Structure and topography of the synaptic V-ATPase-synaptophysin complex.

Synaptic vesicles are organelles with a precisely defined protein and lipid composition1,2, yet the molecular mechanisms for the biogenesis of synaptic vesicles are mainly unknown. Here, we discovered a well-defined interface between the synaptic vesicle V-ATPase and synaptophysin by in situ cryo-electron tomography and single particle cryo-electron microscopy of functional synaptic vesicles isolated from mouse brains3. The synaptic vesicle V-ATPase is an ATP-dependent proton pump that establishes the protein gradient across the synaptic vesicle, which in turn drives the uptake of neurotransmitters4,5. Synaptophysin6 and its paralogs synaptoporin7 and synaptogyrin8 belong to a family of abundant synaptic vesicle proteins whose function is still unclear. We performed structural and functional studies of synaptophysin knockout mice, confirming the identity of synaptophysin as an interaction partner with the V-ATPase. Although there is little change in the conformation of the V-ATPase upon interaction with synaptophysin, the presence of synaptophysin in synaptic vesicles profoundly affects the copy number of V-ATPases. This effect on the topography of synaptic vesicles suggests that synaptophysin assists in their biogenesis. In support of this model, we observed that synaptophysin knockout mice exhibit severe seizure susceptibility, suggesting an imbalance of neurotransmitter release as a physiological consequence of the absence of synaptophysin.

Structure-based design of a SARS-CoV-2 Omicron-specific inhibitor.

The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) introduced a relatively large number of mutations, including three mutations in the highly conserved heptad repeat 1 (HR1) region of the spike glycoprotein (S) critical for its membrane fusion activity. We show that one of these mutations, N969K induces a substantial displacement in the structure of the heptad repeat 2 (HR2) backbone in the HR1HR2 postfusion bundle. Due to this mutation, fusion-entry peptide inhibitors based on the Wuhan strain sequence are less efficacious. Here, we report an Omicron-specific peptide inhibitor designed based on the structure of the Omicron HR1HR2 postfusion bundle. Specifically, we inserted an additional residue in HR2 near the Omicron HR1 K969 residue to better accommodate the N969K mutation and relieve the distortion in the structure of the HR1HR2 postfusion bundle it introduced. The designed inhibitor recovers the loss of inhibition activity of the original longHR2_42 peptide with the Wuhan strain sequence against the Omicron variant in both a cell-cell fusion assay and a vesicular stomatitis virus (VSV)-SARS-CoV-2 chimera infection assay, suggesting that a similar approach could be used to combat future variants. From a mechanistic perspective, our work suggests the interactions in the extended region of HR2 may mediate the initial landing of HR2 onto HR1 during the transition of the S protein from the prehairpin intermediate to the postfusion state.

Neutral lysophosphatidylcholine mediates α-synuclein-induced synaptic vesicle clustering.

α-synuclein (α-Syn) is a presynaptic protein that is involved in Parkinson's and other neurodegenerative diseases and binds to negatively charged phospholipids. Previously, we reported that α-Syn clusters synthetic proteoliposomes that mimic synaptic vesicles. This vesicle-clustering activity depends on a specific interaction of α-Syn with anionic phospholipids. Here, we report that α-Syn surprisingly also interacts with the neutral phospholipid lysophosphatidylcholine (lysoPC). Even in the absence of anionic lipids, lysoPC facilitates α-Syn-induced vesicle clustering but has no effect on Ca2+-triggered fusion in a single vesicle-vesicle fusion assay. The A30P mutant of α-Syn that causes familial Parkinson disease has a reduced affinity to lysoPC and does not induce vesicle clustering. Taken together, the α-Syn-lysoPC interaction may play a role in α-Syn function.

Structural conservation among variants of the SARS-CoV-2 spike postfusion bundle.

