I. Regulation of carbon-nitrogen metabolism balance in cyanobacteria

As the oldest photosynthetic autotrophic organisms on Earth, cyanobacteria have evolved a carbon concentration mechanism (CO₂-concentrating mechanism, CCM). Through a series of carbon transporters, they efficiently transport and absorb CO₂ and highly concentrate it in carboxysomes. The carboxysome is a nearly icosahedral carbon-fixing machine with a size of about 100-200 nm and a total molecular weight of more than 100 MDa. It is currently the largest known super-large protein machine with extremely complex structure and assembly. Some cyanobacteria can also convert various forms of nitrogen sources (including nitrogen in the air) into combined nitrogen. Therefore, cyanobacteria are ideal model organisms for studying the basic biological issue of carbon-nitrogen metabolism balance. We continuously focus on the working mechanism of the cyanobacterial carbon concentration mechanism and the molecular mechanism of carbon-nitrogen metabolism balance regulation. By means of structural biology, biochemistry, microbial genetics, and synthetic biology, we elucidate the molecular mechanism of the orderly self-assembly and efficient carbon fixation of carboxysomes. Through multi-omics and metabolic flux and other means, we mine new and efficient carbon fixation modules and key regulatory elements related to carbon-nitrogen metabolism in cyanobacteria, and analyze their fine regulatory mechanisms. We rationally design and optimize efficient carbon fixation and nitrogen fixation modules and pathways, and study the interaction network and adaptation mechanism of these modules in cyanobacteria, laying a foundation for creating new and efficient carbon-fixing crops and microalgae and applying them in the fields of crop improvement and biological carbon neutrality.

We have revealed the molecular mechanism by which cyanobacteria efficiently absorb, concentrate, and fix CO2 through the carbon concentration mechanism. We have analyzed the multi-level fine dynamic regulation process of the assembly and maturation of the photosynthetic carbon-fixing enzyme RuBisCO (Nature Plants 2020; Cell Discovery 2022), and revealed the fine assembly mode and efficient carbon fixation mechanism of the carbon-fixing machine carboxysome (Nature Plants 2024; Structure 2024; Protein Science 2021). We have discovered a new mechanism for the regulation of carbon-nitrogen metabolism balance in cyanobacteria. We have revealed the mechanism by which multiple transcription factors respond to different metabolic small molecules and precisely regulate carbon-nitrogen metabolism balance (PNAS 2018; PNAS 2010; J Mol Biol 2010). We have proposed a new model of dual transcription factors synergistically regulating gene transcription by changing DNA conformation (Nat Struct Mol Biol 2024). We have revealed a new mechanism by which the signal protein PII regulates the activity of carbon-nitrogen transporters and thereby maintains carbon-nitrogen balance (PNAS 2024; Cell Discovery 2021). These research results have revealed the multi-level regulatory network of cyanobacterial photosynthetic carbon fixation and carbon-nitrogen metabolism balance, laying a foundation for the application of cyanobacteria in the fields of biological carbon fixation and crop improvement.

As one of the five major freshwater lakes in China, Lake Chaohu is an important freshwater resource and ecological wetland in the middle and lower reaches of the Yangtze River. However, in recent years, cyanobacterial blooms in Lake Chaohu have frequently erupted, which not only causes deterioration of lake water quality and ecological imbalance but also endangers the safety of humans and livestock and affects the social and economic development of the Lake Chaohu Basin. Just as there are phages wherever there are bacteria, there are cyanophages wherever there are cyanobacteria. Cyanophage is a virus that specifically infects cyanobacteria and is widely distributed in fresh water and seawater. It participates in the macroscopic regulation of the abundance and population balance of host cyanobacteria in water bodies and may be a potential new means of intervening in cyanobacterial blooms. At present, only 27 strains of freshwater cyanophages have been isolated and sequenced (except in our laboratory), and there are few studies on the assembly mechanism and omics of cyanophages at home and abroad. The study of the three-dimensional structure of cyanophages will help to elucidate their self-assembly mechanism and the molecular mechanism of specifically recognizing cyanobacteria during the infection of hosts. The study of cyanophage omics will help to enrich the library of freshwater cyanophages and reveal their seasonal fluctuation pattern with host cyanobacteria, providing a theoretical basis for the application of cyanophages in the early warning and intervention of cyanobacterial blooms.

