Blog

Cancer-immunity cycle

Shen XIaoxi, 4 Dec 2025

The cancer-immune cycle describes how the body recognizes and eliminates cancer cells, functioning as a closed loop from antigen release to repeated T-cell activation. Tumor cells first release antigens as a result of mutations or abnormal protein expression. These antigens are captured and processed by dendritic cells, which present them via MHC I and MHC II molecules to CD8⁺ and CD4⁺ T cells, respectively, thereby initiating cytotoxic and helper immune responses. Dendritic cells then migrate to the lymph nodes and activate naive T cells through three essential signals: TCR recognition of antigen–MHC complexes, co-stimulatory signaling, and the cytokine environment. During this stage, CTLA-4 functions as an early “brake” by inhibiting excessive activation. Once activated, effector T cells proliferate and enter circulation, migrating precisely to the tumor through selectin-mediated rolling, chemokine-triggered integrin activation, and firm adhesion to ICAM-1/VCAM-1 on endothelial cells. After infiltrating the tumor tissue, effector T cells recognize tumor-presented antigens and execute killing through multiple mechanisms, including perforin/granzyme release, Fas–FasL signaling, and secretion of IFN-γ. This process induces tumor cell apoptosis and simultaneously releases new antigens, further amplifying the immune cycle through “antigen spreading.”

Figure 1. The seven steps of the cancer-immunity cycle.

However, most tumors interrupt this cycle at various stages, creating immune-evasive barriers that result in three immune phenotypes, among which the “immune-desert” and “immune-excluded” types are considered “cold tumors.” Immune-desert tumors often arise from insufficient antigen release or impaired antigen presentation, preventing T-cell priming. Immune-excluded tumors are characterized by T cells trapped in the stromal periphery due to abnormal vasculature, lack of chemokines, or dense extracellular matrix. Even when T cells infiltrate the tumor, the microenvironment often suppresses their activity through immunosuppressive cells and molecules such as IL-10, TGF-β, IDO, adenosine, as well as hypoxia and acidic metabolism. Additionally, high PD-L1 expression on tumor cells binding to PD-1 on T cells induces functional exhaustion.

Figure 2. Reversing a cold into a hot tumor, and which step of the anti-cancer immune response is not functional in cancers.

T cell exhaustion

YUAN Dingdong, 11 Nov 2025

T cell exhaustion refers to a state of dysfunction exhibited by CD8+ T cells under chronic antigen stimulation. It mainly occurs in chronic viral infections and cancer. Exhausted T cells demonstrate reduced production of partial cytokines, weakened cytotoxicity, high expression of inhibitory receptors, decreased or completely lost proliferative ability, and eventually apoptosis.

T cell exhaustion results from the combined action of multiple factors (Figure 1), mainly including: 1. Persistent antigen stimulation, which is the main driver of T cell exhaustion; 2. Tumor microenvironment, where immunosuppressive factors such as IL10 and TGFβ promote T cell exhaustion. Additionally, hypoxia and nutrient deprivation further exacerbate T cell exhaustion; 3. Intercellular interactions, regulatory cells like Tregs play an important role in T cell exhaustion. Beyond Tregs, other regulatory cell types like immunoregulatory APCs, MDSCs, NK cells, and even CD8+ regulatory populations may influence viral persistence in chronic infections, directly or indirectly promoting T cell exhaustion. 4. High expression of inhibitory receptors such as PD1, TIM3, CTLA-4, and LAG3.

Figure 1. Overview of mechanisms of T cell exhaustion.

Inhibitory receptors regulate T cell exhaustion by influencing T cell molecular signaling pathways. Typically, T cell activation requires three conditions: binding of TCR to the MHC antigen complex on APCs; 2. Co-stimulatory signals from CD28 and CD80/CD86 on APCs; and cytokine IL2. The inhibitory receptor PD1, possessing an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM) in its intracellular region, recruits SHP2 to inhibit CD28 and TCR signaling upon binding to PDL1 or PDL2. Another inhibitory receptor, CTLA-4, competitively binds to CD80/CD86 with CD28 to suppress CD28 signaling. CTLA-4’s intracellular region contains a tyrosine inhibitory motif YVKM, which is phosphorylated after binding to CD80/CD86, recruiting SHP2 to inhibit TCR signaling. Other inhibitory receptors, such as LAG3 and TIM3, regulate T cell exhaustion by suppressing TCR or CD28 signaling through various mechanisms.

Figure 2. Molecular pathways of inhibitory receptors associated with T cell exhaustion.

Cytokines

CHANG Jingyue, 14 Oct 2025

Cytokines are small molecule proteins secreted by various cells during immune responses, mainly used for intercellular signal transmission and for regulating immune and inflammatory responses by controlling cell proliferation, differentiation, and antibody secretion by immune cells. The signal transmission of cytokines can be classified into three modes: autocrine, paracrine, or endocrine (Figure 1).

Figure 1. Signal transmission of cytokines.

Generally speaking, cytokines can be distinguished based on their molecular structure, source and function. In terms of structure, cytokines can be classified into hematopoietic cytokine family, interferon family, chemokine family or tumor necrosis factor (TNF) family. In terms of molecular function, different structures of cytokines exhibit different functions, such as regulation of immune cells, anti-viral activity, allergic reactions, cell apoptosis and transport. At the immune level, cytokines can be classified into pro-inflammatory and anti-inflammatory factors, although some cytokines simultaneously exhibit both characteristics under different conditions. Another typical way to distinguish cytokines is through different receptors, as the receptors corresponding to different cytokines show significant differences. Figure 2 shows the structures of different typical cytokine receptors.

Figure 2. Classification of cytokine receptors.

Figure 2. Classification of cytokine receptors.