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge currently available COVID-19 vaccines and monoclonal antibody therapies due to structural and dynamic changes of the viral spike glycoprotein (S). The heptad repeat 1 (HR1) and heptad repeat 2 (HR2) domains of S drive virus­host membrane fusion by assembly into a six-helix bundle, resulting in delivery of viral RNA into the host cell. We surveyed mutations of currently reported SARS-CoV-2 variants and selected eight mutations, including Q954H, N969K, and L981F from the Omicron variant, in the postfusion HR1HR2 bundle for functional and structural studies. We designed a molecular scaffold to determine cryogenic electron microscopy (cryo-EM) structures of HR1HR2 at 2.2­3.8 Å resolution by linking the trimeric N termini of four HR1 fragments to four trimeric C termini of the Dps4 dodecamer from Nostoc punctiforme. This molecular scaffold enables efficient sample preparation and structure determination of the HR1HR2 bundle and its mutants by single-particle cryo-EM. Our structure of the wild-type HR1HR2 bundle resolves uncertainties in previously determined structures. The mutant structures reveal side-chain positions of the mutations and their primarily local effects on the interactions between HR1 and HR2. These mutations do not alter the global architecture of the postfusion HR1HR2 bundle, suggesting that the interfaces between HR1 and HR2 are good targets for developing antiviral inhibitors that should be efficacious against all known variants of SARS-CoV-2 to date. We also note that this work paves the way for similar studies in more distantly related viruses.

Nanomolar inhibition of SARS-CoV-2 infection by an unmodified peptide targeting the prehairpin intermediate of the spike protein.

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge currently available coronavirus disease 2019 vaccines and monoclonal antibody therapies through epitope change on the receptor binding domain of the viral spike glycoprotein. Hence, there is a specific urgent need for alternative antivirals that target processes less likely to be affected by mutation, such as the membrane fusion step of viral entry into the host cell. One such antiviral class includes peptide inhibitors, which block formation of the so-called heptad repeat 1 and 2 (HR1HR2) six-helix bundle of the SARS-CoV-2 spike (S) protein and thus interfere with viral membrane fusion. We performed structural studies of the HR1HR2 bundle, revealing an extended, well-folded N-terminal region of HR2 that interacts with the HR1 triple helix. Based on this structure, we designed an extended HR2 peptide that achieves single-digit nanomolar inhibition of SARS-CoV-2 in cell-based and virus-based assays without the need for modifications such as lipidation or chemical stapling. The peptide also strongly inhibits all major SARS-CoV-2 variants to date. This extended peptide is ∼100-fold more potent than all previously published short, unmodified HR2 peptides, and it has a very long inhibition lifetime after washout in virus infection assays, suggesting that it targets a prehairpin intermediate of the SARS-CoV-2 S protein. Together, these results suggest that regions outside the HR2 helical region may offer new opportunities for potent peptide-derived therapeutics for SARS-CoV-2 and its variants, and even more distantly related viruses, and provide further support for the prehairpin intermediate of the S protein.

In Situ Polymerization of Hydrogel Electrolyte on Electrodes Enabling the Flexible All-Hydrogel Supercapacitors with Low-Temperature Adaptability.

All-hydrogel supercapacitors are emerging as promising power sources for next-generation wearable electronics due to their intrinsic mechanical flexibility, eco-friendliness, and enhanced safety. However, the insufficient interfacial adhesion between the electrode and electrolyte and the frozen hydrogel matrices at subzero temperatures largely limit the practical applications of all-hydrogel supercapacitors. Here, an all-hydrogel supercapacitor is reported with robust interfacial contact and anti-freezing property, fabricated by in situ polymerizing hydrogel electrolyte onto hydrogel electrodes. The robust interfacial adhesion is developed by the synergistic effect of a tough hydrogel matrix and topological entanglements. Meanwhile, the incorporation of zinc chloride (ZnCl2) in the hydrogel electrolyte prevents the freezing of water solvents and endows the all-hydrogel supercapacitor with mechanical flexibility and fatigue resistance across a wide temperature range of 20 °C to -60 °C. Such all-hydrogel supercapacitor demonstrates satisfactory low-temperature electrochemical performance, delivering a high energy density of 11 mWh cm-2 and excellent cycling stability with a capacitance retention of 90% over 10000 cycles at -40 °C. Notably, the fabricated all-hydrogel supercapacitor can endure dynamic deformations and operate well under 2000 tension cycles even at -40 °C, without experiencing delamination and electrochemical failure. This work offers a promising strategy for flexible energy storage devices with low-temperature adaptability.

