed the newly-generated InDel genotype data from 628 Dingjie Sherpa people by merging with 4222 worldwide InDel profiles and collected genome-wide SNP data (approximately 600K SNPs) from 3324 individuals in 382 modern and ancient populations to explore and reconstruct the fine-scale genetic structure of Sherpas and their relationships with nearby modern and ancient East Asians based on the shared alleles and haplotypes. The forensic parameters of 57 autosomal InDels (A-InDels) included in our used new-generation InDel amplification system showed that this updated InDel panel is informative and polymorphic in Sherpas, suggesting that it can be used as the supplementary tool for forensic personal identification and parentage testing in the highland East Asians. Descriptive findings from the PCA, ADMIXTURE and TreeMix-based phylogeny suggested that Sherpas showed excess allele sharing with neighboring Tibeto-Burman Tibetans. Furthermore, patterns of allele sharing in f-statistics demonstrated that Sherpa people had a different evolutionary history compared with their neighbors from Nepal (Newar and Gurung) but showed genetic similarity with 2700-year-old Chokhopani and modern Tibet Tibetans. QpAdm/qpGraph-based admixture sources and models further showed that Sherpa, core Tibetans and Chokhopani formed one clade which could be fitted as having the main ancestry from late Neolithic Qijia millet farmers and other deep ancestries from early Asians. Chromosome painting profiles and shared IBD fragments inferred from FineStructure and ChromoPainter not only confirmed the abovementioned genomic affinity patterns but also revealed the fine-scale microstructures among Sino-Tibetan speakers. Finally, natural-selection signals revealed via iHS, nSL, and iHH12 showed signatures associated with disease susceptibility in Sherpa people. Generally, we provided the comprehensive landscape of admixture and evolutionary history of Sherpa people based on the shared alleles and haplotypes from the low-density forensic markers and high-density genome-wide SNP data. The more detailed genetic landscape of Sherpa people should be further confirmed and characterized via ancient genomes or single-molecule real-time sequencing technology.

genetics↗

bioRxiv2021

Molecular insights into differentiated ligand recognition of the human parathyroid hormone receptor 2

The parathyroid hormone receptor 2 (PTH2R) is a class B1 G protein-coupled receptor (GPCR) involved in regulation of calcium transport, nociception mediation, and wound healing. Naturally occurring mutations in PTH2R were reported to cause hereditary diseases, including syndromic short stature. Here we report the cryo-electron microscopy structure of PTH2R bound to its endogenous ligand, tuberoinfundibular peptide (TIP39), and a heterotrimeric Gs protein at a global resolution of 2.8 [A]. The structure reveals that TIP39 adopts a unique loop conformation at N terminus and deeply inserts into the orthosteric ligand-binding pocket in the transmembrane (TM) domain. Molecular dynamics (MD) simulation and site-directed mutagenesis studies uncover the basis of ligand specificity relative to three PTH2R agonists, TIP39, PTH, and PTH-related peptide (PTHrP). We also compare the action of TIP39 with an antagonist lacking six residues from the peptide N terminus, TIP(7-39), which underscores the indispensable role of the N terminus of TIP39 in PTH2R activation. Additionally, we unveil that a disease-associated mutation G258D significantly diminished cAMP accumulation induced by TIP39. Together, these results not only provide structural insights into ligand specificity and receptor activation of class B1 GPCRs, but also offer a foundation to systematically rationalize the available pharmacological data to develop novel therapies for various disorders associated with PTH2R.

biochemistry↗

bioRxiv2021

Molecular insights into ago-allosteric modulation of the human glucagon-like peptide-1 receptor

The glucagon-like peptide-1 (GLP-1) receptor is a validated drug target for metabolic disorders. Ago-allosteric modulators are capable of acting both as agonists on their own and as efficacy enhancers of orthosteric ligands. However, the molecular details of ago-allosterism remain elusive. Here, we report three cryo-electron microscopy structures of GLP-1R bound to (i) compound 2 (an ago-allosteric modulator); (ii) compound 2 and GLP-1; and (iii) compound 2 and LY3502970 (a small molecule agonist), all in complex with heterotrimeric Gs. The structures reveal that compound 2 is covalently bonded to C347 at the cytoplasmic end of TM6 and triggers its outward movement in cooperation with the ECD whose N terminus penetrates into the GLP-1 binding site. This allows compound 2 to execute positive allosteric modulation through enhancement of both agonist binding and G protein coupling. Our findings offer the structural basis of ago-allosterism at GLP-1R and new knowledge to design better therapeutics.

molecular biology↗

bioRxiv2021

Transcriptomic Insights into the Responses in Leaves Storing Lipid Organelles under Different Irradiances

