GEFs play an important role in the control of small G proteins
QIU Jiaming, 5 May 2022
1. Rho superfamily
Rho GTPases are molecular switches that control a wide variety of signal transduction pathways inside cells. The Ras superfamily consists of more than 150 family members and can be divided into five categories, including five conserved subfamilies – Ras, Rho, Ran, Rab, and Arf(see Figure 1) – each with its own characteristics in terms of sequence, structural similarity, and cellular function. The Ras subfamily contains 36 members responsible for regulating signaling pathways involved in cell proliferation and for regulating signaling pathways involved in cell proliferation, morphology, differentiation, and cellular functions. The largest subfamily of the Ras superfamily is Rab, which consists of more than 60 members involved in vesicle and protein transport in cells. 30 members of the Arf family act as regulators of intracellular transport. The Ras subfamily consists of 36 members and is responsible for regulating signaling pathways involved in cell proliferation. The 20 members of the Rho subfamily are responsible for the regulation of signaling pathways involved in cell proliferation, transport and differentiation, and cell survival. The 20 members of the Rho subfamily are key regulators of actin organization, gene expression and cell cycle progression. The largest one subfamily, Rab, consists of more than 60 members and is involved in vesicle and protein transport. involved in vesicle and protein transport. There are 30 members of the Arf protein members that are also regulators of intracellular transport.Ran subfamily. The Ran subfamily has only one protein, which is the most abundant small GTPase in the cell. It is the most abundant small GTPase in the cell and is involved in nuclear transport.
2. GEFs and GAPs
Guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) regulate the activity of small guanine nucleotide-binding (G) proteins for the purpose of regulating cellular functions (See Figure2). In general, GEFs regulate signaling by catalyzing the exchange of G protein-bound GDP to GTP, whereas GAPs terminate signaling by inducing GTP hydrolysis. Recent evidence suggests that these proteins may be potential therapeutic targets for the development of drugs to treat a variety of diseases, including anticancer antibacterial. GEFs contain a variety of species, all of which activate Cdc42 by mediating the exchange of GDP with GTP in Cdc42. during activation, when receiving external signals, GEFs such as Dbl bind to inactive Cdc42, forming a Cdc42-GEF binary complex, causing GDP binding to fall off from small G proteins, followed by the release of GTP from Cdc42 due to the cell membrane when GTP can be more than ten times that of GDP. GEF binds with low affinity to active Cdc42 and is therefore released from Cdc42-GTP. Active Cdc42 interacts with downstream effectors such as N-WASP to produce biological functions such as actin polymerization. GTPase activating proteins or GAPs promote GTP hydrolysis of Cdc42 and return Cdc42 to its basal inactive state
Human Cdc42 is a small GTPase of the Rho family that plays a very important role in the cellular life cycle, including the regulation and control of intracellular signaling pathways such as cell morphology, cell migration, cell polarization, and cell cycle progression. Rho GTPases are key factors in regulating intracellular actin cytoskeleton assembly and reorganization, which can regulate cell function by regulating is cell adhesion and migration, as well as being major players in pathogen infestation of host cells such as EPEC. Activation of Cdc42 requires regulation by members such as GEFs to undergo conformational changes, and from cdc42GDP to cdc42GTP transformation, which initiates the reorganization of actin and regulates the adhesion, migration and invasion of cells as well as pathogenic microorganisms. Thus, the regulation of cdc42 affects numerous physiological functions of cells and has become a hot topic of research.
- Takai et al. Small GTP-Binding Proteins. Physiol. 2001
- Jeffrey R et al. Proceedings of the National Academy of Sciences, 2001
- Soniya Sinha, Wannian Yang. Cellular signalling. 2008
Emerging New Concepts of Degrader Technologies
YUAN Dingdong, 28 April 2022
Human genetic studies have shown that many novel target proteins cause disease by acquiring functional toxicity. Traditional drug development strategies need to occupy binding sites to inhibit the functional activity of target proteins, which makes disease-related scaffold proteins, transcription factors, and other non-enzymatic proteins “undruggable”. These “undruggable” disease-causing proteins can be corrected or degraded by enhancing protein quality control systems.
Selective degradation of pathogenic protein levels is a more effective strategy. Nucleic acid-based RNA- or DNA-targeting agents for gene silencing are one approach, while gene therapy is promising, it still faces some key challenges such as being difficult to deliver and prohibitively expensive. Therefore, inducing degradation of target proteins is more feasible and can meet clinical needs (Fig. 1).1
- 2. Total synthesis of quinine
Small molecules are able to induce selective degradation of the protein of interest (POI) by adding tags recognized by the degradation machinery. In 2011-2012, Crews and colleagues reported POI degradation induced by small molecule hydrophobic tags (HyT). HyT is achieved by a molecule composed of a hydrophobic segment and a ligand that binds to POI. Thus, HyT can add hydrophobic fragments to the POI, resulting in structural changes of the target protein and subsequent proteolysis (Fig. 1).1
At present, the main method of small molecule-induced protein degradation is the protein degradation targeted chimera (PROTAC) technology. The PROTAC molecule increases the ubiquitination efficiency of the target protein by shortening the distance between the target protein and E3 ligase, thereby inducing the proteasome system to degrade the target protein. However, PROTAC has certain limitations. For example, this method relies on a specific E3 ligase, which limits its application in certain cells. Its molecular weight is generally too large, and it cannot target the secret proteins and membrane proteins (Fig. 1).1
LYTAC (Lysosome Targeted Chimera) technology is a method that utilizes the endosome/lysosome pathway to degrade target proteins. PROTAC technology mainly targets intracellular proteins, while LYTAC can act on membrane proteins and extracellular proteins that are resistant to PROTAC (such as EGFR). LYTAC molecules consist of antibodies against a specific target protein and are covalently linked to mannose 6-phosphate (M6P). Antibodies are recognized by endogenous systems, and their bound target proteins are transported to lysosomes for degradation. The advantage of LYTAC technology is that it utilizes the endogenous degradation pathway to degrade extracellular proteins and membrane proteins. The main disadvantage is that the molecular weight is large, and the antibody or polypeptide in the molecule may induce an immune response (Fig. 2).2
In March 2021, they published a new work in the journal Nature Chemical Biology, revealing a LYTAC technology that utilizes the asialoglycoprotein receptor (ASGPR). Since ASGPR is a liver-specific lysosomal targeting receptor, LYTAC technology utilizing ASGPR is able to degrade extracellular proteins in a cell-type-restricted manner. Specifically, the study conjugated the binder triantenerrary N-acetylgalactosamine (tri-GalNAc) of ASGPR to the target protein through a linker. Based on these findings, the researchers believe that GalNAc-LYTAC represents a cell-type-restricted lysosomal protein degradation pathway that further paves the way for the clinical translation of LYTAC technology (Fig. 2).3
Autophagy-targeted chimeras (AUTACs) have a similar design to PROTACs and are well suited to degrade cytoplasmic target proteins resistant to PROTAC molecules. POIs are first phagocytosed to form autophagosomes, and then autophagy receptors such as SQSTM1/p62 recognize Lys63 (K63) polyubiquitinated POIs and transfer them to autophagosomes for degradation. Both AUTAC and PROTAC molecules act through ubiquitination, but AUTAC molecules induce target degradation by triggering K63 polyubiquitination. The study found that AUTAC molecules can degrade target proteins and damaged organelles such as mitochondria (Fig. 3).4
Autophagosome-binding compounds (ATTECs) are a more direct approach to utilize autophagy to degrade target proteins. Unlike PROTAC and AUTAC, ATTEC molecules are not associated with ubiquitination. ATTEC molecules can directly bind the target protein to LC3 and promote the phagocytosis of the target protein by autophagosomes. mHTT (mutated mHTT) is the main cause of Huntington’s disease (HD), and this protein has repeat-expanded polyglutamine (polyQ). Studies have shown that autophagosome-conjugated compounds can degrade mHTT in cells or animal models and attenuate HD-related phenotypes. In vitro experiments showed that ATTEC molecules can interact specifically with polyQ to degrade mHTT without affecting the level of wtHTT selectively. ATTEC molecules are also able to degrade other disease-causing polyQ proteins. By directly interacting with the autophagosome protein LC3 and bypassing the ubiquitination process, ATTEC molecules have great potential to degrade DNA/RNA molecules, damaged organelles, and other non-protein cargoes recognized by autophagy. Whether ATTEC molecules affect overall autophagic activity remains to be investigated. Compared with the molecular weights of PROTAC, LYTAC, and AUTAC, the molecular weight of ATTEC targeting mHTT is very small, which may have better drug properties (Fig. 4).5
The project team designed ATTEC compounds (LD-ATTEC) with lipid droplets, the intracellular fat-storing organelles, as degradation targets. Excessive accumulation of lipid droplets may be associated with various diseases, such as obesity, non-alcoholic fatty liver disease, and neurodegeneration. Therefore, the degradation of lipid droplets may have beneficial effects on related diseases, and LD-ATTEC can also be used as a tool compound to study the causal association of lipid droplets with these diseases (Fig. 5).6
The lysosomal pathway can theoretically degrade various biological macromolecules and even organelles. On the other hand, lysosomes can also degrade proteins that were originally degraded by the proteasome. Therefore, lysosome-targeted degradation technology may theoretically degrade various pathogenic proteins, protein aggregates, DNA/RNA, organelles, pathogens, lipids, peroxisomes, and other disease-related substances.
LYTAC, AUTAC, and ATTEC, these lysosome targeting technologies, have different principles. LYTAC mainly utilizes the endosome-lysosome pathway, which is suitable for extracellular and cell membrane proteins. AUTAC is based on selective autophagy mediated by K63 ubiquitination and can act on intracellular proteins that K63 can ubiquitinate. ATTEC directly binds the target to be degraded to the autophagosome, so the target range that can be degraded is not limited to proteins.
- Ding, Y., Fei, Y., & Lu, B. (2020). Emerging New Concepts of Degrader Technologies. Trends In Pharmacological Sciences, 41(7), 464-474. doi: 10.1016/j.tips.2020.04.005
- Banik, S., & Bertozzi, C. (2020). Lysosome-targeting chimaeras for degradation of extracellular proteins. Nature, 584(7820), 291-297. doi: 10.1038/s41586-020-2545-9
- Roggenbuck, D., Mytilinaiou, M., Reinhold, D., & Conrad, K. (2012). Asialoglycoprotein receptor (ASGPR): a peculiar target of liver-specific autoimmunity. Autoimmunity Highlights, 3(3), 119-125. doi: 10.1007/s13317-012-0041-4
- Takahashi, D., Moriyama, J., Itto-Nakama, K., & Arimoto, H. (2019). AUTACs: Cargo-Specific Degraders Using Selective Autophagy. Molecular Cell, 76(5), 797-810.e10. doi: 10.1016/j.molcel.2019.09.009
- Li, Z., Wang, C., Wang, Z., Ding, C., Ding, Y., Fei, Y., & Lu, B. (2019). Allele-selective lowering of mutant HTT protein by HTT–LC3 linker compounds. Nature, 575(7781), 203-209. doi: 10.1038/s41586-019-1722-1
- Fu, Y., Chen, N., Ding, Y., & Lu, B. (2021). Degradation of lipid droplets by chimeric autophagy-tethering compounds. Cell Research, 31(9), 965-979. doi: 10.1038/s41422-021-00532-7
Story of Quinine: Structure, History, and Total Synthesis
BAO Yishu, 21 April 2022
1. Discovery of quinine
Tonic water glows faintly in sunlight and this phenomenon is even more apparent under ultraviolet light irradiation. It arises from tonic water’s main ingredient, quinine. The glow of a quinine solution was firstly noticed by the polymath Sir John Herschel.1 A few years later, this phenomenon was called “fluorescence” by the physicist and mathematician Sir George Stokes.