Deep learning-based pseudo-mass spectrometry imaging analysis for precision medicine.

Liquid chromatography-mass spectrometry (LC-MS)-based untargeted metabolomics provides systematic profiling of metabolic. Yet, its applications in precision medicine (disease diagnosis) have been limited by several challenges, including metabolite identification, information loss and low reproducibility. Here, we present the deep-learning-based Pseudo-Mass Spectrometry Imaging (deepPseudoMSI) project (https://www.deeppseudomsi.org/), which converts LC-MS raw data to pseudo-MS images and then processes them by deep learning for precision medicine, such as disease diagnosis. Extensive tests based on real data demonstrated the superiority of deepPseudoMSI over traditional approaches and the capacity of our method to achieve an accurate individualized diagnosis. Our framework lays the foundation for future metabolic-based precision medicine.

WeChat usage and preservation of cognitive functions in middle-aged and older Chinese adults: indications from a nationally representative survey, 2018-2020.

PURPOSE: To investigate the associations between the most popular social media platform WeChat usage and cognitive performance among the middle-aged and older Chinese population using data from a nationally representative survey. METHODS: In total, 17,472 participants (≥ 45 years old) from the China Health and Retirement Longitudinal Study (CHARLS, Wave 4, 2018) were analyzed. Cognitive performance including episodic memory and executive function was assessed using Mini-Mental Status Examination (MMSE). Other confounding variables included socio-economic characteristics, medical status, and lifestyle-related information. Multiple linear regression models were used to test the association between cognitive performance and WeChat usage by introducing covariates hierarchically. Subgroup analyses of age and gender were conducted to estimate the robustness of the primary findings. RESULTS: After adjusting for multiple confounders across all linear models, WeChat usage is significantly associated with executive function, episodic memory, and global cognitive performance (all p values<0.05). Such results remained robust in subgroup analyses, stratified by age and gender, and also verified according to longitudinal analyses. Compared to 'Chat-only' users who only used WeChat for online interpersonal communication, further usage of WeChat functions such as using 'Moments' appeared to be significantly associated with better cognitive performance, especially for episodic memory. CONCLUSION: Social media usage is significantly and positively associated with better cognitive performance among the middle-aged and older Chinese population. Along with point-to-point messaging, using 'Moments' and extended social media platform functions may correlate to better cognitive performance.

Mechanistic basis for receptor-mediated pathological α-synuclein fibril cell-to-cell transmission in Parkinson's disease.

The spread of pathological α-synuclein (α-syn) is a crucial event in the progression of Parkinson's disease (PD). Cell surface receptors such as lymphocyte activation gene 3 (LAG3) and amyloid precursor-like protein 1 (APLP1) can preferentially bind α-syn in the amyloid over monomeric state to initiate cell-to-cell transmission. However, the molecular mechanism underlying this selective binding is unknown. Here, we perform an array of biophysical experiments and reveal that LAG3 D1 and APLP1 E1 domains commonly use an alkaline surface to bind the acidic C terminus, especially residues 118 to 140, of α-syn. The formation of amyloid fibrils not only can disrupt the intramolecular interactions between the C terminus and the amyloid-forming core of α-syn but can also condense the C terminus on fibril surface, which remarkably increase the binding affinity of α-syn to the receptors. Based on this mechanism, we find that phosphorylation at serine 129 (pS129), a hallmark modification of pathological α-syn, can further enhance the interaction between α-syn fibrils and the receptors. This finding is further confirmed by the higher efficiency of pS129 fibrils in cellular internalization, seeding, and inducing PD-like α-syn pathology in transgenic mice. Our work illuminates the mechanistic understanding on the spread of pathological α-syn and provides structural information for therapeutic targeting on the interaction of α-syn fibrils and receptors as a potential treatment for PD.