B-Cell Receptor (BCR) and Antibody (Immunoglobulin)

LI Biquan, 12 Sept 2025

B lymphocytes develop from lymphoid stem cells in the bone marrow of mammals or the bursa of Fabricius in birds, hence the name B cells. Mature B cells primarily reside in the lymphoid follicles of peripheral lymphoid organs, accounting for approximately 20% of the total peripheral lymphocyte population. B cells contribute to specific humoral immunity by producing antibodies, serve as antigen-presenting cells, and participate in immune regulation. B cells develop in the bone marrow and migrate to peripheral lymphoid organs, where antigens can activate them (Figure 1). B-lineage cell development progresses through several stages marked by the rearrangement and expression of immunoglobulin genes, as depicted in Figure 2.

Figure 1. Development of B Cells.

The B-cell receptor complex is made up of cell-surface immunoglobulin with one each of the invariant signaling proteins Igα and Igβ. The immunoglobulin recognizes and binds an antigen but cannot itself generate a signal. It is associated with antigen-nonspecific signaling molecules—Igα and Igβ. Each has a single ITAM (immunoreceptor tyrosine-based activation motif) in its cytosolic tail that enables it to signal when the B-cell receptor is ligated with antigen. Igα and Igβ form a disulfide-linked heterodimer that is non-covalently associated with the heavy chains.

Figure 2. Heavy Chain (H Chain) Locus Rearrangements in the Development of B Cells.

Figure 3. B-cell Signaling after Activation by Antigen in Peripheral Lymphoid Organs.

T-cell signaling pathways

JIANG Hao, 4 Sept 2025

The ability of the T cell to respond to foreign pathogens appropriately depends heavily on signaling by cell-surface receptors, whose abnormal behavior would cause many diseases including immunodeficiency diseases and autoimmune diseases.

Figure 1. Overview of the T-cell signaling pathways.

The whole network of signaling pathways is highly complicated, efficiently organized and strictly regulated. Although many various pathways are involved, they share some common features and strategies, including the generation of second messengers such as calcium and phosphoinositide, the activation of both serine/threonine and tyrosine kinases, recruitment of signaling proteins to the plasma membrane and the assembly of multiprotein signaling complexes.

Figure 2. Simplified scheme about multiple signaling pathways necessary for IL-2 expression.

Protein Folding and diseases

Ryan Pak Hong Yip, 27 Aug 2024

Normally, misfolded proteins are either refolded or degraded as part of the cell’s quality control mechanisms. However, in cases where protein folding goes awry, it can lead to the development of diseases. Two notable examples are cystic fibrosis and prion diseases. Cystic fibrosis is a genetic disorder caused by mutations affecting the folding of the cystic fibrosis transmembrane conductance regulator protein (CFTR). Misfolded CFTR leads to the accumulation of thick, sticky mucus in the body’s airways and digestive system, particularly impacting the lungs. Prion diseases, such as Creutzfeldt-Jakob Disease (CJD), Kuru, mad cow disease, and scrapie, result from the misfolding of prion proteins. Unlike cystic fibrosis, there are currently no effective treatments or vaccines for prion diseases. These conditions can lead to spongiform encephalopathy, characterized by brain shrinkage, dementia, loss of appetite, and aggressive behaviors. Unfortunately, patients affected by prion diseases typically face a fatal outcome.

Figure 1. The CDJ disease.

Prion diseases exhibit symptoms akin to other neurodegenerative conditions but stand out as contagious illnesses that progress rapidly and often culminate in death. The misfolded prion protein PrP serves as an infectious agent, acting as a chaperone capable of propagating by binding to normal PrP and inducing its transformation into a hazardous conformation mirroring its own. The accumulation of these misfolded proteins leads to the onset of spongiform encephalopathy.

Figure 2. Misfolding of prion leads to neuronal degeneration.

Introduction to RNA-editing

XU Zhiyi, 13 Aug 2024

RNA Editing Therapy is a Rising Star in Medicine

RNA editing technology has evolved significantly over the past few decades. Initially, the focus was on understanding the basic mechanisms of RNA editing, particularly the adenosine-to-inosine (A-to-I) and cytidine-to-uridine (C-to-U) conversions. These discoveries laid the groundwork for developing therapeutic applications. Recently, RNA editing has gained momentum, with several therapies entering clinical trials. The COVID-19 pandemic highlighted the potential of RNA-based technologies, further accelerating research and development in this field. On June 18th, 2024, Roche payed Ascidian Therapeutics $42M to develop novel medicines for difficult to treat neurological diseases. The future of RNA editing looks promising, with ongoing advancements expected to enhance its precision, efficiency, and safety, potentially making it a cornerstone of personalized medicine.

Figure 1. Nature news of RNA-editing therapy

Mechanism of RNA Editing
RNA editing involves post-transcriptional modifications that alter the nucleotide sequence of RNA molecules. The most common types of RNA editing are A-to-I and C-to-U conversions. These processes are catalyzed by specific enzymes: adenosine deaminases acting on RNA (ADARs) for A-to-I editing and apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) for C-to-U editing. ADARs deaminate adenosine to inosine, which could pair with Cytidine and read as guanosine during translation, thereby altering the protein sequence. This A-to-I editing helps human body to distinguish between viral RNA and the host’s own RNA. ADAR1 could edit endogenous double-stranded RNA (dsRNA) by converting adenosine (A) to inosine (I), which is then recognized as self-RNA by the immune system. Compared with A-to-I editing, C-to-U editing is a less common but significant type of RNA editing in the human body. This kind of editing could change a codon or even create a premature stop codon during the translation to produce different proteins with one genes for different functions. One of the best-characterized examples of C-to-U RNA editing occurs in the mRNA of apolipoprotein B (ApoB). In the small intestine, a cytidine is edited to uridine (U), converting a CAA codon to a UAA stop codon. This results in the production of a shorter protein, ApoB48, which is essential for the assembly and secretion of chylomicrons. These mechanisms allow for the correction of genetic mutations at the RNA level without altering the underlying DNA sequence.