Quinine is an alkaloid extracted from the bark of Cinchona trees indigenous to South American forests, which attracted intense interest worldwide on account of its prophylactic characteristics. Its name is derived from quina-quina and it can be roughly translated as bark of barks. Native South Americans used it to alleviate shivering, fever, and pain.2 Later, they found that quinine also promptly relieved the symptoms of malaria and quinine became well-known around the world for it. Before artemisinin was discovered, quinine was the drug of choice for the treatment of malaria (Figure 1).
- 2. Total synthesis of quinine
The chemical structure of quinine is complex and its total synthesis confounded chemists for over a century since its first isolation from the bark in 1820 by two French pharmacists. In 1856, Sir William Henry Perkin who was only 18 years old, attempted to synthesize quinine by oxidizing N-allyltoluidine. He failed but he generated mauveine which is the first synthetic organic dye to be synthesized on a large scale for industrial and commercial purposes (Figure 2).3 Subsequent attempts by Paul Rabe and Karl Kindler in the early twentieth century. This sequence involved: (1) oxidation of d-quinotoxine with sodium hypobromite to produce N-bromoquinotoxine; (2) base-mediated cyclization to produce quininone; and (3) aluminum powder reduction of quininone to produce quinine and quinidine (Figure 3).4
Regarding the breakthrough progress in the total synthesis of quinine, we must mention an organic chemistry legend, Robert Burns Woodward. Woodward was born in Boston in 1917. In 1933, 16-years-old Woodward enrolled as an undergraduate at Massachusetts Institute of Technology (MIT). In 1936, he received a Bachelor of Science degree. Only one year later, MIT awarded him a doctorate. In 1944, World War II broke out, which made the spread of malaria especially fast, resulting in a short supply of the quinine. At this time, Woodward made an amazing decision to totally synthesize quinine. He and his postdoctoral researcher William von Eggers Doering reported the synthesis of quinine, starting from using 3-hydroxybenzaldehyde, through 55 reactions including condensation, cyclization, alkylation, reduction, acetylation, oxidation, and so on. The final step is the Claisen condensation and finally, the generation of the racemic quinidone after acid treatment (Figure 4).5
In addition to quinine, Woodward and his research group’s achievements in total synthesis are breathtaking: cholesterol, cortisone, lanosterol, strychnine, tetracylines, colchicine, reserpine, lysergic acid and ergonovine, chlorophyll, cephalosporin C, erythromycin, and vitamin B12 (the last, with Albert Eschenmoser). Those are just a few among the herd of his synthetic accomplishments. In 1965, Woodward won the Nobel Prize in Chemistry for his outstanding achievements in organic synthesis.
However, the total synthesis of quinine reported by Woodward and Doering did not address the stereoselective problem. In 2001, Gilbert Stork reported the first entirely stereoselective total synthesis of (−)-quinine (Figure 5).6 The Staudinger reaction involved in the synthetic route proposed by Stork was developed by Bertozzi’s group as a bioorthogonal reaction, the Staudinger coupling reaction in 2000 (Figure 6).7 the Staudinger ligation is a modification of the classical Staudinger reaction in which an electrophilic trap is placed on the triaryl phosphine. In aqueous media, the aza-ylide intermediate rearranges, to produce an amide linkage and the phosphine oxide, and is so named the Staudinger ligation because it ligates two molecules together, whereas, in the classical Staudinger reaction, the two products are not covalently linked after hydrolysis. Staudinger ligation has been used for the labeling of glycans, lipids, DNA, and proteins.
In summary, the total synthesis of quinine is too complicated. Still, the main source of commercial quinine today remains Cinchona bark. Quinine serves to both treat and prevent malaria, but how precisely it exerts its killing effect on the virulent Plasmodium falciparum parasite has so far remained elusive. There is much evidence to indicate that it interferes with the parasite’s haemoglobin degradation pathway. In addition, a recent study used a cellular thermal shift assay coupled with mass spectrometry to identify a protein target, P. falciparum purine nucleoside phosphorylase (PfPNP) (Figure 7).8
Nowadays, the Cinchona alkaloid is still ubiquitous. It continues to be clinically used as a medicine to treat severe babesiosis, its blue glow serves as a fluorescence standard, and it is a useful ligand in asymmetric organocatalysis. It also remains a key ingredient in tonic water.
- Herschel, J. F. W. Phil. Trans. R. Soc. Lond. 1845, 135, 143-145.
- Kaufman, T. S. Rúveda, E. A. Angew. Chem. Int. Ed. 2005, 44, 854-885.
- Perkin, W. H. J. Chem. Soc. Trans. 1896, 69, 596-637.
- Smith, A. C. Williams, R. M. Angew. Chem. Int. Ed. 2008, 47, 1736-1740.
- Woodward, R. B. Doering, W. E. J. Am. Chem. Soc. 1945, 67, 860-874.
- Stork, G. Niu, D. Fujimoto, R. A. Koft, E. R. Balkovec, J. M. Tata, J. R. Dake, G. R. J. Am. Chem. Soc. 2001, 123, 3239-3242.
- Saxon, E. Bertozzi, C. R. Science 2000, 287, 2007-2010.
- Dziekan, J. M. Yu, H. Chen, D. Dai, L. Wirjanata, G. Larsson, A. Prabhu, N. Sobota, R. M. Bozdech, Z. Nordlund, P. Sci. Transl. Med. 2019, 11, eaau3174.
Photobiocatalysis-a new way for transformation
CHEN Hongfei, 14 April 2022
1. The era of antibiotic discovery
Biocatalysis reaction especially enzyme reaction has advantages in high chemical selectivity and good stereoselectivity. However, this kind of reaction always has limited substrates scope. On the other hand, photocatalysis reaction which using visible light as energy source can generate open-shell intermediates for different reactions. But the poor stereoselectivity is one of the drawbacks of this kind of reaction. Recent years, researchers combine the biocatalysis and photocatalysis together for new transformations which based on visible light and enzymes. The photobiocatalysis are divided into three main parts: a) natural photoenzymes; b) new reactivity of cofactor dependent enzymes; c) synergistic combination of photocatalyst and enzymes (Fig.1.).
The first kind of photobiocatalysis is natural photoenzyme. A recent example is the discovery and characterization of a fatty acid photodecarboxylase (CvFAP). CvFAP was first reported by Beisson and co-workers in 2017. This enzyme can use FAD and light to produce alkanes and alkenes from long-chain fatty acids (Fig.2.). [2,3]
Since the discovery of the newly characterized CvFAP, a variety of photoenzymatic transformations have been disclosed by tuning substrate specificity and function. Very recent studies of engineering CvFAP for a photodecarboxylative deuteration and for a selective photodecarboxylation of trans fatty acids were reported by Wu and co-workers. Given the growing number of applications using this newly discovered CvFAP, it is sure that the discovery of other new photoenzymes will enable the development of new to- nature chemical transformations.
The second kind of photobiocatalysis is new reactivity of cofactor dependent enzymes. The redox active cofactors of these enzymes can form electron donor−acceptor (EDA) complexes with unnatural substrates which can generate radical species under mild conditions through single-electron transfer (SET) events. In 2016, Hyster and co-workers demonstrated the first example of the use of photoexcited enzyme−substrate complexes for achieving non-natural reactivity in enzymes. They discovered NADPH-dependent ketoreductases, which normally catalyze the nonlight-driven reduction of carbonyl functional groups, could catalyze the radical dehalogenation of halolactones through the photoexcitation of a halolactone-NADPH EDA complex. Very recently, they expanded the scope of the reductive cyclization to chiral tertiary amine synthesis by altering the radical acceptor from a tethered C=C double bond to an oxime (Fig.3.). [5,6,7]
The third kind of photobiocatalysis is synergistic combination of photocatalyst and enzymes. Currently, there are three strategies to synergize external photoredox catalysts and enzymes for abiological transformations. In each strategy, the enzyme is used to control selectivity of the reaction, and the external photoredox catalyst can be used for (I) selective single-electron reduction of the enzyme-bound substrate, (II) substrate racemization enabled by the photoinduced SET, or (III) E/Z-isomerization of alkenes via energy transfer. In the last five years, researchers have leveraged the reactivity of photocatalysts and selectivity of enzymes for difficult asymmetric syntheses. It is expected that emerging strategies for dual photocatalysis, and enzyme catalysis can be applied to enzymes without light absorbing cofactors and that the compatibility of photocatalysts with enzyme cofactors could be further explored for abiological transformations. [8,9,10]
Compared to the booming development of photocatalysis and biocatalysis, the application of photobiocatalysis for the discovery and development of non-natural chemical transformations is still developing at a much slower pace. In the future, more chemical reactions can be discovered and potential interest in exploring the scalability of photobiocatalytic processes for industrial manufacturing.
 Wesley Harrison, Xiaoqiang Huang, and Huimin Zhao. Photobiocatalysis for Abiological Transformations. Acc. Chem. Res. 2022, 55, 8, 1087–1096
 Sorigué, D.; Légeret, B.; Cuiné, S.; Blangy, S.; Moulin, S.; Billon, E.; Richaud, P.; Brugière, S.; Couté, Y.; Nurizzo, D.; Müller, P.; Brettel, K.; Pignol, D.; Arnoux, P.; Li-beisson, Y.; Peltier, G.; Beisson, F. An Algal Photoenzyme Converts Fatty Acids to Hydrocarbons. Science 2017, 357, 903−907
 Sorigué, D.; Hadjidemetriou, K.; Müller, P.; Beisson, F. Mechanism and Dynamics of Fatty Acid Photodecarboxylase. Science 2021, 372, eabd5687.
 Li, D.; Han, T.; Xue, J.; Xu, W.; Xu, J.; Wu, Q. Engineering Fatty Acid Photodecarboxylase to Enable Highly Selective Decarboxylation of trans Fatty Acids. Angew. Chem., Int. Ed. 2021, 60, 20695−20699.
 Emmanuel, M. A.; Greenberg, N. R.; Oblinsky, D. G.; Hyster, T. K. Accessing Non-Natural Reactivity by Irradiating Nicotinamide- Dependent Enzymes with Light. Nature 2016, 540, 414−417.
 Biegasiewicz, K. F.; Cooper, S. J.; Gao, X.; Oblinsky, D. G.; Kim, J. H.; Garfinkle, S. E.; Joyce, L. A.; Sandoval, B. A.; Scholes, G. D.; Hyster, T. K. Photoexcitation of Flavoenzymes Enables a Stereoselective Radical Cyclization. Science 2019, 364, 1166−1169.
