Viruses are significant pathogens involved in human diseases throughout human history. Many of these virus-related human diseases have zoonotic origins, such as AIDS, bird flu, and severe acute respiratory syndrome (SARS). We focus on the essential stages of the viral life cycle: cellular entry, replication and transcription of the viral genome, and virion assembly and release to identify the pivotal targets for drug development. 

To study the underlying mechanisms of interplay between viruses and their hosts will also help reveal new drug targets. Innate host immunity is the first line of defense against pathogen infection, playing a critical role in host resistance to pathogenic microorganisms. Interestingly, the viruses have evolved a variety of strategies to antagonize host restriction and escape the innate immune response. The interaction between host antiviral proteins and viral antagonizing proteins constitutes a dynamic and sophisticated network. Thus, strategies that boost the roles of host restriction while diminishing viral antagonism may lead to new therapies against viral infection.

Mycology, Biotechnology, and Synthetic Biology

Many valuable microbes are not usually evolved to produce desired products and this necessitates the need to improve/maximize their metabolic and regulatory networks through genetic engineering. The main aim of our research in Mario's laboratory of yeast synthetic biology is to re-engineer some key metabolic pathways from filamentous fungi into Saccharomyces cerevisiae (yeast) cells in other to enhance its abilities to synthesis natural products (NPs). Metabolic pathways associated with NPs are usually encoded into clusters of genes (biosynthetic gene clusters—BGCs) while the traditional methods for the integration of genes into the yeast genome rely on homologous recombination at the loci of auxotrophic markers.  Our project is designed to establish a reliable protocol for the integration of multiple genes into the yeast genome in a single “shot” through CRISPR-Cas9 or CRISPR-Cas12a system with which allows multiple-integration protocol. This will allow us to build a different type of synthetic gene circuits such as complex digital transcription networks for application as biosensors, molecular classifiers, and DNA computing. 

      We are considering a pilot comparative study by assembling in yeast, the “natural” and a “retrosynthetic” gene cluster for a well-known NPs from different filamentous fungi, which grow in extreme conditions. We are also looking at a different way to enhancing their production. In this case, new transcriptional activators for these NPs are designed via the CRISPR-dCas9 system, where dCas9 means “deficient Cas9” i.e. a Cas9 stripped of its nuclease activity. Guide RNA molecules would be designed such that they bind only in the proximity of the target promoter without any off-target effects that could lead even to cell death. This issue might force us to consider other nuclease-deficient proteins (e.g. dCas12a) that bind the DNA in the presence of protospacer adjacent motifs (PAMs) distinct from those recognized by dCas9 (NGG and NAG). Finally, activation of transcription is optimized by fusing different activation domains, such as VPR and VP64, to the chosen deficient Cas protein.

The research of the Gao group covers medicinal chemistry and molecular targeting, synthetic chemistry and organo catalysis, and computer-aided drug design, aimed at the discovery of functional drug delivery carriers and understanding mechanisms of molecular targeting. Specific areas include a) strategies for development of small molecular anti-cancer drugs for targeted therapy, b) design and development of actively transportable small molecule drugs or protein-drug conjugates, c) discovery and development of novel drug-delivery carriers and pharmaceutics based on supramolecular chemistry, d) computer aided molecular design and modeling for innovative drug discovery and mechanistic study of drug transporters.

The research of the Huang group encompasses the following main areas:  

1) Molecular design (AIDD & Chiral Catalyst/ligand Design)

3) New Chemical Space Exploration

Paramagnetic NMR, Pseudocontact Shifts, MS Spectrometry.

Structure and function research of ion channels that act as insecticide targets. Nowadays, ion channels remain the primary targets of many the small molecule insecticides. Voltage-gated sodium (NaV) channels are responsible for the generation and propagation of action potentials in excitable cells and play an important role in insect physiology. However, so far, the binding sites and the molecular mechanism for the modes of actions of these pesticides remain elusive, hindering the improvement of the pesticides based on the rational design. In 2017, Nieng Yan’s group has been solved the structure of a putative NaV channel from American cockroach at 3.8 angstrom resolution by Cryo-EM . The near–atomic resolution structure of this eukaryotic NaV channel establishes an important foundation for investigating the function and disease mechanisms of NaV channels and for structure-guided drug development . Although atomic structures of NaV provides a structural template for the modeling and virtual docking of known pesticides, the accurate binding modes are still not clear. In my future project, I plan to study the complex structures of the insect sodium channel bound with known pesticides by cryo-EM. 

    We’re interested in solving important and interesting scientific problems with structural biology and protein engineering techniques. Currently, our research has been focused on three directions: 1) Molecular mechanism underlying the regulation of vesicle trafficking. 2) Structure based semi-rational engineering of enzymes. 3)Orthogonal system in yeast cells.

1)  Molecular mechanism underlying the regulation of vesicle trafficking.