Figure 2. Mechanism of A-to-I and C-to-U RNA editing.

Aging of phase-separated proteins: liquids, gels, or solids

HAN Yongxu, 12 July 2024

The cellular environment includes various membraneless compartments, it can form and dissolve in response to cellular signals. The composition of condensates is specific. a cell can trigger phase separation of specific proteins, certain clients partition into these condensates, but others are kept out. Many of these compartments appear with phase transition. They have material properties of liquids, gels, or solids. Material properties are linked to distinct functions and pathologies. Condensate assembly is tightly regulated in the cellular environment, and failure to control condensate properties, formation, and dissolution can lead to protein misfolding and aggregation, human genetics indicate a strong link between condensate-forming proteins and ageing-related diseases such as neurodegeneration and cancer.

Fig. 1 (a) Functions of condensates and (b) Aging may be associated with aberrant condensates. [1]

In brief, Monomers can assemble into liquid-like condensates by phase separation. Liquid-like droplets can subsequently harden into a gel-like or glass-like state. They can further convert into solid-like fibrils. The ageing process is accompanied by condensate shrinking, suggesting that the molecular components increasingly interact and jam.

Fig. 2 Process of protein aging [1]

PTMs can modulate the phase transition. It can affect the affinity of the binding elements and dynamics of IDRs to regulate assembly and disassembly. Apart from the PTM, the aggregation of proteins like tau is highly dependent on its surrounding conditions. The negative charge of polyanions can compensate for the positive charge of tau, leading to an increase in aggregate. The metal ions can lead to oxidative stress with the generation of ROS and thus make protein aggregate. Fatty acids, as precursors of phospholipids in cellular membranes, can act as inducers of tau aggregation. The amphiphilic nature of fatty acids allows them to generate micelles. The anionic surfaces are enhanced by fatty acid micelles and could serve as nucleation points for tau fibrillization through charge neutralization effects.

Fig. 3 The mechanisms and processes regulating the conversion of condensates. [2, 3]

Furthermore, pH, temperature, ionic strength, organic solvents, molecular crowders, and osmolytes can alter tau’s aggregation pathways and kinetics.

Recent Work about Targeted Protein Degradation

YUAN Dingdong, 5 July 2024

In 2001, Kathleen Sakamoto, Craig Crews and Ray Deshaies reported the first PROTAC, the PROTAC technology has been developed to degrade many kinds of proteins. The targeted protein degradation can be briefly divided into two categories by two degradation pathways: the first one is the traditional PROTAC small molecules which focus on the intracellular protein degradation using the proteasome pathway (Figure 1 left), and the other one is the cell-surface and secreted protein degradation using the lysosome pathway such as the LYTAC (Figure 1 right). [1 and 2] Many methods have been developed for different protein degradation using different strategies, but there are still many problems. For example, most methods had low selectivity to cancer cells (targeted cells), many proteins are hard to degrade, and the biosafety of the PROTACs is always judged. Therefore, people are still trying to develop novel methods to solve these problems. Here I hope to introduce three novel works on targeted protein degradation using different kinds of strategies.

Figure 1. (left) PROTACs based on proteasome pathway. (right) PTD based on the lysosome pathway.

The first one is the TransTAC (Transferrin Receptor Targeting Chimeras). TransTAC is a bispecific antibody with one head that can bind to the targeted proteins and another head that can bind to the transferrin receptor 1(TfR 1). The TfR is required for iron import from transferrin into cells by endocytosis. Iron is an essential element for cells, and its transportation is facilitated by TfR 1. Rapidly dividing cells, such as cancer cells and activated immune cells, exhibit substantially increased TfR1 expression compared to non- or slow-dividing cells due to their high demand for iron. As a result, TfR1 is an attractive target for modulating these cell types. Several studies have investigated TfR1 as a therapeutic target for cancer with promising outcomes, as well as for tumor imaging and targeted drug delivery (Figure 2). [3]

Figure 2. Overview of the TransTAC technology and Transferrin Receptor 1 (TfR1) expression analysis. (a) Schematic of TransTACs. TransTAC induces close proximity of TfR and a protein of interest (POI) at the cell surface. (b) Illustration of an example TransTAC protein. (c) Relative cell surface TfR1 expression levels across various non-tumorigenic and cancer cell lines characterized by flow cytometry. (d) Relative TFR1C RNA expression levels in primary tumor compared to normal tissues based on the MERAV database.

Then the authors designed and synthesized three bispecific antibodies for three cell surface proteins: PD-L1, ERFR, and CD20. From the western blot (WB) results, TransTACs all perform very well for the targeted protein degradation. Consequently, they developed TransTACs that may enable selective targeting of cancer cells while minimizing toxicity to normal cells, addressing the off-target effects commonly associated with traditional cancer treatments (Figure 3).

Figure 3. Developing TransTACs degraders for various proteins (a) Schematic of membrane proteins targeted by TransTACs in the present study. (b) PD-L1 degradation by TransTACs in MDA-MB-231 breast cancer cells analyzed by Western blot. A scFv or Fab format of atezolizumab is used as the PD-L1 binding moiety. (c) EGFR degradation by TransTAC in A549 lung carcinoma cells. An affibody is used as the EGFR binding moiety. (d) CD20 degradation by TransTAC. A Fab format of rituximab is used as the CD20 binding moiety.