 Black, M. J.; Biegasiewicz, K. F.; Meichan, A. J.; Oblinsky, D. G.; Kudisch, B.; Scholes, G. D.; Hyster, T. K. Asymmetric Redox- Neutral Radical Cyclization Catalysed by Flavin-Dependent ‘Ene’- Reductases. Nat. Chem. 2020, 12, 71−75.
 Biegasiewicz, K. F.; Cooper, S. J.; Emmanuel, M. A.; Miller, D. C.; Hyster, T. K. Catalytic Promiscuity Enabled by Photoredox Catalysis in Nicotinamide-Dependent Oxidoreductases. Nat. Chem. 2018, 10, 770−775
 DeHovitz, J. S.; Loh, Y. Y.; Kautzky, J. A.; Nagao, K.; Meichan, A. J.; Yamauchi, M.; MacMillan, D. W. C.; Hyster, T. K. Static to Inducibly Dynamic Stereocontrol: The Convergent Use of Racemic β-Substituted Ketones. Science 2020, 369, 1113−1118
 Litman, Z. C.; Wang, Y.; Zhao, H.; Hartwig, J. F. Cooperative Asymmetric Reactions Combining Photocatalysis and Enzymatic Catalysis. Nature 2018, 560, 355−359.
Computer-aided engineering of enzymatic selectivity
WANG Yue, 7 April 2022
Life has evolved an astonishing array of enzymes that play a wide range of roles in vital biological processes. Intriguingly, enzymes with such remarkable power, selectivity, and sustainability strongly encourage biochemists to supplement or even replace some traditional chemical synthesis routes on a large scale. Although the trend toward biosynthesis of natural products or blockbuster drugs in diverse industries strongly drives the development of enzymes that are capable of catalyzing challenging reactions, even non-natural ones, there is still a mismatch between the inefficiency of enzymes toward non-native substrates and the need for complex chemicals in diverse industries.
The development of credible mechanistic insights requires a high-quality structure in knowledge-based protein engineering. Compared to decades ago, enzyme crystallization has advanced significantly, but is still a much slower process than sequence accumulation. As a result, a variety of computer-based prediction approaches are available that can effectively provide acceptable enzyme structures, which are mainly homology alignments based on templates and conformational sampling strategies without templates (Kuhlman and Bradley, 2019). Meanwhile, machine learning has also been used to explore deeper buried information in structure databases to improve the accuracy of structure prediction, especially in the case of the latest alphafold2 in CASP14 (Service, 2020).
By interacting with the substrate, enzymes perform their catalytic function. As a fundamental tool to predict the optimal binding mode of ligands and enzymes, molecular docking was inspired by “lock-and-key” (Fischer, 1894), “induced-fit” (Koshland, 1958), and “configurational selection” (Kumar et al., 2000) models. These approaches included rigid docking, semi-flexible docking, flexible docking, and the latest CaverDock, which considered substance transport in enzyme channels. The precondition for excellent molecular docking results is the use of high-quality structures, which can be obtained either experimentally or computationally.
Static snapshot structures are often inadequate to analyze dynamic enzyme-catalyzed processes, including substrate binding, chemical bond cleavage, and product release. Dynamic conformational ensembles are continuously sampled using MD simulation, a powerful computational scheme based on classical mechanics (Hollingsworth and Dror, 2018). Additionally, MD simulations of large biosystems are approaching experimentally relevant timescales, raising the possibility of mutually reinforcing experimental and theoretical findings, providing new opportunities in rationalizing and engineering enzymes (Cerutti and Case, 2019; Franz et al., 2020; Huggins et al., 2019; Surpeta et al., 2020). At present, various useful MD methods have been developed, such as MD simulations incorporating enhanced sampling to reveal ligand migration (Rydzewski and Nowak, 2017) and coarse-grained MD simulations to cope with multiscale biological processes (Kmiecik et al., 2016; Souza et al., 2020).
Kuhlman, B., Bradley, P., 2019. Advances in protein structure prediction and design. Nat. Rev. Mol. Cell Biol. 20 (11), 681–697.
Service, R.F, 2020. The game has changed. AI triumphs at solving protein structures. Science.
Fischer, E., 1894. Einfluss der configuration auf die wirkung der enzyme. Berichte der deutschen chemischen Gesellschaft 27 (3), 2985–2993.
Koshland, D.E., 1958. Application of a theory of enzyme specificity to protein synthesis. Proc. Natl. Acad. Sci. U. S. A. 44 (2), 98–104.
Kumar, S., Ma, B., Tsai, C.-J., Sinha, N., Nussinov, R., 2000. Folding and binding cascades: dynamic landscapes and population shifts. Protein Sci.9 (1), 10–19.
Hollingsworth, S.A., Dror, R.O., 2018. Molecular dynamics simulation for all. Neuron 99(6), 1129–1143.
Cerutti, D.S., Case, D.A., 2019. Molecular dynamics simulations of macromolecular crystals. WIREs Comput. Mol. Sci 9 (4).
Franz, F., Daday, C., Gr¨ater, F., 2020. Advances in molecular simulations of protein mechanical properties and function. Curr. Opin. Struct. Biol. 61, 132–138.
Huggins, D.J., Biggin, P.C., D¨amgen, M.A., Essex, J.W., Harris, S.A., Henchman, R.H., Khalid, S., Kuzmanic, A., Laughton, C.A., Michel, J., Mulholland, A.J., Rosta, E., Sansom, M.S.P., van der Kamp, M.W., 2019. Biomolecular simulations: from dynamics and mechanisms to computational assays of biological activity. WIREs Comput. Mol. Sci 9 (3).
Surpeta, B., Sequeiros-Borja, C.E., Brezovsky, J., 2020. Dynamics, a powerful component of current and future in silico approaches for protein design and engineering. Int. J. Mol. Sci. 21 (8), 2713.
Rydzewski, J., 2020. maze: heterogeneous ligand unbinding along transient protein tunnels. Comput. Phys. Commun 247, 106865.
Kmiecik, S., Gront, D., Kolinski, M., Wieteska, L., Dawid, A.E., Kolinski, A., 2016. Coarsegrained protein models and their applications. Chem. Rev. 116 (14), 7898–7936.
Souza, P.C.T., Thallmair, S., Conflitti, P., Ramírez-Palacios, C., Alessandri, R., Raniolo, S., Limongelli, V., Marrink, S.J., 2020. Protein–ligand binding with the coarse-grained Martini model. Nat. Commun. 11 (1), 3714.
Antibiotic resistance crisis-the sword of Damocles hanging in the human head
Chunjian Wu, 24 March 2022
1. The era of antibiotic discovery
For most of human history, bacterial pathogens have been a major cause of disease and mortality. In thepre-antibiotic era of the early 1900s, people had no medicines against these common germs and as a result, human suffering was enormous. Death from infection could follow from something as minor as a scratch. But this situation changes after the first antibiotic—penicillin was discovered in 1928 by Scottish scientist Alexander Fleming. Suddenly, the morbidity and mortality sharply were reduced by this“miracle drugs”, which also raises a lot of attention all over the world. Shortly afterward, the golden era of antibiotic launched, and the main classes of antibiotics were discovered in a fairly short period of time (Figure 1).
Antibiotics have revolutionized the field of medicine. They have saved millions of lives each year, alleviated pain and suffering, and have even been used prophylactically for the prevention of infectious diseases. What is more, antibiotics have been crucial in the increase in life expectancy in the United States from 47 years in 1900 to 74 years for males and to 80 years for females in the year 2000 (The Journal of antibiotics, 70(5), pp.520-526.). But the discovery of antibiotic resistance appeared as a disturbing storm-cloud in this clear sky.
The crisis of antibiotic resistance
In fact, such a continuous progress in discovery of new antibiotics in the 1940–1980 period was mostly fostered by the necessity of “fighting new antibiotic resistances,” where the new discoveries compensated for the emergence of new resistances in bacterial pathogens. The golden era ended rather abruptly in the early 1960s (Figure 1). Infectious disease is now the second leading killer in the world, number three in developed nations and fourth in the United States (The Journal of antibiotics, 70(5), pp.520-526.). For example, current worldwide deaths attributable to antimicrobial resistance have been estimated at ~700,000 per year, rising to 10 million per year by 2050 if present trends continue (Science, 2021, 373(6554), pp.471-471.).
Despite the fact that the spread of antibiotic-resistant bacteria poses a substantial threat to morbidity and mortality worldwide, pharmaceutical research and development has failed to meet the clinical need for new antibiotics. In the past 20 years, only two new antibiotic classes (lipopeptides and oxazolidinones) have been developed and approved by international drug agencies (US Food and Drug Administration and European Medicines Agency)—both of which provide coverage against Gram-positive bacteria. More sadly, the drugs against Gram-negative bacteria have not been developed after the last novel drug, quinolones , discovered in 1962
The World Health Organization (WHO) recently introduced a list of priority pathogens (The Lancet Infectious Diseases, 18(3), pp.318-327.) and noted those of “critical priority”-drug-resistant Enterobacteriaceae (E. coli, Salmonella typhimurium, Klebsiella pneumoniae, Enterobacter), Pseudomonas aeruginosa, and Acinetobacter baumannii, which are responsible for a global health problem. All of the critical priority pathogens are Gram-negative bacteria. The antibiotic resistance crisis threatens the return of the pre-antibiotic era of epidemics and pandemics. It is important to note that antibiotics not only save lives, but enable modern medicine. Without antibiotics, surgery, chemotherapy, or organ transplantation become highly problematic.
Targets of antibiotics targets and resistance mechanisms
Antibiotics are available that effectively inhibit bacterial cell wall synthesis, protein synthesis, and DNA replication (Figure 3a). But bacteria have evolved an array of mechanisms that enable them to resist the inhibitory action of antibiotics. There exists by now an extensive body of knowledge regarding the nature of different mechanistic classes of antibiotic resistance (Figure 3a). Most such mechanisms serve either to reduce antibiotic concentration in the vicinity of the molecular target (through active efflux or chemical inactivation of the antibiotic) or involve a fundamental change in the nature of the target that reduces the antibiotic’s inhibitory effect (for example, as a consequence of mutation or chemical modification). Recent work has revealed that target protection is a key mechanistic player in clinically significant antibiotic resistance that affects diverse classes of antibacterial drugs and is prevalent in bacterial pathogens.
The spread of antibiotic resistance in bacterial pathogens
The emergence of antimicrobial resistant bacteria is not limited to humans or human medicine only, but is influenced by multiple direct and indirect pathways, that form a complex web of interactions that contribute to the selection and spread of resistance in the human/animal microbiota and the environment (Figure 4)
WHO and numerous other groups and researchers agree that the spread of bacterial antimicrobial resistance is an urgent issue requiring a global, coordinated action plan to address（The Lancet. 2022, 399 (10325): 629-655）. Understanding how antibiotic resistance evolves and spreads is key to improving antibiotic treatment strategies.