   Vesicle trafficking is a fundamental cellular process by which membrane-encapsulated vesicles transport materials between different cellular compartments and between a cell and its environment. Malfunction of vesicle trafficking will cause a broad spectrum of severe diseases, such as cancer, diabetes, immune deficiency and neuropathy. Vesicle trafficking occurs in four steps including vesicle biogenesis, transport, docking and fusion with the target membrane, each of which is mediated by a specific concert of protein families. Membrane fusion is mediated by the SNAREs and docking is manly mediated by the MTCs (Multi-subunit tethering complexes). MTCs not only mediate vesicle docking via tethering the trafficking vesicles to the target membrane, but also interact with the SNAREs to promote membrane fusion between them (Figure 1). However, the molecular mechanisms underlying these cellular processes are largely unknown.

Figure 1. MTCs and SNAREs in different stages of vesicle trafficking. MTCs are shown in light blue boxes, and SNAREs in white boxes.

With multidisciplinary strategies including structural biology, biochemistry, cell biology and yeast genetics, we have uncovered the molecular mechanism of exocyst-assembly (Figure 2, ref 1). We continue to work on the functional mechanism of other MTCs, as well as the interaction between MTCs and SNAREs.

Figure 2. The hierarchical assembly of the exocyst complex. The eight exocyst subunits share similar structural folds, with an N-terminal CorEx motif (the blue coiled-coil) followed by a long helical rod to the C-terminus (top left). Sec3 and Sec5, Sec6 and Sec8, Sec10 and Sec15, Ex70 and Exo84 form heterodimers through the pairing of their CorEx motifs (down left). Then Sec3, Sec5, Sec6 and Sec8 form the Subcomplex I via four helix bundle formation of their CorEx motifs; Sec10, Sec15, Exo70 and Exo84 form the Subcomplex II in the same manner (down right). Finally, Subcomplex I and Subcomplex II clap into each other to form the fully assembled exocyst complex (top right).

2) Structure based semi-rational engineering of enzymes.

     The biosynthesis of many important nature products, e.g. ginsenoside, is mediated by plant enzymes. Since the production of these nature products from native plants are often quite low, it is necessary to improve their biosynthesis via bio-techniques. Structure based semi-rational design is a good way to achieve it. Key residues of the enzymes involved in catalysis of substrates could be analyzed based on the structure or predicted structure and known knowledge. And improved enzymes may be obtained via a series of mutations of these residues. In this field, our research includes both structure determination and structure-based engineering of interested enzymes (Figure 3, ref 2).

Figure 3. Semi-rational design of Pq3-O-UGT2. A) The structure model of Pq3-O-UGT2 is superimposed onto crystal structure of UGT74AC1. Mutant residues of engineered UGT74AC1 that highly improved its activities and corresponding residues of Pq3-O-UGT2 are indicated. B) Catalytic conformations of Pq3-O-UGT2 (WT)-UDPG-Rh2 (top) and Pq3-O-UGT2 (S49R/I50M/H85Y, Mutant)-UDPG-Rh2 (bottom) after MD simulations. The mutations improve the interaction between Rh2 and Pq3-O-UGT2.

3) Orthogonal system in yeast cells.

   Recently, a group has reported application of linear plasmids in budding yeast to obtain autonomous hypermutation of interested proteins, including nanobodies and enzymes (Figure 4, ref 3). We are very interested in this powerful and cost-effective system. Thus, right now, we are trying to set up this system in our lab and using it to obtain desired mutations of proteins we are interested in.

Figure 4. An orthogonal replication system based on the p1/2 replication system. The replication of p1/2 linear plasmids is mediated by TP-DNAP1 and TP-DNAP2, which is independent of host DNA polymerases. Thus, mutations could be introduced to p1/2 via TP-DNAP1 during proliferation of the host cells. 

Reference

1. Mei K, Li Y, Wang S, Shao G, Wang J, Ding Y, Luo G, Yue P, Liu JJ, Wang X, Dong MQ, Wang HW, Guo W. Cryo-EM structure of the exocyst complex. Nat Struct Mol Biol. 2018 Feb;25(2):139-146.

2. Yao L, Zhang H, Liu Y, Ji Q, Xie J, Zhang R, Huang L, Mei K, Wang J, Gao W. Engineering of triterpene metabolism and overexpression of the lignin biosynthesis gene PAL promotes ginsenoside Rg3 accumulation in ginseng plant chassis. J Integr Plant Biol. 2022 Jun 22. doi: 10.1111/jipb.13315. Epub ahead of print.

3. Ravikumar A, Arrieta A, Liu CC. An orthogonal DNA replication system in yeast. Nat Chem Biol. 2014 Mar;10(3):175-7. 

The Nakamura group focuses on the regulation of the cellular actin cytoskeleton and in particular on the molecular mechanisms of chemical and mechanical signal transduction (mechanotransduction), a conversion of mechanical forces into cellular biochemical signals. Mechanotransduction is essential for many physiological processes in diverse organisms during development and maintenance of all tissues. Defects in mechanotransduction, often caused by mutations or deregulation of proteins that disturb cellular or extracellular mechanics, are implicated in the development of various diseases, ranging from muscular dystrophies and hypertension-induced vascular and cardiac hypertrophy to cancer progression and metastasis. Despite its importance, little is known about the underlying mechanisms of mechanotransduction. The group uses a wide range of techniques including proteomics, microscopy, molecular biology, and cell biology, and appreciate collaboration with expertises in structural biology, mechanical engineering, single molecular analysis, computer simulation, and drug design.