The mouse nicotinamide mononucleotide adenylyltransferase chaperones diverse pathological amyloid client proteins.

Molecular chaperones safeguard cellular protein homeostasis and obviate proteotoxicity. In the process of aging, as chaperone networks decline, aberrant protein amyloid aggregation accumulates in a mechanism that underpins neurodegeneration, leading to pathologies such as Alzheimer's disease and Parkinson's disease. Thus, it is important to identify and characterize chaperones for preventing such protein aggregation. In this work, we identified that the NAD+ synthase-nicotinamide mononucleotide adenylyltransferase (NMNAT) 3 from mouse (mN3) exhibits potent chaperone activity to antagonize aggregation of a wide spectrum of pathological amyloid client proteins including α-synuclein, Tau (K19), amyloid ß, and islet amyloid polypeptide. By combining NMR spectroscopy, cross-linking mass spectrometry, and computational modeling, we further reveal that mN3 uses different region of its amphiphilic surface near the active site to directly bind different amyloid client proteins. Our work demonstrates a client recognition mechanism of NMNAT via which it chaperones different amyloid client proteins against pathological aggregation and implies a potential protective role for NMNAT in different amyloid-associated diseases.

massDatabase: utilities for the operation of the public compound and pathway database.

SUMMARY: One of the major challenges in liquid chromatography coupled to mass spectrometry data is converting many metabolic feature entries to biological function information, such as metabolite annotation and pathway enrichment, which are based on the compound and pathway databases. Multiple online databases have been developed. However, no tool has been developed for operating all these databases for biological analysis. Therefore, we developed massDatabase, an R package that operates the online public databases and combines with other tools for streamlined compound annotation and pathway enrichment. massDatabase is a flexible, simple and powerful tool that can be installed on all platforms, allowing the users to leverage all the online public databases for biological function mining. A detailed tutorial and a case study are provided in the Supplementary Material. AVAILABILITY AND IMPLEMENTATION: https://massdatabase.tidymass.org/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Therapeutic effects of atorvastatin on doxorubicin-induced hepatotoxicity in rats via antioxidative damage, anti-inflammatory, and anti-lipotoxicity.

Doxorubicin (DOX), is a high efficiency anthracycline antitumor drug. However, the clinical application of DOX is limited mainly by dose-related adverse drug reactions. Currently, the therapeutic effects of Atorvastatin (ATO) on DOX-induced hepatotoxicity were studied in vivo. The results indicated that DOX impaired hepatic function, as measured by an increased levels of liver weight index and serum concentrations of aspartate transaminase and alanine transaminase, as well as alteration of hepatic histology. In addition, DOX increased the serum levles of triglyceride (TG) and nonestesterified fatty acid. ATO prevented these changes. Mechanical analysis revealed that ATO restored the changes of malondialdehyde, reactive oxygen radical species, glutathione peroxidase and manganese superoxide dismutase. Additionally, ATO inhibited the increased expression levels of nuclear factor-kappa B and interleukin 1ß, hence suppressing inflammation. Meanwhile, ATO inhibited cell apoptosis by dramatically decreasing the Bax/Bcl-2 ratio. In addition, ATO mitigated the lipidtoxicity by inhibiting the adipolysis of TG and accelerating hepatic lipid metabolism. Taken together, the results suggest ATO has therapeutic effect on DOX-induced hepatotoxicity via inhibition of oxidative damage, inflammatory and apoptosis. In addition, ATO attenuates DOX-induced hyperlipidemia via modulation of lipid metabolism.