Pathogenic bacteria acquire resistance through two fundamentally different genetic mechanisms: conjugation and transduction (Figure 5). Resistance genes to clinically relevant antibiotics are often carried on plasmids (circular pieces of DNA) that can be transferred between different types of bacteria. Dissemination of drug resistance is mainly due to the ability of bacteria to acquire genes through horizontal transfer mechanisms, primarily by bacterial conjugation. During conjugation, DNA is transferred from a donor to a recipient cell by direct contact. The resulting transconjugant cell will, in turn, disseminate the conjugative DNA, generally as a plasmid that carries all the genes required for its maintenance and transfer. For example, bacteria that are exposed to antibiotics can survive if they have already received a plasmid with an antibiotic resistance gene from another bacterium.
Collaborative action is critical for dealing with antibiotic resistance
COVID-19 has shown that it is possible to create robust public-private partnerships across research, industry, and public health that accelerate research and clinical trials and spur proactive regulation in the context of a global public health threat (Science, 373(6554), pp.471-471.). Collaborative action is equally necessary to battle antimicrobial resistant bacteria.
The scientific community should leverage lessons learned from COVID-19 to unite academia, industry, government, and policy-makers toward preserving the benefits of modern medicine. Continued procrastination will only lead to countless lives lost to antimicrobial resistant bacteria.
- Lewis, K., 2020. The science of antibiotic discovery. Cell, 181(1), pp.29-45.
- Martens, E. and Demain, A.L., 2017. The antibiotic resistance crisis, with a focus on the United States. The Journal of antibiotics, 70(5), pp.520-526.
- Kwon JH, Powderly WG. The post-antibiotic era is here. Science. 2021 Jul 30;373(6554):471-.
- Laxminarayan, R., Van Boeckel, T., Frost, I., Kariuki, S., Khan, E.A., Limmathurotsakul, D., Larsson, D.J., Levy-Hara, G., Mendelson, M., Outterson, K. and Peacock, S.J., 2020. The Lancet Infectious Diseases Commission on antimicrobial resistance: 6 years later. The Lancet Infectious Diseases, 20(4), pp.e51-e60.
- Boolchandani, M., D’Souza, A.W. and Dantas, G., 2019. Sequencing-based methods and resources to study antimicrobial resistance. Nature Reviews Genetics, 20(6), pp.356-370.
- Murray, C.J., Ikuta, K.S., Sharara, F., Swetschinski, L., Aguilar, G.R., Gray, A., Han, C., Bisignano, C., Rao, P., Wool, E. and Johnson, S.C., 2022. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. The Lancet.
- Flandroy, L., Poutahidis, T., Berg, G., Clarke, G., Dao, M.C., Decaestecker, E., Furman, E., Haahtela, T., Massart, S., Plovier, H. and Sanz, Y., 2018. The impact of human activities and lifestyles on the interlinked microbiota and health of humans and of ecosystems. Science of the total environment, 627, pp.1018-1038.
- MacLean, R.C. and San Millan, A., 2019. The evolution of antibiotic resistance. Science, 365(6458), pp.1082-1083.
Production of Semi-synthetic Artemisinin in engineered yeast
Jiang Hao, 17 March 202
1. Artimisinin, a key in the battle against malaria
In 2016, malaria killed an estimated 445,000 people, 90% of them in Africa. That is, one person dies every 71 seconds, caused by preventable and treatable diseases. Despite a 25% drop in deaths since 2010, the World Health Organization recently warned that progress was stalling and called for a redoubled effort to combat the scourge.
Some of the most critical weapons in this battle are artemisinin-based combination therapy (ACT), considered the most effective antimalarial drug. They contain derivatives of artemisinin, a molecule found in the sweet wormwood (Artemisia annua) plant, which grows mainly in China and Vietnam.
But the world supply of artemisinin has been very unstable over the past 15 years. As more farmers grow artemisinin to meet growing demand, the supply of artemisinin has increased and pushed prices down. So, farmers turned to more profitable crops. The supply of artemisinin then went down, and prices soared again. The artemisinin supply roller coaster has hindered efforts to make ACTs available to everyone who needs them.
There are various methods of introducing foreign nucleic acids into a eukaryotic cell: some rely on physical treatment; others rely on chemical materials or biological particles (viruses) that are used as carriers. There are many different methods of gene delivery developed for various types of cells and tissues, from bacterial to mammalian. Generally, the methods can be divided into three categories: physical, chemical, and biological .
2. Commericial production of semi-synthetic artemisinin
Alternative sources of artemisinin can solve this problem. Back in 2004, the Bill & Melinda Gates Foundation funded a project to develop a genetically engineered yeast that could make artemisinic acid, a compound just steps away from artemisinin itself. Sanofi eventually commercialized the process in 2013 to produce semi-synthetic artemisinin (SSA), capable of meeting about one-third of global demand.
To reach this achievement, we must fully understand the biosynthetic pathway of artemisinin. It is mainly divided into four parts:
Formation of farnesyl pyrophosphate FPP through both mevalonate pathway (MVA) and non-mevalonate pathway (DXP).
Under the action of amorpha-4,11-diene synthase, FPP is cyclized to form amorphine-4,11-diene, the intermediate of artemisinin.
Under the action of amorpha-4,11-diene oxidase, amorpha-4,11-diene is further oxidized to form artemisinol and artemisinaldehyde, and then artemisinic acid are synthesized.
In short, beer yeast (Saccharomyces cerevisiae) was designed to overexpress both the enzyme of the mevalonate pathway and A. annua amorphadiene synthase, resulting in over 40 g/L of amorphadiene (a hydrocarbon precursor of artemisinic acid) in fed-batch fermenters fed with ethanol. Cytochrome P450 enzymes (CYP71AV1) and their homologous reductase (AaCPR) are responsible for the oxidation of amorphadiene, but their expression in yeast results in low conversion of amorphadiene to artemisinic acid. High-level production of artemisinic acid (25 g/L) by yeast fermentation on a 2 L scale was achieved by reducing AACRP expression to alter the stoichiometry of CYP71AV1:AaCPR interactions, as well as co-expression of other enzymes (Cytochrome b5 and two dehydrogenases) are involved in three oxidation reactions that convert amorphadiene to artemisinic acid. Commercial production of semi-synthetic artemisinin began in 2013 with the development of an industrial process for the chemical conversion of artemisinin to artemisinin.
The SSA accounted for about 8% of the global supply of artemisinin in 2013, but fell to zero in 2015. It’s mainly because of its relatively high costs and other ACT manufacturers were reluctant to buy SSA from its rival Sanofi. The following year, sanofi sold its Garesio plant to Huvepharma. There is now a search for new ideas that could “provide a truly sustainable supply of low-cost semi-synthetic artemisinin.”
3. Recent development in increasing the activity of Cryp71Av1
At current production levels, the concentration of amorphadiene in the fermenter is close to 200 mM, well beyond the solubility limit. While CYP71AV1 is able to achieve such high conversions at extremely high concentrations of amorphadiene, methods are still needed to design improved P450 conversions and yield higher, economically relevant artemisinic acid titers.
Direct engineering of catalytic systems can be accomplished in several ways. Based on previous success in titrating AaCPR and cytochrome b5 expression levels, we know that regulation of enzymes indirectly involved in catalysis can have dramatic effects. In addition to designing the active site of P450, engineering how P450 interacts with the yeast endoplasmic reticulum has been fruitful for efforts such as heterologous hydrocodone production. Manipulation of the yeast genome may also be a means of increasing heterologous P450 activity, for example, it was recently shown that a mutation that causes yeast endoplasmic reticulum expansion (Δpah1) results in increased heterologous production of triterpenoid saponins.
- Peplow, M. “Looking for cheaper routes to malaria medicines.” Chem Eng News 96.11 (2018): 29-31.
- Ro, Dae-Kyun, et al. “Production of the antimalarial drug precursor artemisinic acid in engineered yeast.” Nature 440.7086 (2006): 940-943.
- Kung, Stephanie H., et al. “Approaches and recent developments for the commercial production of semi-synthetic artemisinin.” Frontiers in plant science 9 (2018): 87.
- Arendt, Philipp, et al. “An endoplasmic reticulum-engineered yeast platform for overproduction of triterpenoids.” Metabolic engineering 40 (2017): 165-175.
- Tu, Youyou. “Artemisinin—a gift from traditional Chinese medicine to the world (Nobel lecture).” Angewandte Chemie International Edition 55.35 (2016): 10210-10226.
- Hale, Victoria, et al. “Microbially derived artemisinin: a biotechnology solution to the global problem of access to affordable antimalarial drugs.” The American journal of tropical medicine and hygiene 77.6_Suppl (2007): 198-202.
- Paddon, Chris J., and Jay D. Keasling. “Semi-synthetic artemisinin: a model for the use of synthetic biology in pharmaceutical development.” Nature Reviews Microbiology 12.5 (2014): 355-367.
Chemical-based Transfection and Its Applications in Vaccines
Li Biquan, 10 March 2022
1. Introduction to Transfection
Transfection, which is the process of introducing foreign nucleic acids into host cells, is one of the most powerful molecular biology tools currently in use. In animal cells, transfection is the preferred term. Additionally, we should pay attention to the differences of the concept between transfection, transformation, and transduction.
Generally, we can divide transfection into categories, stable and transient transfection, which differ in their long-term effects on a cell. A stably-transfected cell will continuously express transfected DNA and pass it on to daughter cells, while a transiently-transfected cell will express transfected DNA for a short amount of time and not pass it on to daughter cells. From Figure 1, we could compare the differences between stable and transient transfection, and we can note that in the process of stable transfection, the DNA would be integrated into the gene of the host cell, while transient transfection doesn’t experience this step .
There are various methods of introducing foreign nucleic acids into a eukaryotic cell: some rely on physical treatment; others rely on chemical materials or biological particles (viruses) that are used as carriers. There are many different methods of gene delivery developed for various types of cells and tissues, from bacterial to mammalian. Generally, the methods can be divided into three categories: physical, chemical, and biological .
2. Chemical-based Transfection
In this abstract, we are mainly to introduce chemical-based transfection. As shown in Figure 2, Chemical-based transfection can be divided into several kinds: calcium phosphate, cationic polymers, liposomes (lipofection) and, nanoparticles etc .
One of the cheapest methods uses calcium phosphate, originally discovered by F. L. Graham and A. J. van der Eb in 1973 . This process has been a preferred method of identifying many oncogenes and proved that the complex access to the cell through endocytosis. However, the transfection efficiency strongly depends on the cell constitution, the pH, and the quality and the amount of the used nucleotides, and it is harmful and therefore not suitable for most sensitive primary cell lines .
Another method is the use of cationic polymers such as DEAE-dextran or polyethylenimine (PEI). The negatively charged DNA binds to the polycation and the complex is taken up by the cell via endocytosis. This method can be transiently transfected into eukaryotic cells in an inexpensive and simple manner and is able to be easily reproduced, but the efficiency in a number of cell types is low (including primary cells) and it is cytotoxic and therefore not suitable for sensitive cells .