My research involvs the study of insecticide action/ resistance on agricultural crop pest, Diamond back moth (Plutella zylostella). To accomplish this, I am focussing on the structural aspects of insect ion channels (RyR), FKBP and Cytochrome P450 proteins.

The research in the group of Srinivasan encompasses two main areas, 1) Developing new reaction methodologies: The research topics under this area include bioorthogonal reactions, late-stage modification of advanced chemical entities, C-H activation, and high-throughput amenable synthesis – aiming at advancing the way organic molecules are made for drug discovery and chemical biology applications. 2) Inhibitor discovery based on fragment-based approaches: Design and synthesis of ‘unconventional’ fragments with rich structural diversity. These fragments will be used as a starting point towards novel inhibitors for unexplored biological targets such as the AurB-INCENP interaction.

Wei’s research addresses mechanisms of drug activity with associated drug design.  Computational approaches (e.g., molecular docking, pharmacophore modeling, quantitative structure-activity relationship (QSAR), molecular dynamics) are used to identify and characterize putative ligand binding sites, elucidate binding mechanisms, and guide rational design of potentially new drugs.

The research in the Woycechowsky group focuses on the supramolecular chemistry of proteins. In particular, we are interested in proteins that assemble into symmetrical, closed-shell, polyhedral capsid structures. Protein capsids can act as molecular containers and delivery vehicles for a variety of molecular cargoes, and therefore are useful for bionanotechnological applications, such as drug delivery, catalysis, and materials synthesis. Protein engineering strategies are used to explore and exploit the supramolecular chemistry of protein capsids. This approach is inherently interdisciplinary, utilizing methods from biochemistry, biophysics, molecular biology, organic chemistry, and cell biology. Research projects in our lab fall into three main areas, including 1) capsid self-assembly, 2) molecular encapsulation, and 3) drug delivery.

The Zhang lab identifies and characterizes new enzymes and new metabolic pathways in nature using a combination of bioinformatics, genetic, biochemical and biophysical methods.  In particular, the Zhang lab has a long term interest in metal trafficking, metalloenzymes. and their catalytic mechanisms. Other projects in the Zhang lab include synthetic biology, and immuno-based human disease diagnosis.

For more information about the Zhang Lab, please visit

http://zhangyanlab.org 

The research of Yuchi’s group centers on the structure and function of ion channels. Ion channels are the second largest target class for approved drugs. Drugs targeting ion channels are used to treat arrhythmia, neuropathic pain, epilepsy, anxiety and more. The ultimate goal of our group is to understand the physiological and pathological roles of ion channels at the molecular level. The specific questions we are tackling include: 1) the interaction network and regulation of ion channels involved in heart and muscle diseases; 2) how disease-causing mutations perturb the structure and function of critical ion channels; 3) how to target insect ion channels to develop novel biopesticides. To answer these questions, our lab combines a variety of complementary techniques, including X-ray crystallography, electrophysiology, calorimetry, in-silico drug screening, as well as many other biochemical, biophysical and computational methods.

For detailed information, check our lab website @ www.yuchilab.com 

Channel Regulation at High Resolution

Disease-causing Mutations in Ion Channel

The research in the group of Zhang is encompassed in the areas of chiral separation and proteomics analysis.

    Our group research interest lies in investigating basic mechanism of bio-active molecular (including nitric oxide, hydride and methyl et al.) transfer reaction in enzyme or in solution, with a goal of 1) design of more efficient chemical catalysis as well as the designed enzyme; 2) rational design of corresponding inhibitor/drug basing on the mechanism exploration.  This inspiring research area requires the combination the application of physical organic chemistry, biochemistry, chemical biology and molecular biology. 

   The most recently work refers to the understanding the role of compaction in methyl transfer reactions with the target of finding how the molecular motion in enzymes would affect the catalytic ability in the methyl transfer reaction.  This study about the methyl transfer system had/will extended from catechol-O-methyltransferase (COMT) to glycine-N-methyltransferase (GNMT), Nicotinamide N-Methyltransferase (NNMT) and DNA\RNA demethylation with various experimental approaches such kinetic isotope effect, binding isotope effect, time-resolved spectrometers, hydrogen deuterium exchange with mass spectrometry (HDX-MS) as well as computational simulation. 

The research in the group of Zhou encompasses three main areas, including, 1) Investigation of molecular pathogenesis of diseases and cell signaling pathways, and pharmacological mechanism of drug action, 2) Development of new small molecule based targeting anticancer drugs, e.g., TRAF6 as a new target of anti-tumor therapy, 3) Development of cells and C. elegans models, for high-throughput screenings, e.g., anti-aging drugs.