The second one is the TransMoDEs. For most of the PROTACs, it is hard to cross the blood-brain barrier (BBB). But there are still many proteins that need to be degraded in the brain. TransMoDEs consist of two parts, the target binding ligand and the angiopep-2 peptide. The Angiopep-2 is a peptide derived from the Kunitz domain, which shows a high brain penetration capability to overcome the BBB. The angiopep-2 can help the TransTAC enter the brain cells and exocytosis the brain cells (Figure 4). [4]

Reactive oxygen species-triggered prodrug delivery strategies for cancer chemotherapy

KONG Hao, 24 June 2024

Cancer is still one of the deadliest diseases that have not been conquered. During the long history of fighting against cancers, several therapeutic methods have been developed, including surgery, radiation therapy, chemotherapy, immunotherapy and so on (Figure 1). Among these options, chemotherapy that uses highly cytotoxic small-molecule drugs remains a dominant method. By systemic administration of drugs, the proliferation of tumor cells can be inhibited, especially those in advanced or metastatic stages. Chemotherapy can be curative for certain types of cancers, especially in early stages. Besides, chemotherapy can be combined with other treatment methods like radiation therapy or targeted therapy to enhance effectiveness. Despite the great progress that has been made in small molecule drugs, they still suffer from challenges mainly associated with inherent small molecule characteristics, including low water solubility, poor targeting and short circulation in vivo.[1]

Figure 1. Options of cancer treatment methods that have been developed and the pros and cons of chemotherapy.

A series of strategies have been explored to overcome the drawbacks of chemotherapy, for example, the construction of stimuli-responsive activable prodrugs. Prodrugs are derivatives of drug molecules. The activity of the drugs is temporarily blocked by modifying the active sites to lower toxicity in normal tissues and can be activated in targeted cells by intracellular or extracellular stimuli like ROS, pH, enzymes, light or ultrasound, depending on the chemical structure of the cleavable linker (Figure 2A).[2-3] Moreover, encapsulation of small molecule prodrugs within various organic or inorganic nanocarriers has recently emerged as a highly potential strategy, as nanocarriers effectively shield the small-molecule drugs, resulting in an improvement in their stability, solubility, bioavailability and biodistribution. Drugs release can be triggered by various stimuli that are consistent with the prodrug activation (Figure 2B).[4]

Figure 2. Overview of the prodrug construction strategies (A) and the nano-systems used for prodrug delivery (B).

Among these stimuli, reactive oxygen species (ROS) is a typical activation factor. ROS are transient oxygen intermediates produced by successive energy, electron or proton transfer of O2 (Figure 3A). Intracellular ROS are generated mainly in the mitochondria, peroxisome and endoplasmic reticulum, and in addition by some metabolic enzymes. Environmental factors such as radiation and xenobiotics can also cause ROS production (Figure 3B). ROS function as important signaling messengers in the regulation of cell functions, but excessive ROS production can cause cell damage and are associated with several diseases. For example, ROS levels in tumor cells are significantly higher than those in normal cells, which makes ROS as ideal markers for active targeting and inducing the controlled release of drugs.[5-7] Here we will list four common ROS-induced cleavage reactions and their applications in typical strategies for the construction of prodrug delivery systems.

Figure 3. Production of ROS and molecular orbital of ROS (A) and sources and functions of intracellular ROS (B).

The first ROS-induced cleavage reactions used for prodrug construction is a 1O2 or H2O2 activated cleavage of thioketal linkers. Drugs can be totally released by subsequent reactions (Figure 4A). Xing’s group reported a cancer cell and mitochondria dual-targeting polyprodrug nanoreactor (DT-PN) that can achieve mtROS burst and induce cancer cell apoptosis. In this nanoreactor, Camptothecin (CPT) was selected as a model drug, which can act as a cellular respiration inhibitor to stimulate endogenous mtROS production and can promote cell apoptosis (Figure 4B).[8] The macromolecule PDMA-b-PCPTSM was covalently modified with a high content of repeating CPP units through the thioketal linker. Besides, cell-targeting cyclic RGD (cRGD) and mitochondria-targeting TPP were modified at the other side of the macromolecules. These modified macromolecules can co-assemble into nanoparticles, enter αvβ3 positive cancer cells and locate at mitochondria. Upon active targeting to mitochondria, endogenous upregulated mtROS in cancer cells can induce initial CPT release. In situ released CPT further triggers the circulating increase of mtROS, achieving subsequent amplification of high-dosage CPT release and a final mtROS burst, which are favorable for long-term high oxidative stress to efficiently eliminate cancer cells. Experimental results confirmed the CPP release from the nanoparticles, ROS burst in mitochondria both in vitro and in vivo. Also, these nanoparticles can target αvβ3 positive 4T1 cells and showed significant therapeutic effect on subcutaneous tumor in mice.

Phase Separation in Neurodegenerative Diseases

CHENG Kai 24 June 2024

Growing evidence has revealed that neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Alzheimer’s disease (AD), and Parkinson’s disease (PD), are related to pathological protein aggregates. And abnormal liquid-liquid phase separation (LLPS) can cause protein aggregates. [1]

Figure 1. Four neurodegenerative diseases related to the different areas of the brain.

TDP-43 is one of the major pathological signature proteins in ALS disease[2]. Under normal physiological conditions, TDP-43 can participate in the formation of stress particles in response to changes in the cellular environment. Stress particles form membraneless structures through reversible LLPS, but mis-aggregation can lead to irreversible amyloid precipitation. The LLPS of TDP-43 mainly depends on the interaction of its carbon terminal helical structure domain. When this domain is destroyed and becomes unstable, normal LLPS cannot occur, protein aggregates are formed, then diseases further occur. Therefore, understanding the formation process and structural characteristics of these TDP-43 protein precipitates will provide new ideas for drug development in ALS diseases.