Lipofection (or liposome transfection) is a technique used to inject genetic material into a cell by means of liposomes, which are vesicles that can easily merge with the cell membrane since they are both made of a phospholipid bilayer . Lipofection generally uses a positively charged (cationic) lipid (cationic liposomes or mixtures) to form an aggregate with the negatively charged (anionic) genetic material . This transfection technology performs the same tasks as other biochemical procedures utilizing polymers, DEAE-dextran, PEI and, calcium phosphate . This method is easy to apply and yields highly reproducible results of transient and stable transfections, but the transfection efficiency strongly depends on the cell type. Another disadvantage is that he viability after the transfection process might be decreased due to the high cellular sensitivity, especially for primary and non-dividing cells. It is worth noting that lipofection is based on endosomal molecule uptake, and also can easily merge with the cell membrane since they are both made of a phospholipid bilayer .
The last method we’d like to introduce and use in vaccines is nanoparticle-based transfection. Some cells are relatively easy to transfect, whereas others, such as stem cells, suspension cells, or primary cells show poor transfection results. This is where nanoparticles, each behaving as a single unit, come into the play, owing to their ultra-miniaturized size (ranging from 1 to 100 nm) [1, 7]. This method is used in many examples, such as AuNPs-based CRISPR delivery  and LNPs-based mRNA (vaccine) delivery [9, 10], which is shown in Figure 3.
LNP-based delivery is one of the most and main ways in mRNA vaccines delivery, other major delivery methods for mRNA vaccines Commonly used delivery methods and carrier molecules for mRNA vaccines are shown in Figure 4. 
5. Assembly of mRNA Lipid Nanoparticles (Vaccines)
The current methods of mRNA lipid nanoparticle assembly is achieved by rapid mixing in a microfluidic or T-junction mixer of four lipids (ionizable lipid, DSPC, cholesterol, PEG–lipid) in ethanol with mRNA in an aqueous buffer near pH is 4. When the ionizable lipid meets the aqueous phase, it becomes protonated at a pH ~5.5, which is intermediate between the pKa of the buffer and that of the ionizable lipid. The ionizable lipid then electrostatically binds the anionic phosphate backbone of the mRNA while it experiences hydrophobicity in the aqueous phase, driving vesicle formation and mRNA encapsulation. After initial vesicle formation, the pH is raised by dilution, dialysis or filtration, which results in the neutralization of the ionizable lipid, rendering it more hydrophobic and thereby driving vesicles to fuse and causing the further sequestration of the ionizable lipid with mRNA into the interior of the solid lipid nanoparticles. The PEG–lipid content stops the fusion process by providing the LNP with a hydrophilic exterior, determining its thermodynamically stable size, and the bilayer forming DSPC is present just underneath this PEG–lipid layer . (shown in Figure 5 (a))
6. COVID-19 mRNA Vaccination Mechanism
Initially, the mRNA vaccine is injected by intramuscular route, typically into the deltoid muscle. Subsequently, the lipid coat vehicle (lipid nanoparticles) around the mRNA allows for the vaccine to enter the cytosol of the cell, typically Dendritic cells (DCs), antigen-presenting cells. And then, the ribosomes translate the mRNA into spike proteins, and the injected mRNA subsequently degrades. The spike proteins are released from the cell and initiate an adaptive immune response. Eventually, through various activation pathways, immune cells mount a cell-mediated and antibody-mediated immunity against the spike protein of the SARS-CoV-2 virus . (shown in Figure 5 (b))
 Kim TK, Eberwine JH.Mammalian cell transfection: the present and the future. Analytical and Bioanalytical Chemistry. Anal Bioanal Chem, 2010, 397: 3173-3178.
 Graham FL, van der Eb AJ. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973, 52 (2): 456-67.
 Christina Ehrhardt, Mirco Schmolke, Andreas Matzke, Alexander Knoblauch, Carola Will, Viktor Wixler, and Stephan Ludwig. Polyethylenimine, a cost-effective transfection reagent. Signal Transduction, 2006, 6: 179-184.
 Felgner PL, Gadek TR, Holm M, Roman R, Chan HW, Wenz M, Northrop JP, Ringold GM, Danielsen M. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proceedings of the National Academy of Sciences of the United States of America. 1987, 84 (21): 7413-7.
 Felgner JH, Kumar R, Sridhar CN, Wheeler CJ, Tsai YJ, Border R, Ramsey P, Martin M, Felgner PL. Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations. The Journal of Biological Chemistry. 1994, 269 (4): 2550-61.
 P L Felgner, T R Gadek, M Holm, R Roman, H W Chan, M Wenz, J P Northrop, G M Ringold, M Danielsen. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc. Nati. Acad. Sci., 1987, 84: 7413-7417.
 Rein Verbeke, Ine Lentacker, Stefaan C. De Smedt, Heleen Dewitte. Three decades of messenger RNA vaccine development. Nano Today, 2019, 28: 100766.
 Reza Shahbazi, Gabriella Sghia-Hughes, Jack L. Reid, Sara Kubek, Kevin G. Haworth, Olivier Humbert, Hans-Peter Kiem and Jennifer E. Adair. Targeted homology-directed repair in blood stem and progenitor cells with CRISPR nanoformulations. Nature Materials, 2019, 18, 1124-32.
 Na-Na Zhang, Xiao-Feng Li, Yong-Qiang Deng, You-Chun Wang, Bo Ying, Cheng-Feng Qin. A Thermostable mRNA Vaccine against COVID-19. Cell, 2020, 182, 1271-83.
 Michael D. Buschmann, Mohamad Gabriel Alameh, Manuel J. Carrasco, Suman Alishetty, Mikell Paige, and Drew Weissman. Vaccines, 2021, 9 (65): 1-30.
 Yang Wang, Ziqi Zhang, Jingwen Luo, Xuejiao Han, Yuquan Wei and Xiawei Wei. mRNA vaccine: a potential therapeutic strategy. Mol Cancer, 2021, 20 (33): 1-23.
 Pratibha Anand and Vincent P. Stahel. The safety of Covid-19 mRNA vaccines: a review. Anand and Stahel Patient Safety in Surgery, 2021, 15 (20):1-9.
Proximity labeling: a new tool to explore molecular interactions
Zhang Yu, 17 Feb. 2022
Many biological processes are regulated through the molecular interactions of proteins and nucleic acids. In the past several years, affinity purification and yeast two-hybrid have been widely applied to discover potential molecular interactions. (1,2) Antibody-based affinity purification, in combination with mass spectrometry–based proteomics, allows the enrichment and identification of stable interaction partners of specific proteins of interest. The main limitation of affinity purification, however, is that weak or transient interactions are often lost during cell lysis and the subsequent washing steps. Moreover, affinity purification is challenging to apply to insoluble targets or protein baits.
Yeast two-hybrid and other protein complementation assays represent another approach for mapping protein–protein, protein–RNA and protein-DNA interactions in living cells. (1) These approaches are often high throughput, enabling the screening of thousands to millions of potential molecular interactions. (3) However, many proteins complementation assays have cell type and organelle type restrictions.
Then, with the development of the molecular biology, a brand-new technology was developed—the proximity labelling. Proximity labeling (PL) is a technology used to find the interaction partners of ‘baits’ proteins by fusing ‘baits’ proteins with enzymes who can modified proximity molecules. Tagged molecules that interact with baits can then be enriched and identified by mass spectrometry or nucleic acid sequencing. (4) The predecessor of the proximity labelling is DamID. It is a method developed in 2000 by Steven Henikoff for identifying parts of the genome proximal to a chromatin protein of interest. (5) DamID relies on a DNA methyltransferase fusion to the chromatin protein to nonnaturally methylate DNA, which can then be subsequently sequenced to reveal genome methylation sites near the protein. Researchers were guided by the fusion protein strategy of DamID to create a method for site-specific labeling of protein targets, culminating in the creation of the biotin protein labelling-based BioID in 2012. (6) Alice Ting and the Ting lab at Stanford University have engineered several proteins that demonstrate improvements in biotin-based proximity labeling efficacy and speed.
For now, several enzymes were selected as tagging enzymes to fuse with the ‘baits’ proteins. (Table 1) Each of them has its own advantages and disadvantages according to the application scenario. Just take APEX for an example, the requirement of the toxic H2O2 and the short labelling time make it a good option in the dead cells experiments. BASU provides a possible tagging scheme in living cells with a disadvantage of long labelling time. None of them can be applied to all situations. Besides, avoiding background from endogenously biotinylated proteins to improve accuracy for PL in vivo and developing more PL enzymes which will boost sensitivity and analysis of transcriptomes and genomes in distinct cell populations is still a long but meaningful work for researchers.
In conclusion, proximity labelling is a burgeoning field with lots of chances and challenges. Continuing development of increasingly sophisticated PL technology may vastly expand the range of PL-based discoveries and address more challenging questions, such as determining the affinity, stoichiometry and contact sites of molecular interactions.
1. Brückner, A., Polge, C., Lentze, N., Auerbach, D. & Schlattner, U. Yeast two-hybrid, a powerful tool for systems biology. Int. J. Mol. Sci. 10, 2763–2788 (2009).
2. Dunham, W. H., Mullin, M. & Gingras, A. C. Affinity-purification coupled to mass spectrometry: basic principles and strategies. Proteomics 12, 1576–1590 (2012).
3. Trigg, S. A. et al. CrY2H-seq: a massively multiplexed assay for deep-coverage interactome mapping. Nat. Methods 14, 819–825 (2017).
4. Qin, W., Cho, K. F., Cavanagh, P. E., Ting, A. Y., Nat. Methods 18, 133-143 (2021).
5. Steensel, B., Henikoff, S. Identification of in vivo DNA targets of chromatin proteins using tethered Dam methyltransferase. Nat Biotechnol 18, 424–428 (2000).
6. Roux KJ, Kim DI, Raida M, Burke B. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. J Cell Biol. 196, 801-810 (2012).
Potential of AlphaFold in Determining Protein Structure
Han Yongxu, 10 Feb. 2022
Centra dogma uncovers the production of protein which was translated from mRNA caring the genetic information of DNA. Protein-related research is the most fertile areas in chemical biology and medical biology. Function of protein is determined by its conformation which is complicated and variable, thus deciphering how protein worked entails the illustration of the structure.
The determination of protein structures can be dated from 60 years ago. John Kendrew, as a biologist from England, was awarded the Nobel Prize in chemistry in 1962 due to the contribution on the determination of myoglobin which containing heme with x-ray crystallography. Several scientist were also granted the Nobel Prize for the discovery on the conformation of proteins like virus particles, rhodopsin protein, aquaporins and ion channel protein and so forth in the following years. X-ray diffraction crystallography, NMR and cryo-EM were major methods to explore the protein structures. Searching from the Protein Data Bank (PDB), we can found that there are about 180,000 released entries for protein structures, among of them, more than 80% were determined from X-ray crystallography, 13,448 were solved by NMR and no more than 10, 000 were from cryo-EM (1). X-ray diffraction crystallography was one of the first experimental methods used for structural resolution, however, X-ray diffraction cannot be applied to larger proteins (2). NMR was generally believed to be able to describe the real structure of proteins in solutions rather than crystal structure. Moreover, NMR analysis can obtain the structural position of hydrogen atom. However, due to the unstable structures of proteins in solution sometimes, it is difficult to capture stable signals, thus computer modeling and other methods are supplemented for the structure determination (3). Cryo-EM method in resolving bound ligands and delineating structured water molecules makes it possible to obtain detailed information relevant to molecular mechanisms (4).