Our laboratory takes Lake Chaohu as the research object and regularly collects water samples from its 10 inlets, 1 outlet, and the centers of the east and west parts of the lake to screen for dominant populations of cyanobacteria in blooms and their corresponding freshwater cyanophages. We isolated a long-tailed cyanophage Mic1 that specifically infects Microcystis aeruginosa FACHB 1339 from Lake Chaohu. We used cryo-electron microscopy to resolve the three-dimensional structure of its head and elucidated the head assembly mechanism based on structural analysis and comparison (Structure 2019). At the same time, through systematic analysis and comparison of the genome sequence of Mic1, we found that it is a new type of freshwater long-tailed cyanophage (Frontiers in Microbiology 2020). We isolated two strains of Pseudanabaena mucicola Chao 1806 and Pseudanabaena sp. Chao 1811 from Lake Chaohu and screened 10 cyanophages with different tail types that use these two cyanobacteria as hosts: Pam1~Pam5 and Pan1~Pan5. Using cryo-electron microscopy combined with crystallography technology, we resolved the near-atomic resolution complete structure of the freshwater short-tailed cyanophage Pam1, revealed its fine assembly mechanism, and found that the head reinforcement protein of Pam1 has dual functions of stabilizing the head and recognizing the host. The right-handed helical rifling channel formed by its connector protein can promote the injection of DNA into the host cell (Structure 2022). Based on comparative genomics and metagenomics analysis, we revealed the evolutionary diversity of Pam1~Pam5 and Pan1~Pan5. Through phylogenetic analysis of the auxiliary metabolic gene phoH, we provided direct evidence for the inter-phylum horizontal gene transfer mediated by phages between cyanobacteria and α-proteobacteria, and proposed a high-throughput strategy for identifying new cyanophages based on existing metagenomic data and the reference genomes of a limited number of experimentally isolated cyanophages (Microbiome 2022，Environmental Microbiome 2023). We resolved the complete cryo-electron microscopy structure of Pam3, which contains a head with a diameter of 680 Å, a three-jointed neck, an 840 Å long contractile tail, and the simplest base plate known to be composed of only five proteins, revealing the assembly mechanism of myoviridae cyanophages (PNAS 2023). Combined with the structure of the complex of the tail fiber protein and molecular chaperone of Pam3, we proposed the molecular mechanism of molecular chaperone-assisted folding and assembly of tail fibers (Viruses 2022). On the basis of previously resolving the icosahedral capsid structure (Journal of Virology 2021), we further resolved the complete structure of the tail machine of the myoviridae cyanophage A-1(L) that specifically infects Anabaena variabilis PCC 7120 by cryo-electron microscopy. Combined with a series of biochemical experiments, we identified multiple key functional modules for recognizing and hydrolyzing the host, and reannotated its genome based on structural information, revealing the structural basis and molecular mechanism of the interaction between A-1(L) and its specific host cyanobacteria (Nature Communications 2024). In addition, we used transcriptomics and genome resequencing methods to study the interaction characteristics and coevolution relationship between Microcystis aeruginosa FACHB 1339 and cyanophage Mic1 (Microbiology Spectrum 2024).

11 strains of freshwater cyanophages (December 2022)

Three-dimensional structure of cyanophages (December 2022)

III. Membrane transport proteins related to major diseases

The cell membrane acts as a barrier between the cytoplasm and the extracellular microenvironment. It provides selective permeability through transmembrane proteins to maintain the homeostasis of the intracellular environment. Ion channels and facilitators can transport solutes across the membrane along the electrochemical potential gradient. This transport process does not require energy and is therefore also called passive transport. In contrast, active transport can transport and enrich solutes against the concentration gradient, which requires energy consumption. Organisms have evolved a series of active transport proteins for the transport of different substances during long-term evolution to meet a variety of physiological needs. Among them, ABC transporters are the largest superfamily of transporters. ABC transporters transport substrates across the membrane against the chemical potential energy by hydrolyzing adenosine triphosphate (ATP). Almost all molecules with physiological functions are substrates of ABC transporters, so they are widely involved in various physiological processes. Human ABC transporters are involved in many physiological processes including the clearance of xenobiotics, nutrient uptake, resistance to exogenous substances, antigen presentation, cholesterol and lipid transport, and stem cell formation. The loss of their functions is closely related to many human diseases.

We focus on membrane proteins related to major human diseases, especially the structural and functional studies of membrane proteins at key nodes of the enterohepatic circulation pathway and the molecular mechanisms of disease occurrence. Enterohepatic circulation refers to the circulation process in which cholates, bilirubin, drugs or other substances are excreted from the liver to the bile duct, then enter the small intestine and are absorbed by intestinal wall cells, and then reach the portal vein through the bloodstream and return to the liver. Cholate is the main component of bile and a surfactant derived from cholesterol. It is an essential small molecule for digesting lipids and fat-soluble vitamins. In the metabolic and circulatory pathway of cholate, a series of ABC transporters such as ABCB4, ABCB11, ABCC2, ABCC3, ABCG1, etc., and other passive transport membrane transporters such as NTCP, OATP, etc. need to work together in relays. The functional imbalance of these key transporters will lead to various human diseases such as cholestasis, Dubin-Johnson syndrome and cholangiocarcinoma.