Figure 2 Liquid-liquid phase separation of TDP protein

The second, Fused in sarcoma (FUS) protein is an RNA-binding protein involved in RNA transcription, splicing, transport, and translation[3]. The occurance and progression of FTD disease are closely related to the abnormality of FUS. We know that FUS undergoes rapid, physiologically reversible phase separation between dispersed, liquid droplet, and hydrogel states. Research have showed that N-terminal low complexity domain of FUS is necessary and sufficient for phase separation and gelation of FUS, and does so by forming intermolecular β-sheet-rich fibrils. In this low complexity domain, cation-π interaction is considered to be a major phase separation driving force. Namely, carbon terminal arginine and nitrogen terminal tyrosine in FUS proteins can form phase separation through cation-π interaction. These dynamic phase structures take up, sequester, transport, and then release key RNA and protein cargos that regulate local RNA and protein metabolism in subcellular niches, such as axon terminals and dendrites. With the increase of cation-π interaction, the FUS tend to aggregate, the above processes may go awry, and disease will be triggered.

Figure 3 Liquid-liquid phase separation of the RNA-binding protein FUS

The third, Tau protein is the major constituent of neurofibrillary tangles in AD[4]. It have been proposed that LLPS of phosphorylated tau is a cell biologically relevant event, and tau droplet formation may resemble the initial step for aberrant tau aggregation in the brain. When phosphorylated and detached from the microtubules, the intraneuronal tau proteins can undergo LLPS to form droplets, and the transition from microtubule-bound and soluble monomeric/dimeric tau into tau droplets is facilitated by tau phosphorylation and the normal intracellular molecular crowding. When neurons are in a healthy state, LLPS may be a normal process of containing, condensing, and providing tau locally in the axon, and soluble and phase-separated tau exist in balance in the cytosol. When tau proteins are hyper-phosphorylated, equilibrium will shift in the direction of tau aggregation, these effects can cause high levels of short-term and long-term stress and eventually lead to neuronal death.

Formation and Applications of Metal-Polyphenol Networks

XIAO Kemeng 18 June 2024

Polyphenols, the organic molecules containing phenolic hydroxyl groups, are ubiquitous in natural plants and have demonstrated beneficial effects on human health, such as anti-inflammatory, antimicrobial, and anti-cancer properties. The common polyphenols are summarized in Figure 1. Among these, tannic acid (TA) is of particular interest, as its adhesive capabilities enable it to readily deposit on a wide range of inorganic, organic, and biological substrates. The abundant di- and/or trihydroxy phenyl units in TA facilitate its easy surface modification. For instance, TA can serve as a hydrogen donor to form stronger hydrogen bonds with molecules containing hydrogen acceptors. Furthermore, the deprotonated phenolic groups in TA can establish robust associations with metal cations, an attribute that has garnered significant attention due to the attractive characteristics of these TA-metal complexes, such as structural flexibility, pH-responsiveness, and thermal stability. Notably, among various metal ions, Fe3+ ions, with their high binding affinity and low biotoxicity, have been a focus of particular interest in this regard. The pioneering work on TA-Fe assemblies, which successfully achieved versatile film and particle engineering, was first reported in 2013.[1] Building on this research, TA has since been coupled with various metals to enable self-assembly and subsequent coating of different materials. [2]

Figure 1. Chemical structures of common polyphenols.[3]

The TA-Fe coordination can be achieved through various methods (Figure 2). One approach involves the simple mixing of iron and polyphenols in the presence of a substrate, which is a straightforward, cost-effective, and scalable process. Alternatively, a multistep method can be employed, where polyphenols are first incubated with a template (e.g., polystyrene), followed by the addition of metal ions to the resulting polyphenol-terminated template. Repeating this two-step sequence leads to the deposition of multiple layers of metal-polyphenol networks (MPNs) on the template. MPNs prepared via the multistep method tend to have a higher metal ion proportion compared to the simple mixing approach. Additionally, the sol-gel method has also been utilized for MPN assembly. In this technique, the polyphenols are first pre-crosslinked by formaldehyde in alkaline water/ethanol solutions to fabricate phenolic oligomers, after which metal ions are added to the suspension of these polyphenol oligomers to form MPN nanospheres.

Figure 2. Different method for fabrication of MPN.[4]

The applications of Fe-TA networks are further explored, with several representative works highlighted. Firstly, Fe-TA networks have been utilized for the organized assembly of particles into superstructures (Figure 3). These superstructures are of great interest across a variety of fields due to their distinct mechanical, electronic, magnetic, and optical properties. This assembly method involves a two-step process: first, the particle surfaces are functionalized with polyphenol moieties, a step that has been shown to be largely independent of the substrate and/or particle surface chemistry, largely owing to the multidentate binding capabilities of polyphenols. This is followed by the directed assembly of the functionalized particles, driven by interfacial molecular interactions and interparticle locking mediated by metal ions. The right-hand figure demonstrates the successful assembly of a superstructure using small sphere-shaped building blocks.

Figure 3. Schematic of versatile materials with different shapes, structures, compositions and functionalities to form superstructures via TA-Fe networks. Right: the images of formed superstructures on SiO2.[5]

A second application of Fe-TA networks is in the context of semi-artificial photosynthesis (Figure 4). Semi-artificial photosynthesis involves the integration of inorganic-biological hybrid systems, which offer the potential to serve as sustainable, efficient, and versatile chemical synthesis platforms. This approach combines the light-harvesting properties of semiconductors with the synthetic capabilities of biological cells. In one example, light-harvesting indium phosphide (InP) nanoparticles were independently prepared and coated with Fe-TA networks, which were then adhered to yeast cells. Owing to the close contact between the nanomaterials and the yeast cells, upon illumination, the nanomaterials could produce photoelectrons that were then transferred to the yeast cells. This, in turn, facilitated the regeneration of NADPH within the yeast cells, which was then harnessed to drive the biosynthesis of shikimic acid (SA).