AlphaFold known from Critical Assessment of Structure Prediction (CASP) conference is a neural-network-based approach to predicting protein structures with high accuracy. Recently, the source code and almost 350,000 protein models within AlphaFold 2 from various species, including human and bacterial have been made public (5).
The network of AlphaFold can directly predict 3D coordinates of all heavy atoms for a given protein using the primary amino acid sequence and aligned sequences of homologues as inputs, then apply multiple sequence alignments (MSAs) and pairwise features to illustrate a new architecture. There are two index including the predicted local-distance difference test (pLDDT) and template modelling score (TM-score) to judge the prediction efficiency. pLDDT can reliably predict the Cα local-distance difference test (lDDT-Cα) accuracy of the corresponding prediction, and the latter one presents the global superposition metric template modelling score (6).
Apart from the predication of unknown structures of proteins, AlphaFold can also predict certain classes of protein–protein interfaces (7). Researchers have established 3D models for 106 previously unidentified complexes and 806 that have not been structurally characterized from yeast with the combination of RoseTTAFold and AlphaFold (8). In addition, one report on drug discovery by Alphafold have been published. The authors shown that they can screen a target molecule to CDK20 for the treatment of HCC within 30 days combining the AI engines PandaOmics and Chemistry42 (9).
In conclusion, AlphaFold 2 open the door to the application for protein conformational predication with integrating novel neural network architectures and training processes according to the evolutionary, physical and geometric constraints of protein structures (6,10). It is undoubtedly that AlphaFold 2 makes contribution to the medicine discovery and clarification of protein interaction which is related to the cell process at molecular pathway, and thus giving perspective of biological function. However, some scientists think that the protein-folding problem by AlphaFold 2 has not been solved yet. In addition, AlphaFold 2 cannot predicate the complicated protein structures in the dynamic process which is universal in a real cell. Proteins in allosteric states where it is bonded to partners like ligand, cofactor, DNA or other macromolecules can varied dramatically, thus it is difficult to predicate the accurate protein structures at specific moment. Although innovative work are entailed to address the mentioned above limitations, the introduction of AlphaFold 2 herald a new era in the development of structural biology and medical biology.
- Subramaniam, Sriram, and Gerard J. Kleywegt. A paradigm shift in structural biology. Nature methods 19.1 (2022): 20-23.
- Schotte, Friedrich, et al. Watching a protein as it functions with 150-ps time-resolved x-ray crystallography. Science 300.5627 (2003): 1944-1947.
- Sakakibara, Daisuke, et al. Protein structure determination in living cells by in-cell NMR spectroscopy. Nature 458.7234 (2009): 102-105.
- Yip, Ka Man, et al. Atomic-resolution protein structure determination by cryo-EM. Nature 587.7832 (2020): 157-161.
- Tunyasuvunakool, Kathryn, et al. Highly accurate protein structure prediction for the human proteome. Nature 596.7873 (2021): 590-596.
- Jumper, John, et al. Highly accurate protein structure prediction with AlphaFold. Nature 596.7873 (2021): 583-589.
- Baek, Minkyung, et al. “Accurate prediction of protein structures and interactions using a three-track neural network.” Science 373.6557 (2021): 871-876.
- Humphreys, Ian R., et al. Computed structures of core eukaryotic protein complexes. Science 374.6573 (2021): eabm4805.
- Ren, Feng, et al. AlphaFold Accelerates Artificial Intelligence Powered Drug Discovery: Efficient Discovery of a Novel Cyclin-dependent Kinase 20 (CDK20) Small Molecule Inhibitor. arXiv preprint arXiv:2201.09647 (2022).
- Senior, Andrew W., et al. Improved protein structure prediction using potentials from deep learning. Nature 577.7792 (2020): 706-710.
Protein Liquid-Liquid Phase separation
Liu Min, 20 Jan. 2022
Since 2009, when Hyman’s group discovered the liquid property of P granules from C. elegans, most of the research hotspots focused on the protein condensate. Actually, since 2000, many scientists have discovered various membrane-less structures. Most of these structures are 100-1000 nm diameters and contain a large amount of protein and RNA, which are involved in the normal physiological activities of cells. For example, stress granules are structures associated with translation regulation, while dvl2 puncta are associated with cell signaling.
Liquid-liquid separation is a very common phenomenon in the field of physical chemistry. Many substances are single phase in the monomer stage, and when polymerized they form two phases. The protein phase separation is more complicated than polymer, and the two most important driving forces are the disorder of the protein sequence and the multivalent effect. For example, simply increasing the concentration of a protein component can see phase separation, increasing the valency of a component can also promote phase separation, or reducing the solubility of a component can also help phase separation.
In 2012, Rosen’s group from Southwestern Medical Center reconstituted the phase-separated system for the first time in vitro. They successfully realized the observation of phase-separated droplets by increasing the valency of the two interacting proteins. Afterwards, Professor Clifford P. Brangwynne from Princeton did a lot of interesting work on the phase separation system from discovery, establishment to application, which are related to the transcription and translation of genes.
Phase-separated systems have now expanded into many applications. For example, in enzymatic reactions, it can be used as a concentrated enzyme and substrate to facilitate the rapid progress of the reaction. In addition, in signal transduction, it can function as a sequestration to achieve organelle-like functions. There are also many people who have made some organizational hubs to achieve some separated functions through a phase-separated system.
In conclusion, phase separation is a complex system integrating biology, physics and chemistry, and its functions and applications are still being explored. In future research, we expect more valuable discoveries and phenomena.
- Brangwynne, Clifford P., et al. “Germline P granules are liquid droplets that localize by controlled dissolution/condensation.” Science 324.5935 (2009): 1729-1732.
- Li, Pilong, et al. “Phase transitions in the assembly of multivalent signalling proteins.” Nature 483.7389 (2012): 336-340.
- Feric, Marina, et al. “Coexisting liquid phases underlie nucleolar subcompartments.” Cell 165.7 (2016): 1686-1697.
- Kedersha, Nancy, and Paul Anderson. “Mammalian stress granules and processing bodies.” Methods in enzymology 431 (2007): 61-81.
- Schwarz-Romond, Thomas, Ciara Metcalfe, and Mariann Bienz. “Dynamic recruitment of axin by Dishevelled protein assemblies.” Journal of cell science 120.14 (2007): 2402-2412.
- Shin, Yongdae, and Clifford P. Brangwynne. “Liquid phase condensation in cell physiology and disease.” Science 357.6357 (2017).
The virulence mechanisms of EPEC
Qiu Jiaming, 13 Jan. 2022
Enteropathogenic Escherichia coli (EPEC) is an attaching and effacing (A/E) pathogen, which can cause significant paediatric morbidity and mortality all over the world, especially in developing countries. The infection of EPEC involves four stages: attachment, sending in effector proteins, actin reorganization, and pedestal formation and includes various elements which play a vital role in EPEC infection.
Firstly, EPEC uses locus of enterocyte effacement (LEE) pathogenicity island, which encodes a type III secretion system (T3SS) and various cognate effectors (Tir, Map, EspF, EspG, EspH, and EspI) to infect host cells. Through the integration of coordinated intracellular and extracellular cues, the T3SS is assembled within the bacterial cell wall, as well as the plasma membrane of the host cell. T3SS construction includes four stages: (i) assembly of the basal body and export apparatus, (ii) assembly of the inner rod and needle, (iii) assembly of the filament and translocon, and (iv) secretion of effectors. And along with the formation of injectisome, EPECs can complete the infection.
Intimin and Tir
Secondly, the especially feature of A/E bacterial pathogens is the formation of pedestal beneath tightly adherent bacteria and localized disruption of the brush border of the intestine. And in this process, the intimin which belongs to bacterial outer membrane adhesin, is required for the production of A/E lesions and diarrhea. As we known, EPEC use T3SS to translocate their own intimin receptor, Tir, to the membranes of mammalian cells. The translocated Tir triggers additional host signaling events and actin nucleation. Actin nucleation is critical for lesion formation. Current studies have demonstrated that when the IBD domain of Tir binds to the D3 domain of the EPEC intimin protein on the mammalian cell surface, the bacteria are primed to remain on the mammalian cell surface.
N-WASP is best known as an Arp2/3 complex activator in processes like actin nucleation and has been reported to regulate actin nucleation and polymerization for multiple cell activities. It was found that at the carboxyl terminus of N-WASP, there is a conserved VCA region, which is composed of the verprolin homology region (V), the cofilin homology region (C) and an acidic region (A). The acidic region and the cofilin homology region bind to the actin-associated complex Arp2/3, and upon binding, promote the conversion of F-actin to G-actin, which will eventually cause the polymerization of actin leading to the formation of pedestal.The amino-terminal region of N-WASP has an EVH1 (WH1) structural domain immediately followed by a guanosine triphosphatase (GTPase) binding domain. The GTP hydrolase binding domain binds to the GTPase Cdc42 and can activate the function of N-WASP. Between the amino- and carboxy-terminal structural domains of N-WASP is a proline-rich sequence, and this structural domain can bind to the Src homology (SH)3 structural domains of signaling proteins such as Grb2 and Nck, as well as cytoplasmic tyrosine kinases.
In conclusion, EPEC infection is an extremely complex physiological and biochemical process, citing various proteins and physiological processes such as T3SS, Intirmin, Tir, Nck, N-WASP, Arp2/3, and actin, all of which play an important and irreplaceable role in the EPEC infection process. It is also only when we fully understand the functions and mechanisms of these proteins and interactions that we can more effectively research antibacterial drugs and contribute to human health.
- Todd, E. C. Epidemiology of foodborne diseases: a worldwide review. World Health Stat Q. 1997;50(1-2)
- Yu Luo et al. Crystal structure of enteropathogenic Escherichia coli intimin–receptor complex. Nature. 2000 Jun 29;405(6790)
- Sabrina L Slater et al. The Type III Secretion System of Pathogenic Escherichia coli. Curr Top Microbiol Immunol. 2018;416(51-72).
- Neal M Alto et al. Identification of a bacterial type III effector family with G protein mimicry functions. Cell. 2006 Jan 13;124(1)
- Teemu Kallonen , Christine J Boinett. EPEC: a cocktail of virulence. Nat Rev Microbiol. 2016 Apr;14(4):196.
- J Fawcett , T Pawson. AN-WASP Regulation–the Sting in the Tail. Science. 2000 Oct 27;290(5492):725-6
Yuan Dingdong, 22 Dec. 2021
Bioorthogonal chemistry means any chemical reaction that may occur inside living systems without interfering with native biochemical processes. This concept was coined by Carolyn R. Bertozzi in 2003. The applications of bioorthogonal chemistry are diverse and include genetic code expansion and metabolic engineering, drug target identification, antibody–drug conjugation and drug delivery. Key reactions include native chemical ligation and the Staudinger ligation, copper-catalysed azide–alkyne cycloaddition, strain-promoted [3 + 2] reactions, tetrazine ligation, metal-catalysed coupling reactions, oxime and hydrazone ligations as well as photoinducible bioorthogonal reactions (Fig. 2) .