Based on the structural and mechanistic study of ABCB11, an important transporter of cholate, a new transport mechanism of ABCB11 was revealed. It can couple substrate concentration gradient diffusion and ATP hydrolysis energy to achieve substrate transport (Cell Research 2020 & 2022). In the study of the three-dimensional structure and activation mechanism of ATP8B1, a type IV P-type ATPase that regulates the function of ABCB11, and its accessory protein CDC50 complex, a unique molecular mechanism of self-inhibition and activation by cholate was discovered (PNAS 2022). The above research results respectively elucidate the pathogenic mechanisms of type I and type II cholestasis. When cholate accumulates abnormally in the liver, ABCC2, ABCC3 and ABCC4 can transport cholate out of liver cells to alleviate cholate accumulation. ABCC2 can also excrete conjugated bilirubin into the bile ductules, which is the rate-limiting step in bilirubin metabolism. The functional defect of ABCC2 will cause a transport disorder of conjugated bilirubin, leading to Dubin–Johnson syndrome. We resolved the structures of ABCC2 in the apo-form and in three states of binding to substrate analogs and ATP/ADP respectively, and proposed a unique molecular mechanism in which the regulatory domain finely regulates the substrate recognition and transport of ABCC2 (Nature Communications 2024). We resolved the structures of human ABCC3 in the apo-form and bound to physiological substrates estradiol glucuronide and dehydroepiandrosterone sulfate, and proposed the common characteristics of the substrate binding pockets of multidrug resistance proteins of the same family, providing a basis for the rational design of inhibitors of MRPs (EMBO Journal 2023). By resolving the six complex structures of ABCC4 in the states of binding to physiological substrates and drugs, it was proved that both platelet activator TXA2 and antagonist aspirin are substrates of ABCC4. At the same time, it was found that dipyridamole is a strong competitive inhibitor of ABCC4, and the molecular mechanism of its clinical combination with aspirin was elucidated (Nature Cardiovascular Research 2023). This work was highlighted and highly evaluated by Professor John Hwa of Yale University School of Medicine in the current issue: "For the first time, the platelet substrate and drug transport mechanism is elucidated, providing a theoretical basis for the development of specific platelet antagonists." Cholesterol is the precursor of cholate biosynthesis. Based on the structure of cholesterol transporter ABCG1, the molecular mechanism of its mediated transmembrane transport of cholesterol was revealed, and it was found that high-density lipoprotein and sphingomyelin molecules are very important for the transport function of ABCG1. The research results provide a theoretical basis for the clinical treatment of atherosclerotic diseases (Cell Reports 2022). In addition, we also resolved the structures of human very long chain fatty acid transporter ABCD1 (Nature Communications 2022) and branched chain fatty acid transporter ABCD3 (Cell Discovery 2024), and proposed a general transport cycle mechanism model applicable to ABCD subfamily ABCD1-3. Based on the cultivation and accumulation in the field of human ABC transporters, we were invited to write a review in 2022 (Proteins 2022).

We will continue to use structural biology, enzymology and cell biology methods to study the transport and metabolism of physiological substrates and drugs in the entire enterohepatic circulation. Based on the systematic resolution of the three-dimensional structures of key transporters in the whole pathway, a high-resolution global molecular map of enterohepatic circulation will be constructed; combined with a dual or multiple coupled biochemical and physiological activity detection system, the molecular mechanism of specific transport of various substrates will be revealed, and the molecular mechanism of transmembrane transport and synergistic action will be elucidated. These studies will provide theoretical guidance for related disease diagnosis and new drug development.

IV. Nucleic acid drug discovery and rational design 

(Joint Laboratory of USTC and Shanghai Hawyee Biotech Co., Ltd.)

The development of traditional vaccines and protein drugs is a complex, expensive, slow, and laborious task that requires a large amount of investment. mRNA drugs are expected to greatly change the traditional development methods. The basic principle is to deliver a transcript (mRNA) encoding one or more proteins (immunogens) into the cytoplasm of host cells to produce target proteins (immunogens) and activate the immune response in the body to combat various pathogens. RNA drugs can express or produce therapeutic target proteins and are suitable for the treatment of diseases with definite genetic targets, including infectious diseases, cancers, immune diseases, and neurological diseases.

The production of mRNA drugs neither involves infectious factors nor poses the risk of stable integration with the genome of host cells. Therefore, compared with traditional methods, mRNA drugs have many advantages in safety, effectiveness, and production. The most prominent constraints in the research and development of mRNA drugs include the following two aspects: (1) How to efficiently obtain high-purity mRNA. At present, the production of mRNA mainly relies on in vitro transcription technology. Many enzymes, including RNA polymerase, capping enzyme, ligase, etc., are needed in the production process. Although most enzymes can already be used in industrial production, there are still some problems, such as the polymerization efficiency and specificity of RNA polymerase, and low production efficiency for ultra-long products. Optimizing the enzymes required in the mRNA production process or developing new enzymes is crucial to solve this practical problem. (2) mRNA itself is not stable enough and is easily decomposed. There are many RNA binding proteins (RBPs) in cells. Their binding can make mRNA more stable or more easily degraded. Studying the specific mechanism of action of these RBPs is conducive to deepening the understanding of RNA degradation pathways in cells and thus guiding the early sequence design. The structural characteristics of mRNA itself will also affect its stability. Using artificial intelligence (AI) technology to analyze the massive natural mRNA structures, stabilities, and expressibilities, and predict relatively stable mRNA sequences suitable for in vivo expression is crucial for the research and development of mRNA drugs. It is expected to develop an algorithm that can accurately predict the stability and expressibility of mRNA and be used to guide the development of mRNA drugs.