Understanding the Electron Transfer Pathway of Cytochrome P450s: A Structural Point of View

WANG Yue, 2 March 2023

Cytochrome P450s are a large and ancient enzyme superfamily found in all kingdoms of life. They have a unique ability to detoxify chemicals in humans, making them an important target in medicinal and pharmaceutical drug discovery. The term “P450” is derived from the spectrophotometric peak at the wavelength of the absorption maximum of the enzyme (450 nm) when it is in the reduced state and complexed with carbon monoxide. The official nomenclature of P450 is based on gene sequence similarity, which assigns a family number (e.g., CYP1, CYP2), followed by a subfamily letter (e.g., CYP1A, CYP2B) and then several individual proteins (e.g., CYP1A1, CYP2B6).

P450 systems mainly consist of two functional parts: the heme-containing P450 domain and redox partners (RPs). The P450 domain contributes to substrate binding and transformation, while the RPs contain redox centers that relay electron equivalents from electron donors (usually NADPH) to activate the dioxygen bound to the P450 domain. Together, these events form an electron transfer (ET) pathway of NADPH-RPs-heme-O2.

Coacervates are condensed liquid-like droplets formed by liquid–liquid phase separation of molecules through multiple weak associative interactions. In recent years it has emerged that not only long polymers, but also short peptides can form simple and complex coacervates. This emerging peptide-based coacervate holds promise as efficacious delivery vehicles for multi-purpose biomedical applications.

The core region of the heme domain of all known P450s folds into a triangular globular domain (Figure 1A). Membrane-bound P450s have an additional N-terminal transmembrane domain and a flexible linker that may influence the localization and orientation in the membrane. The catalytic center of P450 is a Fe ion that is ligated to a protoporphyrin XI macrocycle and two additional axial ligands[1]. The proximal one is a thiolate of the strictly conserved Cys from the enzyme, and the distal one is a ligand that varies during the catalytic cycle. The elongated helix I running through the distal side of the heme contains residues that interact with substrate and dioxygen (Figure 1A). Helix F and G, which lie above helix I, are highly flexible and control the opening of the substrate entry tunnel.[2] The space of the closed-form pocket varies substantially in different P450 domains, determining the enzyme-substrate specificity. The heme-coordinating Cys residue located on the C-terminus of the helix L is situated on the proximal side of the heme group (Figure 1B). The proximal side is considered a portal to accept electrons delivered from RPs, and thus there are fewer structural elements on this side. The Cys sulfur is hydrogen-bonded to the nearby peptide NH groups to render the heme iron a sufficient redox potential.[3-4]

Because P450 domains share a similar fold, using the diverse RP systems used by various P450s is also a common approach to categorize these enzymes. Class II P450s are the most common eukaryotic P450s and are generally found in the endoplasmic reticulum (ER). These P450s employ a two-component system that comprises a membranous P450 domain and NADPH-cytochrome P450 reductase (CPR; Figure 2, class II). In the human liver, class II CYPs and CPRs are anchored on ER membrane by a single TM domain with their catalytic domain facing the cytosol. CPR contains FAD-containing reductase and FMN-binding domains that are linked via a “hinge” region, which is presumed to confer conformational plasticity of two flavin-binding domains.

Peptide-based liquid droplets as emerging delivery vehicles

CHEN Hongfei, 2 Feb 2023

Coacervates are condensed liquid-like droplets formed by liquid–liquid phase separation of molecules through multiple weak associative interactions. In recent years it has emerged that not only long polymers, but also short peptides can form simple and complex coacervates. This emerging peptide-based coacervate holds promise as efficacious delivery vehicles for multi-purpose biomedical applications.

1. Peptides as building blocks for liquid microdroplets
There are mainly four kinds of peptide-based coacervate: simple coacervate formed by peptides and peptide derivatives, polymers formed by repeated peptide motifs, complex of oppositely charged peptides and polyelectrolytes, and polymer–peptide hybrids (Figure 1). [1-4] Simple coacervation of peptides is formed by interactions of a single peptide or peptide derivative in aqueous buffer which often triggered by a change in temperature, pH or salinity. The microdroplets contain most of the peptides originally in solution and a significant amount of hydration water. [1] The engineering peptide polymers containing tandem repeats of sequences can form coacervate more easily. Compared with short peptides, peptide polymers are more prone to phase separation because their mixing entropy per amino acid is smaller. [2] By combining two oppositely charged peptides complex coacervates can be formed. Oligopeptides as short as ten amino acids can form stable salt-responsive complex coacervate microdroplets whose critical salt concentrations can be tuned over two orders of magnitude by changing peptide length and composition. [3] Besides of this, short peptides can be grafted onto a hydrophilic polymer backbone to enhance their hydration and create coacervates that more closely mimic the physicochemical properties of membraneless organelles. The hybrids phase separates into liquid microdroplets resembling membraneless organelles in vitro and in living cells. [4]

2. Peptide-based coacervates as stimuli-responsive delivery vehicles
Peptide-based coacervate droplets, which range from nanometers to micrometers, are promising delivery vehicles. Within the microdroplets, molecules can be hold by weak non-covalent interactions, allowing the packaging of different types of therapeutics. Compared with solid particles that are more commonly used for delivery, such as polymer, inorganic or lipid nanoparticles, coacervates are viscoelastic objects that can quickly recruit cargoes under aqueous conditions, which is especially beneficial for preserving the bioactivity of biomacromolecular therapeutics (Figure 2).