Amongst all bioorthogonal reactions developed to date, the [4+2] cycloaddition of 1,2,4,5-tetrazines (s-tetrazines, Tz) and various dienophiles, referred as inverse electron demand Diels–Alder (IEDDA) reaction, is the one that satisfies most of the bioorthogonal criteria (e.g., fast, selective, biocompatible and catalyst-free) necessary for use in applications from protein labelling to cancer imaging or materials science. Diels–Alder [4+2]-cycloaddition describes the reaction between a diene (e.g., 1,2,4,5-tetrazines) and a dienophile (alkene or alkyne) to form a six-membered ring in a π4s + π2s fashion, via suprafacial/suprafacial interaction of 4π-electrons of the diene with the 2π-electrons of the dienophile (Fig. 3) .
Tetrazine can react with rich and varied strained or unstrained dienophiles, such as trans-cyclooctene (TCO), cyclopropane, norbornene and styrene which can be tuned to reach rate constants from 1 up to 106 M-1s-1 with different types of dienophiles (Fig. 4) .
Tetrazines have become important for bioconjugation strategies also due to their high fluorogenic. Since tetrazines are chromophores absorbing light at around 500–550 nm they can act as quenchers towards a series of fluorophores by Förster resonance energy transfer (FRET) and through-bond energy transfer (TEBT). Through IEDDA reaction between the tetrazine and TCO the structure of the tetrazine will be broken which can turn on the fluorescent moiety. Besides, inspired by previous “click-to-release” strategies of the reaction between tetrazine and vinyl ether, The Devaraj group has developed a tetrazine near-infrared ﬂuorogenic probe through a different quenching mechanism: internal charge transfer (ICT) process (Fig. 5) .
Also, the the aerial oxidation of dihydrotetrazines can be eﬃciently catalyzed by nanomolar levels of horseradish peroxidase under peroxide-free conditions. The dihydrotetrazine/tetrazine pair can be transferred under the GSH or LED light and photocatalyst. Using this feature Fox group have developed a seirs interesting and useful applications in living cell imaging by light control (Fig. 6) .
In summary, tetrazine based IEDDA reaction match the principle of the bioorthognoal reactions perfectly. It has high efficiency and can undergo more chemical reactions due to its unique structure compared with the traditional click reaction between azide and alkynes. In resume, the potential of IEDDA reactions is enormous and suggest a rich future of this bioorthogonal reaction in the field of chemical biology.
- Ellen M. Sletten and Carolyn R. Bertozzi, Angew. Chem. Int. Ed. 2009, 48, 6974 – 6998.
- B. L. Oliveira, Z. Guo and G. J. L. Bernardes, Chem. Soc. Rev. 2017, 46, 4895 – 4950.
- Haoxing Wu, and Neal K. Devaraj, Acc. Chem. Res. 2018, 51, 1249−1259.
- Joseph M. Fox, et al, J. Am. Chem. Soc. 2016, 138, 5978−5983.
Evolution of Directed Evolution Methodology
Liu Miao, 8 Dec. 2021
In 1859, Darwin’s On the Origin of Species founded one of the most important theory in biology, the theory of evolution. Although elution theory itself has experienced an evolutionary process, the basic concept of variation–selection–retention cycle unchanged. Adopted from the evolution theory, evolving proteins by mutagenesis and selection was proposed, and in 1993, Arnold and co-workers turned this concept into practice. Since then directed evolution has been widely used to obtain powerful biomolecules. The general procedure of directed evolution includes two main steps: gene diversification and screening/selection (Figure 1). The improved variant will serve as a new starting point for the next round of gene diversification to form a cycle. With the development of chemistry, biochemistry and molecular biology, strategies used in directed evolution have also been evolved.
1. Development of Gene Diversification
In molecular evolution, gene mutation and recombination are two major forces for diversification. For creating gene mutation, random mutagenesis by error-prone PCR was firstly used as the experiment is convenient to carry out, but high-throughput screening methods are necessary because of its random nature. With more knowledge of the protein structure–function relationships revealed by biochemist, focused mutagenesis like site saturation mutagenesis can be performed on critical residues of enzymes, thus creating a smaller but more efficient library. For gene recombination, various DNA shuffling methods were utilized as more molecular biology tools has been developed to imitate the natural process of homologous recombination.
Beside create mutants in test tube, which is labor and time intensive, in vivo gene diversification methods were also developed as more in vivo gene edit tools were found. Early mutation applications in microorganism use E. coli carrying with gene mutation enzyme plasmid, but their applications were limited since they introduce deleterious mutations in the host genome and result in genetic instability. So the development of in vivo gene mutation focused on finding orthogonal mutator plasmids and strains. For in vivo gene recombination, molecular biology tools like bacteriophage λ Red system and Cre-LoxP system were applied. In recent years, CRISPR-Cas system was proved to be a powerful gene edit tool in prokaryotic and eukaryotic cells, which was also used to introduce gene diversifications in vivo.
2. Development of Screening/Selection
Different from creating gene diversification, which is simply relied on the evolution of molecular biology, development of screening/selection methods were functional-oriented and diversified. Whatever the detection method using, the bottle-neck problem is to improve the throughput and efficiency.
For screening methods, each library variant will be assayed individually by using biochemical or biophysical analytical methods to evaluate the desired property. Traditional analytical methods like microplate or chromatography limit with throughputs, thus digital imaging, droplet microfluidics and fluorescence activated cell sorting were applied to increase analytical throughput. For selection methods, the protein function will be linked to the growth or survival of the host organism. The nonfunctional variants will be automatically eliminated during the selection process. So building a general platform for identifying desired phenotypes efficiently is the key challenge for selection methods, solutions include kinds of display-based methods and functional compartments.
3. Speed-up Direct Evolution Process
Beside the evolution on individual step, efforts have also been made to expedite the whole diversification–selection–amplification cycle. First, the efficiency of directed evolution can be greatly influenced by the quality of the library. At the early stage, most directed evolution experiments relied on random diversification, limiting the library quality. To overcome these limitations, rational design can be introduced to reduce the size of the library and simplify screening. With the increase of directed evolution experiments, a giant experimental database can be generate to link the sequence information with functional information, computational tools have been designed to identify promising initial variants. Moreover, in recent years, artificial intelligence has been introduced, structure prediction program like AlphaFold developed by DeepMind has successfully predicted protein structure, which may lead another evolution in this area.
As directed evolution usually need several iterative cycles to get desired variants, the connection between selection rounds is also a point to be optimized. For example, there are hundreds of data obtained in one round using screening method, while in traditional methods only the most efficient one was picked and others were wasted. With the rapid development of data science, machine learning has been applied to solve the problem, which using all the screening data to obtain a sequence-to-function landscape and guide the next round more efficiently. Another example is continuous evolution in vivo. As mentioned before, both gene diversification and selection can be carried out inside the cell, if two parts can be assembled in one compartment without affect the phenotype, the whole directed evolution process can be automated accomplished. Phage-assisted continuous evolution (PACE) is one of the most successful examples.
- Wang, Y. et al. Chem. Rev. 2021, 121, 20, 12384–12444
- Packer, M. and Liu, D. Nat. Rev. Genet. 2015, 16, 379–394
- Yang, K.K. et al. Nat. Methods 2019, 16, 687–694
Photocatalytic protein reactions
Bao Yishu, 15 Dec. 2021
Light has been wildly used in chemistry for decades. It can promote bond cleavage, such as azobisisobutyronitrile (AIBN)1, and enable thermally-prohibited transformations by promoting alkenes to their excited states, such as cycloadditions reactions.2 Molecules that interact with visible light can act as mediators for chemical transformations.
Photocatalysts and substrates with an appropriately matched redox potential can generate radical species via visible light and subsequently perform chemical transformations (Fig. 1).3 Photoredox catalysis absorbs light and then undergoes electronic excitation. Substrates with a higher redox potential can accept an electron from the excited photocatalyst, while substrates with a lower redox potential can lose an electron to the excited photocatalyst. In this way, organic radicals are formed on the substrates, which can then undergo further transformation. Subsequently, electrons are returned to, or taken from, the intermediate catalyst species to regenerate the ground state catalyst and participate in the next catalytic cycle.
Recent years have witnessed significant progress in photocatalytic reactions for amino acids, peptides, and proteins. For example, MacMillan’s group recently reported a single-site-selective tyrosine modification via photoredox catalysis.4 Owing to the amphiphilicity of phenol, tyrosines often exist in various microenvironments, resulting in different reactivity. Surface-exposed or hydrogen-bond-donating tyrosines are more reactive, while those sterically hindered or engaged in cation-π interactions are deactivated. Based on this microenvironment, the authors designed a bifunctional linchpin molecule that could enable endogenous protein labeling and be suitable for biorthogonal synthesis applications thereafter.
Besides tyrosine, photocatalytic methods are also widely used in cysteine modification. Thiol-ene reactions can be achieved via visible light photocatalysis. In 2017, Wang and co-workers used allyl alcohols and amides as reagents for cysteine and thiosugar modification.5 In 2018, Molander and co-workers developed a method for cysteine arylation using a ruthenium photocatalyst, nickel co-catalyst.6 Protein could also be modified. Wilson and co-workers demonstrated photocatalytic proximity labeling of a native cysteine-containing protein, human MCL-1, using a ruthenium-bipyridyl-modified peptide known to bind MCL-1 in high affinity.7
Compared with previous methods, one advantage associated with photocatalysis is C–H functionalization. Aliphatic residues such as leucine, isoleucine, and glycine, have remained underexploited in amino acid modification due to their inherently inert side chains precluding modifications using traditional methods. Palladium-catalyzed C–H activation is one of the main methods used to target these residues, but many procedures involve biologically-incompatible conditions, such as high temperatures. In contrast, photocatalytic strategies are more suitable for bioconjugation purposes because of the milder conditions.
In summary, photocatalysis shows great promise for amino acids, peptides, and protein modification. Especially, the emergence of C–H functionalization protocols compatible with peptide substrates and capable of modifying aliphatic side chains open a new door for the future of amino acid modification. Despite these great advances, significant challenges remain. Many of the exciting strategies are less effective when applied to larger peptide substrates. In addition, many of the synthesis procedures involve organic solvents, which greatly limits the biological application. For these strategies to be more widely used, biologically compatible reaction conditions need to be further explored.
- D. H. R. Barton, D. Crich, W. B. Motherwell, J. Chem. Soc., Chem. Commun. 1983, 939-941.
- K. Singh, W. Trinh, R. Latifi, J. D. Weaver, Org. Biomol. Chem. 2019, 17, 1854-1861.
- T. A. King, K. J. Mandrup, S. J. Walsh, D. R. Spring, Chem. Soc. Rev. 2021, 50, 39-57.
- B. X. Li, D. K. Kim, S. Bloom, R.Y. Huang, J. X. Qiao, W. R. Ewing, D. G. Oblinsky, G. D. Scholes, D. W. C. MacMillan, Nat. Chem. 2021, 13, 902-908.
- G. Zhao, S. Kaur, T. Wang, Org. Lett. 2017, 19, 3291-3294.
- B. A. Vara, X. Li, S. Berritt, C. R. Walters, E. J. Petersson, G. A. Molander, Chem. Sci. 2018, 9, 336-344.