The role of 20E biosynthesis relative gene Shadow in the reproduction of the predatory mirid bug, Cyrtorhinus lividipennis (Hemiptera: Miridae).

Cytorhinus lividipennis is a natural enemy of rice planthoppers and leafhoppers. Improving the fecundity of C. lividipennis will be helpful to improve its control effect on pests. However, little is known about the hormonal regulatory mechanism of reproduction in C. lividipennis. In the current study, we examined the role of 20-hydroxyecdysone (20E) biosynthesis relative gene Shadow in the reproduction of C. lividipennis. The complementary DNA sequence of ClSad is 2018 -bp in length with an open reading frame of 1398-bp encoding 465 amino acid residues. ClSad was readily detected in nymphal and adult stages, and highly expressed in the adult stage. ClSad was highly expressed in the midgut and ovaries of adult females. Moreover, RNA interference-mediated knockdown of ClSad reduced the 20E titers and ClVg transcript level, resulting in fewer fully developed eggs and a decrease in the number of eggs laid by dsSad-injected adult females within 15 days. These results suggest that ClSad plays a critical role in the reproduction of C. lividipennis. The present study provides insights into the molecular mechanism of the ClSad gene for the reproduction of C. lividipennis.

Experimental Study of Somatic Variants of Osteosarcoma by Whole-Exome Sequencing.

BACKGROUND This study aimed to investigate the role of gene mutation site distribution, biological function, pathway enrichment, and gene association analysis in the occurrence, development, and migration of osteosarcoma. MATERIAL AND METHODS Somatic mutation screening was performed using the whole-exome sequencing of osteosarcoma samples, and the distribution of mutations was demonstrated by Circos diagrams. Metascape was used to analyze the GO and KEGG signal pathway enrichment of the genes harboring protein coding alterations, and GeneMANIA was used to analyze the interaction of mutated genes. RESULTS The results showed that the protein coding alterations were found throughout the whole genome in 3 osteosarcoma samples. A large number of identical or related biological processes and pathways were found in osteosarcoma samples. The GeneMANIA analysis of the 10 mutations shared by 3 samples showed that the target gene minichromosome maintenance complex component 4 (MCM4) and 3 lateral genes were most functional, and were all related to DNA replication. The analysis of GO and KEGG signal pathway enrichment showed that the mutated genes were involved mainly in tumor-related metabolic pathways. Three mutated genes were involved in the cell process, and 2 mutated genes were involved in the metabolic process. Known driver gene mutations were also observed in the samples. CONCLUSIONS The gene analysis confirmed that patients with osteosarcoma had a wide range of common gene mutations related to each other, which are involved in tumor-related metabolic pathways. These findings provide a basis for further gene-targeted therapy and pathway research.

Mechanistic insights into the switch of αB-crystallin chaperone activity and self-multimerization.

αB-Crystallin (αBc) is a small heat shock protein that protects cells against abnormal protein aggregation and disease-related degeneration. αBc is also a major structural protein that forms polydisperse multimers that maintain the liquid-like property of the eye lens. However, the relationship and regulation of the two functions have yet to be explored. Here, by combining NMR spectroscopy and multiple biophysical approaches, we found that αBc uses a conserved ß4/ß8 surface of the central α-crystallin domain to bind α-synuclein and Tau proteins and prevent them from aggregating into pathological amyloids. We noted that this amyloid-binding surface can also bind the C-terminal IPI motif of αBc, which mediates αBc multimerization and weakens its chaperone activity. We further show that disruption of the IPI binding impairs αBc self-multimerization but enhances its chaperone activity. Our work discloses the structural mechanism underlying the regulation of αBc chaperone activity and self-multimerization and sheds light on the different functions of αBc in antagonizing neurodegeneration and maintaining eye lens liquidity.

Identification of rare variants in cardiac sodium channel ß4-subunit gene SCN4B associated with ventricular tachycardia.