Coacervate, developed from thermo-responsive ELPs fused to therapeutics, can greatly prolong retention times of the therapeutics when injected subcutaneously or directly at the tumor sites. Rational molecular design can tune the coacervation temperature which enables the peptides to be injected as a solution at room temperature and then form viscous depots at body temperature. Using this concept, an ELP fused to glucagonlike peptide 1 (a promising drug for treatment of type 2 diabetes) was designed so that it was soluble at ambient temperature for ease of injection, but quickly transitioned at body temperature into a droplet with slow-releasing characteristics. With this strategy, the blood sugar level could be controlled for up to 17 days in a diabetic primate animal model. [5] Peptide microdroplets can also be used to deliver therapeutics inside the cell.

Introduction to Antibody-Drug Conjugates and Recent Advances in Linker

LI Biquan 22 Dec 2022

Antibody-drug conjugates or ADCs are a class of biopharmaceutical drugs designed as a targeted therapy for treating cancer. ADCs combine the targeting properties of monoclonal antibodies with the cancer-killing capabilities of cytotoxic drugs, designed to discriminate between healthy and diseased tissue[1]. Totally, ADCs include three parts: antibody, linker and drug, as shown in Figure 1. Firstly, we could divide the conjugations between antibody and linker to site-specific conjugations and non-specific conjugations. On the other hand, linkers comprise two key parts: the antibody- and the payload-attachment (Figure 1). The bonding between the linker and the mAb is critical since it defines the drug-to-antibody- ratio (DAR, in generally 3 to 4 and maximum would not exceed 8 to 9) and therefore the homogeneity and stability of the ADC. The bonding between the linker and the payload is equally important and determine the releasing mechanisms of the drugs[2-4].

Additionally, we have to make a clear mind at the mechanisms that how ADCs enter the target cells. As shown in Figure 2 (A), endocytosis leads ADCs to the lysosome, a unique compartment with a low pH and high concentration of hydrolytic enzymes. Alternatively, ADC linkers can also be labile to the extracellular tumor microenvironment, according to the non-internalizing mechanism of action previously discussed (Figure 2 (B))[2-3].

For ADCs to be selective and potent, the linker technology employed should strive towards three key properties: (1) High stability in circulation. (2) High water solubility to aid bioconjugation and avoid formation of inactive ADC aggregates. (3) Allow efficient release of a highly cytotoxic payload-linker metabolite[3]. ADC linkers can be classified as ‘cleavable’ or ‘non-cleavable’. For non-cleavable linkers, there is no inbuilt chemical trigger to cleave the linker. Conversely, cleavable linkers exploit specific conditions to release the drug at the target cell. More than 80% of the clinically approved ADCs employ cleavable linkers. In addition, cleavable linkers can be further subcategorized as chemically- cleavable or enzyme-cleavable[3].

Chemically cleavable linkers could be subcategorized as acid cleavable, reducible disulfides and those cleavable by exogenous stimuli, such as extra adding chemicals and photo triggers. On the other hand, enzyme cleavable linkers so far include cathepsins cleavable, glycosidase cleavable sulfatase cleavable and phosphatase cleavable linkers[3-4].

1 Chemically Cleavable Linkers

Acid cleavable linkers aim to exploit the acidity of the endosomes (pH 5.5-6.2) and lysosomes (pH 4.5-5.0), whilst maintaining stability in circulation at pH 7.4. This strategy yielded the earliest clinical success in the field with Pfizer’s gemtuzumab ozogamicin (Mylotarg) ADC and was later employed in Besponsa[5-6] (Shown in Figure 3). In 2019, the group[7] developed a novel silyl ether-based acid-cleavable ADC carrying highly cytotoxic monomethyl auristatin E (Shown in Figure 3). Glutathione (GSH)-cleavable (reducible disulfides) linkers rely on the higher level of glutathione in the cytoplasm (1-10 mmol/L) compared to the blood plasma (5 mmol/L)[8]. Disulfides are the most prominent class of chemically cleavable motifs found in ADC linkers and, alongside hydrazones, they have found clinical success in Pfizer’s Mylotarg and Besponsa. Disulfides are stable at physiologic pH but are susceptible to nucleophilic attack from thiols (Figure 4) [3-4]. The examples are shown in Figure 4. Whilst the cleavage of ADC linkers with endogenous triggers is the simplest method for drug release, external small molecule triggers of extracellular drug release may offer the following advantages: (1) avoidance of any disparity in linker cleavage rates caused by variable biology across patients, and (2) the ADCs can be effective when extracellular concentrations of endogenous triggers are insufficient for efficient payload release[4].

Stenton et al. have described a thioether-containing linker that, upon exposure to Pd (0), releases an amine-linked payload (Figure 5 (A))[9]. Rossin et al. have recently developed an ADC bearing an antibody fragment and a trans-cyclooctene linker that, upon inverse-electron demand Diels–Alder (IEDDA) reaction with an externally administered tetrazine, releases an amine-containing payload in vivo (Figure 5 (B))[10].

The strategy of payload release based on photo-responsive cleavable triggers has gradually emerged in recent years. Photo-responsive cleavable triggers have the following advantages: (1) controlled drug release and effective reduction in off-target toxicity and (2) determination of the cleavage mechanism and no limitation on the intracellular release. Photo- responsive cleavable triggers enjoy the following advantages, including low toxicity, a rapid response, and high sensitivity and specificity[4].