To increase the nutritional value of forage, transgenic ryegrass known as High Metabolizable Energy (HME) were previously generated that co-express cysteine-oleosin and diacylglycerol O-acyltransferase. HME not only accumulate lipids in the leaf but also has elevated CO2 assimilation and increased biomass. Shading is one of the most influencing factors for ryegrass growth environments particularly in swards. The aim of this study, therefore, was to determine the influence of irradiance levels on photosynthesis and gene expression in the HME leaves when compared with their corresponding non-transformant (NT). Under low light (150-250 {micro}mol m-2 s-1) and standard light (600-1000 {micro}mol m-2 s-1), the HME accumulated more lipid than NT. The previously reported elevated photosynthesis and increased biomass was observed when the HME were grown under standard light but not under low light. Under both light conditions, compared to NT, the HME had upregulated a number of transcripts involved in lipid metabolism, light capturing, photosynthesis, and sugar signalling network while downregulated genes participated in sugar and fructan biosynthesis. We further discuss how the HME differentially manipulated several genes other metabolic pathways including maintenance of redox homeostasis. Combined, the data suggests that the increased photosynthesis capacity in the HME likely corresponds to an increase of micro-lipid sink strength; these are influenced by available light energy and may be related to diffusional and biochemical activities of stomata. Overall, this work provides a clearly understanding of the changes in molecular and biochemical mechanisms underlying the carbon storing as leaf lipid sink of the HME ryegrass. One sentence summaryShading led to increase leaf lipid accumulation but limit the greater photosynthesis trait of high lipid ryegrass which may be related to limitation of biochemical activities of stomata.

molecular biology↗

bioRxiv2021

Redetermination of the first unknown protein MicroED structure by high resolution X-ray diffraction

Microcrystal electron diffraction (MicroED) has the potential to considerably impact the field of structural biology. Indeed, the method can solve atomic structures of a wide range of molecules, beyond the reach of single particle cryo-electron microscopy, exploiting crystals too small for X-ray diffraction (XRD) even using X-ray free-electron lasers. However, until the first unknown protein structure - a R2-like ligand-binding oxidase from Sulfolobus acidocaldarius (SaR2lox) - was recently solved at 3.0 [A] resolution, MicroED had only been used to study known protein structures previously obtained by XRD. Here, after adapting sample preparation protocols, the structure of the SaR2lox protein originally solved by MicroED was redetermined by XRD at 2.1 [A] resolution. In light of the higher resolution XRD data and taking into account experimental differences of the methods, the quality of the MicroED structure is examined. The analysis demonstrates that MicroED provided an overall accurate model, revealing biologically relevant information specific to SaR2lox, such as the absence of an ether cross-link, but did not allow to detect the presence of a ligand visible by XRD in the protein binding pocket. Furthermore, strengths and weaknesses of MicroED compared to XRD are discussed in the perspective of this real-life protein example. The study provides fundaments to help MicroED become a method of choice for solving novel protein structures. SynopsisThe first unknown protein structure solved by microcrystal electron diffraction (MicroED) was recently published. The redetermination by X-ray diffraction of this protein structure provides new insights into the strengths and weaknesses of the promising MicroED method.

biochemistry↗

bioRxiv2021

Twelve-month specific IgG response to SARS-CoV-2 receptor-binding domain among COVID-19 convalescent plasma donors in Wuhan

To investigate the duration of humoral immune response in convalescent coronavirus disease 2019 (COVID-19) patients, we conduct a 12-month longitudinal study through collecting a total of 1,782 plasma samples from 869 convalescent plasma donors in Wuhan, China and test specific antibody responses. The results show that positive rate of IgG antibody against receptor-binding domain of spike protein (RBD-IgG) to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the COVID-19 convalescent plasma donors exceeded 70% for 12 months post diagnosis. The level of RBD-IgG decreases with time, with the titer stabilizing at 64.3% of the initial level by the 9th month. Moreover, male plasma donors produce more RBD-IgG than female, and age of the patients positively correlates with the RBD-IgG titer. A strong positive correlation between RBD-IgG and neutralizing antibody titers is also identified. These results facilitate our understanding of SARS-CoV-2-induced immune memory to promote vaccine and therapy development.

immunology↗

bioRxiv2021

Structural insights into hormone recognition by the human glucose-dependent insulinotropic polypeptide receptor

Glucose-dependent insulinotropic polypeptide (GIP) is a peptide hormone that exerts crucial metabolic functions by binding and activating its cognate receptor, GIPR. As an important therapeutic target, GIPR has been subjected to intensive structural studies without success. Here, we report the cryo-EM structure of the human GIPR in complex with GIP and a Gs heterotrimer at a global resolution of 2.9 [A]. GIP adopts a single straight helix with its N terminus dipped into the receptor transmembrane domain (TMD), while the C-terminus is closely associated with the extracellular domain and extracellular loop 1. GIPR employs conserved residues in the lower half of the TMD pocket to recognize the common segments shared by GIP homologous peptides, while uses non-conserved residues in the upper half of the TMD pocket to interact with residues specific for GIP. These results provide a structural framework of hormone recognition and GIPR activation.

molecular biology↗