Ventricular tachycardia (VT) causes sudden cardiac death, however, the majority of risk genes for VT remain unknown. SCN4B encodes a ß-subunit, Navß4, for the voltage-gated cardiac sodium channel complex involved in generation and conduction of the cardiac action potential. We hypothesized that genomic variants in SCN4B increase the risk of VT. We used high-resolution melt analysis followed by Sanger sequencing to screen 199 VT patients to identify nonsynonymous variants in SCN4B. Two nonsynonymous heterozygous variants in SCN4B were identified in VT patients, including p.Gly8Ser in four VT patients and p.Ala145Ser in one VT patient. Case-control association studies were used to assess the association between variant p.Gly8Ser and VT in two independent populations for VT (299 VT cases vs. 981 controls in population 1 and 270 VT patients vs. 639 controls in population 2). Significant association was identified between p.Gly8Ser and VT in population 1 (P = 1.21 × 10-4, odds ratio or OR = 11.04), and the finding was confirmed in population 2 (P = 0.03, OR = 3.62). The association remained highly significant in the combined population (P = 3.09 × 10-5, OR = 6.17). Significant association was also identified between p.Gly8Ser and idiopathic VT (P = 1.89 × 10-5, OR = 7.27). Functional analysis with Western blotting showed that both p.Gly8Ser and p.Ala145Ser variants significantly reduced the expression level of Navß4. Based on 2015 ACMG Standards and Guidelines, p.Gly8Ser and p.Ala145Ser can be classified as the pathogenic and likely pathogenic variant, respectively. Our data suggest that SCN4B is a susceptibility gene for common VT and idiopathic VT and link rare SCN4B variants with large effects (OR = 6.17-7.27) to common VT.

Label- and modification-free-based in situ selection of bovine serum albumin specific aptamer.

Systematic evolution of ligands by exponential enrichment is a traditional approach to select aptamer, which has a great potential in biosensing field. However, chemical modifications of DNA library or targets before selection might block the real recognition and binding sites between aptamers and their targets. In this study, a label- and modification-free-based in situ selection strategy was developed to overcome this limitation. The strategy is an attempt to screen bovine serum albumin aptamers according to the principle of electrophoretic mobility shift assay, and allowed single-stranded DNA sequence to be fully exposed to interact with bovine serum albumin which was mixed with the agarose gel beforehand. After eight rounds of selection, specific aptamer with low dissociation constant (Kd ) value of 69.44 ± 7.60 nM was selected and used for subsequent establishment of fluorescence biosensor. After optimization, the optimal aptasensor exhibited a high sensitivity toward bovine serum albumin with a limit of detection of 0.24 ng/mL (linear range from 1 to 120 ng/mL). These results indicated that the label- and modification-free-based in situ selection strategy proposed in this work could effectively select specific aptamer to develop aptasensor for sensitive detection of bovine serum albumin or other targets in actual complicated samples.

The design of high affinity human PD-1 mutants by using molecular dynamics simulations (MD).

BACKGROUND: Programmed cell death protein 1 (PD-1), a negative co-stimulatory molecule, plays crucial roles in immune escape. Blockade of the interaction between PD-1 and PD-L1 shows exciting clinical responses in a fraction of cancer patients and the success makes PD-1 as a valuable target in immune checkpoint therapy. For the rational design of PD-1 targeting modulators, the ligand binding mechanism of PD-1 should be well understood in prior. METHODS: In this study, we applied 50 ns molecular dynamics simulations to observe the structural properties of PD-1 molecule in both apo and ligand bound states, and we studied the structural features of PD-1 in human and mouse respectively. RESULTS: The results showed that the apo hPD-1 was more flexible than that in PD-L1 bound state. We unexpectedly found that K135 was important for binding energy although it was not at the binding interface. Moreover, the residues which stabilized the interactions with PD-L1 were distinguished. Taking the dynamic features of these residues into account, we identified several residual sites where mutations may gain the function of ligand binding. The in vitro binding experiments revealed the mutants M70I, S87 W, A129L, A132L, and K135 M were better in ligand binding than the wild type PD-1. CONCLUSIONS: The structural information from MD simulation combined with in silico mutagenesis provides guidance to design engineered PD-1 mutants to modulate the PD-1/PD-L1 pathway.