- H. A. Beard, J. R. Hauser, M. Walko, R. M. George, A. J. Wilson, R. S. Bon, Chem. Commun. 2019, 2, 133-142.
Proximal labeling of interacting proteins
CHEN Hongfei, 17 Nov. 2021
Protein-protein interactions (PPIs) play a very important role in biochemical process. Studying PPIs is always a hot topic for many researchers in recent years. Many different approaches such as phage display, yeast hybridization, etc. were developed to illustrate the PPIs, and disclose the composition and organization of protein complexes. However, none of these approaches are based on the real-time and in vivo PPI analysis. Proximity-dependent labeling (PDL) of interacting proteins has been proposed by taking advantage of several enzymes, which can attach the known reactive groups to the nearby proteins covalently.
The mechanism of PDL is to generate a free reactive molecule which can conjugate to nearby proteins during its diffusion process. As shown in the figure (Fig. 1), the protein of interest (bait) is genetically fused to a proximity-based labeling enzyme. The fused enzyme will activate and then release reactive substrates to label proximal proteins. Interacting proteins that are near the bait are more likely to be labeled by the proximity enzyme. Then the quantitative mass spectrometry was used to analyze specific binding partners from background labeling.
Recently, proximity-tagging systems such as BioID1, EMARS2 and APEX3 have been used to identify PPIs. These proximity labeling enzymes—mostly engineered ligase or peroxidase—have different tagging radii and work well in confined compartments. They are very simple and the output information is rich. Just by using more bait proteins or combining with other traditional methods such as the immune precipitation, PDL can be very useful in identifying novel PPIs. Nowadays, these methods have been applied in studying diseases proteins such as Gag protein in HIV4 and LMP1 protein in EBV5. It is undoubtedly that PDL will have a broader application in studying biological processes, diseases, and therapies.
 E. de Boer, P. Rodriguez, E. Bonte, J. Krijgsveld et al. Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice. Proc. Natl. Acad. Sci. USA 2003, 100, 7480
 N. Kotani, J. Gu, T. Isaji, K. Udaka et al. Biochemical visualization of cell surface molecular clustering in living cells. Proc. Natl. Acad. Sci. USA 2008, 105, 7405
 N. M. Bell, A. M. Lever et al. HIV Gag polyprotein: processing and early viral particle assembly. Trends Microbiol. 2013, 21, 136.
 K. Holthusen, P. Talaty, D. N., Jr. Everly et al.Regulation of Latent Membrane Protein 1 Signaling through Interaction with Cytoskeletal Proteins. J. Virol. 2015, 89, 7277.
Cell-cell interaction in microorganisms
LIU Min, 10 Nov. 2021
Quorum sensing was first discovered in Vibrio fischeri by Nealson and colleagues in 1970. They observed that when the bacteria density is low, no fluorescence could be seen. But when the bacteria density reaches a high level, it will show fluorescence. This phenomenon is related to the expression of a fluorescent gene in a bacterium, which is only expressed under the induction of autoinducer. Therefore, bacteria with low population density will not express relevant genes. Autoinducer is a compound that can regulate the expression of specific genes in bacteria. Many autoinducers discovered were derived from molecules with AHL as the backbone. Later, scientists also discovered some oligopeptide autoinducers and other autoinducers with special structures. Under natural conditions, some bacterial cells can gather cooperator and cheater, and the protease secreted by cooperator can enter the symbiotic environment to promote the growth of cheater. In addition, the quorum sensing of Vibrio cholerae is also very common. When Vibrio cholerae exists in the ocean, the density of bacteria is relatively low, so the quorum sensing effect is very weak, which is conducive to the formation of biofilms. When the bacteria enter the host, the density of the bacteria increases and the quorum sensing increases, so the formation of biofilms is inhibited, which is the scenario of vibrio cholerae in the human body.
In synthetic biology, synthetic consortia, a group of bacterial flora, can be used to synthesize natural products, showing advantages of high efficiency, stability, controllability. For example, the Gregory group used the E. coli and yeast multi-host system to produce the precursor of paclitaxel. Besides, the synthetic co-culture controller constructed by the Bentley group could control the growth of bacteria.
In summary, by studying the interaction between microorganisms, we can realize the process from discovery to application. The group effect is still a treasure that the current research can focus on.
Pérez-Velázquez, Judith, Meltem Gölgeli, and Rodolfo García-Contreras. “Mathematical modelling of bacterial quorum sensing: a review.” Bulletin of mathematical biology 78.8 (2016): 1585-1639.
Schaefer, Amy L., et al. “Quorum sensing in Vibrio fischeri: probing autoinducer-LuxR interactions with autoinducer analogs.” Journal of bacteriology 178.10 (1996): 2897-2901.
Stephens, Kristina, and William E. Bentley. “Synthetic biology for manipulating quorum sensing in microbial consortia.” Trends in microbiology 28.8 (2020): 633-643.
Zhou, Kang, et al. “Distributing a metabolic pathway among a microbial consortium enhances production of natural products.” Nature biotechnology 33.4 (2015): 377-383.
Stephens, Kristina, et al. “Bacterial co-culture with cell signaling translator and growth controller modules for autonomously regulated culture composition.” Nature communications 10.1 (2019): 1-11.
Kelly, Robert C., et al. “The Vibrio cholerae quorum-sensing autoinducer CAI-1: analysis of the biosynthetic enzyme CqsA.” Nature chemical biology 5.12 (2009): 891-895.
Synthetic Biology: A glance into the future
Wang Yue, 3 Nov. 2021
It happens that synthetic biology is poised to improve our health in myriads of ways. For example, synthetic biology can produce renewable fuels and manufactured goods, and assist human health in terms of pharmaceutics, nutraceuticals and even therapeutics.
“What I cannot create, I do not understand ”, is a famous saying quoted from Richard Feynman. It is also the essential idea of researchers of synthetic life. In May 2010, John Craig Venter published an article on science describing their research of successfully creating “synthetic cell”. This was accomplished by synthesizing a very long DNA molecule containing an entire mycoplasma genome, and introducing it into another cell. This news has caused a sensation at that time, and people believed that the era of synthetic life will arrive in the near future.
At the same time, Jef Boeke, professor of New York University, was spearheading an ambitious effort to design and build an entire yeast genome from scratch, known as the Sc2.0 project.
As a eukaryote, a category that includes humans and other animals, S. cerevisiae has a more complex genome than mycoplasma. So Jef formed an international consortium to create a synthetic version of the full S. cerevisiae genome within 5 years. Chinese scientists from Tianjin University, Tsinghua University and BGI-Shenzhen have taken part of the work in this project and so far they have assembled four synthetic yeast chromosomes, making China the second country capable of designing and building eukaryotic genomes.
In 2016, researchers led by Craig Venter at the J. Craig Venter Institute in San Diego, California, announced that they had created synthetic “minimal” cells. The genome in each cell contained just 473 key genes thought to be essential for life. The cells were
named JCVI-syn3.0 after the institute and they were able to grow and divide on agar to produce clusters of cells called colonies. But on closer inspection of the dividing cells at the time, Venter and his colleagues noticed that they weren’t splitting uniformly and produce daughter cells of unnatural shapes and sizes. By reintroducing various genes into these synthetic bacterial cells and then monitoring how the additions affected cell growth under a microscope, Strychalski pinpoint seven additional genes required to make the cells divide uniformly. After adding 5 more genes into the JCVI-syn3.0, synthetic cells eventually can grow and divide into cells of uniform shape and size, just like most natural bacterial cells.
Synthetic biology also make the medicines of tomorrow look different from those of today by editing the existing bacteria in vivo or delivering modified bacteria to our bodies.
For example, Eligo Bioscience, based in Paris, is a company that combines gene editing and microbiota modification. Their founders, Dr. Duportet pointed out that in the past few years, there have been multiple validated examples showing the expression of specific bacterial genes and diseases. Eligo’s goal is to use a CRISPR-based system to inactivate these genes-there is a proven link between bacterial genes and disease. Eligo’s drug “Eligobiotics” consists of a viral vector (a phage particle without a phage genome) that contains a gene editing payload and is specific to the target bacteria. Once the CRISPR system is delivered, editing takes place in the body and in the intestines, inactivating genes and destroying bacteria. One candidate for Eligo is addressing antimicrobial resistance (AMR) bacteria in a project supported by the non-profit organization CARB-X. Eligobiotic, known as EB004, aims to remove the AMR gene from the bacteria in the intestines of patients undergoing organ transplantation to prevent bacterial infections. A transplant patient with AMR bacteria in the intestines has a 50% chance of contracting AMR bacteria after transplantation. In the case of infection, the mortality rate jumped to 70%. If AMR bacteria are removed, the mortality rate will drop to 4%.
Based on synthetic biological technology, Synbiotics, a Chinese company based in Beijing, has designed and developed a gene circuit that can accurately identify tumors and improve the killing effect, and insert the gene circuit into the adenovirus vector, forming a method that can accurately identify tumors, improve the immune environment, and effectively improve SynOV system, a new oncolytic virus gene therapy drug platform with tumor killing ability.
Surprisingly, on November 27, 2020, Synbiotics announced that its first gene therapy product, SynOV1.1, developed based on domestically original synthetic biotechnology, has been approved by the US FDA for clinical trials for the treatment of alpha-fetoprotein (AFP), including advanced liver cancer. ) Positive solid tumors, and plans to carry out Phase I/IIa clinical studies at the Memorial Sloan Kettering Cancer Center in the United States in the near future. The application for clinical trials of this drug in China is also underway.
Metabolic engineering is a multidisciplinary engineering toolbox for modern production in daily life. In which，Jay Keasling’s team takes the leading position. In the 1970s, Chinese scientists rediscovered it and identified its active ingredient, artemisinin, and artemisinin is now extracted from sweet wormwood grown commercially in China, Southeast Asia and Africa. However,The quality, supply and cost have been unpredictable and inconsistent.
In 2006, Jay reported to synthesize small amounts of artemisinic acid, which is chemically analogous to the actual drug. Using synthetic biology techniques from Keasling’s lab, Amyris added that gene to yeast along with other plant genes to boost artemisinic acid production by a factor of 15, good enough to interest Sanofi.
Then Keasling has turned to cannabis, where his work endows brewer’s yeast with the capacity to make cannabinoids. Keasling first published a paper on Nature back in 2019, but at this point, it is unclear if the process could be scaled up enough to support a new industry.
 Gibson D G, Glass J I, Lartigue C, et al. Creation of a bacterial cell controlled by a chemically synthesized genome[J]. Science, 2010, 329(5987): 52-56.
 Annaluru N, Muller H, Mitchell L A, et al. Total synthesis of a functional designer eukaryotic chromosome[J]. Science, 2014, 344(6179): 55-58.
 Pelletier J F, Sun L, Wise K S, et al. Genetic requirements for cell division in a genomically minimal cell[J]. Cell, 2021, 184(9): 2430-2440. e16.
 Luo X, Reiter M A, d’Espaux L, et al. Complete biosynthesis of cannabinoids and their unnatural analogues in yeast[J]. Nature, 2019, 567(7746): 123-126.