Selection of DNA aptamers and establishment of an effective aptasensor for highly sensitive detection of cefquinome residues in milk.

Cefquinome (CFQ), which is a fourth-generation cephalosporin approved for veterinary use only, has been widely used for treating porcine or bovine respiratory infection, bovine mastitis and other diseases. However, the antibacterial effect of CFQ is based on the duration of drug concentration remaining in excess of the minimum inhibitory concentration in serum or tissues, thereby inevitably leading to CFQ residues with high levels in animal-sourced food. In this paper, four CFQ-specific ssDNA aptamers were selected via a magnetic bead-based systematic evolution of ligands by the exponential enrichment (SELEX) method. Aptamer W1 with the lowest dissociation constant (Kd) value of 40.13 ± 22.11 nM was chosen for establishing a fluorescence aptasensor based on magnetic separation and release of molecular beacons for detection of CFQ residues. This aptasensor exhibited a high sensitivity toward CFQ with a limit of detection (LOD) of 0.09 ng mL-1 (linear range from 0.5 to 150 ng mL-1). Moreover, the present aptasensor also showed high selectivity against ampicillin and CFQ's structural analogs (i.e., cefpirome sulfate and cefixime). Finally, this aptasensor was used to detect CFQ in real spiked milk. The recovery rate of CFQ from spiked milk samples ranged from 96.6% to 103.2%. These results indicated that the developed aptasensor is a promising, highly sensitive and specific method for CFQ residue detection in animal-sourced food.

Molecular Basis of Gene-Gene Interaction: Cyclic Cross-Regulation of Gene Expression and Post-GWAS Gene-Gene Interaction Involved in Atrial Fibrillation.

Atrial fibrillation (AF) is the most common cardiac arrhythmia at the clinic. Recent GWAS identified several variants associated with AF, but they account for <10% of heritability. Gene-gene interaction is assumed to account for a significant portion of missing heritability. Among GWAS loci for AF, only three were replicated in the Chinese Han population, including SNP rs2106261 (G/A substitution) in ZFHX3, rs2200733 (C/T substitution) near PITX2c, and rs3807989 (A/G substitution) in CAV1. Thus, we analyzed the interaction among these three AF loci. We demonstrated significant interaction between rs2106261 and rs2200733 in three independent populations and combined population with 2,020 cases/5,315 controls. Compared to non-risk genotype GGCC, two-locus risk genotype AATT showed the highest odds ratio in three independent populations and the combined population (OR=5.36 (95% CI 3.87-7.43), P=8.00×10-24). The OR of 5.36 for AATT was significantly higher than the combined OR of 3.31 for both GGTT and AACC, suggesting a synergistic interaction between rs2106261 and rs2200733. Relative excess risk due to interaction (RERI) analysis also revealed significant interaction between rs2106261 and rs2200733 when exposed two copies of risk alleles (RERI=2.87, P<1.00×10-4) or exposed to one additional copy of risk allele (RERI=1.29, P<1.00×10-4). The INTERSNP program identified significant genotypic interaction between rs2106261 and rs2200733 under an additive by additive model (OR=0.85, 95% CI: 0.74-0.97, P=0.02). Mechanistically, PITX2c negatively regulates expression of miR-1, which negatively regulates expression of ZFHX3, resulting in a positive regulation of ZFHX3 by PITX2c; ZFHX3 positively regulates expression of PITX2C, resulting in a cyclic loop of cross-regulation between ZFHX3 and PITX2c. Both ZFHX3 and PITX2c regulate expression of NPPA, TBX5 and NKX2.5. These results suggest that cyclic cross-regulation of gene expression is a molecular basis for gene-gene interactions involved in genetics of complex disease traits.