In 2015, Nani et al. [11] firstly employed a near-infrared (NIR) light photo-caging strategy for ADCs (Figure 6 (A)). More recently, our group initially reported a novel ultraviolet (UV) light-controlled ADC (Figure 6 (B)) [12]. The linkers introduced a UV light-controlled O-nitrobenzyl group as a chemical trigger.

2 Enzyme Cleavable Linkers

In the classical mechanism of action, ADCs are trafficked to the lysosome of a cell where high concentrations of unique hydrolytic enzymes reside, thereby offering an opportunity for enzyme cleavable linkers to be selectively cleaved intracellularly. For selective payload release in the extracellular tumor area, enzymes that are upregulated in tumor microenvironments must be exploited[3].

Clip Chemistry: Diverse (Bio)(macro)molecular and Material Function through Breaking Covalent Bonds

YUAN Dingdong, 27 Oct 2022

The definition of click reaction is “The [click] reaction must be modular, wide in scope, give very high yields, generate only inoffensive byproducts that can be removed by nonchromatographic methods, and be stereospecific (but not necessarily enantioselective). The required process characteristics include simple reaction conditions (ideally, the process should be insensitive to oxygen and water), readily available starting materials and reagents, the use of no solvent or a solvent that is benign (such as water) or easily removed, and simple product isolation.” So, the author borrows this definition and gives the definition of clip reaction “Covalent bond breaking reaction that is modular and wide in scope. It must give very high yields and generate inoffensive byproducts, ideally in a stereo- and regiospecific manner. The process should involve simple reaction conditions and readily available starting materials and reagents.” Here, they give some examples of clip reactions in biomolecular application.

The first type is the Stoichiometric Clip Reaction; stoichiometric clip reactions require the stoichiometric addition of another reagent to trigger bond cleavage. For example, the disulfide reduction reaction. The process is often carried out in the laboratory using a phosphine-reducing agent, as shown in Figure 2a, and in chemical biology, we always used DTT or TCEP as the reductive reagents to reduce their disulfide bond in proteins to break their structure, such as the antibodies, also we use this method to free the cysteine for the next reactions. Also, the silyl ether−fluoride cleavage is another powerful clip reaction, which is most notably known for its role in the protection/ deprotection of alcohols; this method is widely used in organic synthesis; nowadays, this method is also used as the protect part for some biological application such as the drug release (Figure 2b). While the inverse electron demand Diels−Alder reaction of tetrazines and alkenes has been studied since at least the late 1950s, utilized in elegant total syntheses, and more recently popularized as an exceptional click reaction, its development as a clip reaction was only recently reported by Robillard and coworkers. Here, the clip reaction is triggered by a click reaction using modified tetrazines and trans-cyclooctenes and is driven by both dinitrogen extrusion (immediately following the cycloaddition step) and generation of aromaticity (formation of the 1,2-diazine). While these reagents are highly reactive when combined, they are quite stable on their own, giving this system many of the best characteristics of click and clip reactions (Figure 2a and 2c).

Hydrogel in medical biotechnology

HAN Yongxu, 29 Sep 2022

Hydrogels are typically defined as interconnected 3D molecular networks containing a high density of functional groups that enable the absorption and retention of large amounts of water. A wide variety of materials can be used to produce hydrogel networks. These materials can be broadly classified as having either synthetic or naturally occurring components. Synthetic building blocks, including polyacrylic acid, polyethylene glycol and so on. Meanwhile, natural components, including alginate, chitosan, and nucleic acids, in addition to proteins such as collagen, keratin and gelatin that naturally form hydrogels often possess increased biocompatibility and biological functionality when compared to synthetic hydrogels.

Hydrogel in biomedical exploration like drug delivery, tissue engineering, cell delivery, building scaffolds has become increasingly studied. For example, like Figure 2, the author reported a family of synthetic hydrogels that promote extensive organoid morphogenesis through dynamic rearrangements mediated by reversible hydrogen bonding. These tunable matrices are stress relaxing and thus promote efficient crypt budding in intestinal stem-cell epithelia through increased symmetry breaking and Paneth cell formation dependent on yes-associated protein. As such, these well-defined gels provide promising versatile matrices for fostering elaborate in vitro morphogenesis.

Another report shown in Figure 3, demonstrated that silver nanoparticle (AgNP)-based hydrogel enhances immunotherapy via the modulation of oral microbiota. The authors found a genus of bacteria (Peptostreptococcus) that could activate the immune system and improve the prognosis of patients. They had shown that in mice with subcutaneous or orthotopic murine OSCC tumours, combination therapy with the two components (nanoparticle-incorporating hydrogel and exogenous P. anaerobius) synergized with checkpoint inhibition with programmed death-1.

A Brief introduction to electron microscope

JIANG Hao, 19 May 2022

1. The limit of traditional optical microscope

Traditionally, scientists use optical microscope to observe microstructure of biological tissue and cells. But optical microscope have a limit in resolution. The limitation is caused by Rayleigh Criterion. The angular resolution of an optical system can be estimated (from the diameter of the aperture and the wavelength of the light) by the Rayleigh criterion defined by Lord Rayleigh: two point sources are regarded as just resolved when the principal diffraction maximum (center) of the Airy disk of one image coincides with the first minimum of the Airy disk of the other, as shown in Figure. 1.

2. Transmission electron microscope

To reach higher resolution, scientists turn to use electrons to observe the micro world. 1933, Ruska built the first electron microscope that exceeded the resolution attainable with an optical microscope. An electron microscope is a microscope that uses a beam of accelerated electrons as a source of illumination. Electron microscopes use shaped magnetic fields to form electron optical lens systems that are analogous to the glass lenses of an optical light microscope.

The original form of the electron microscope, the transmission electron microscope (TEM), uses a high voltage electron beam to illuminate the specimen and create an image. The electron beam is produced by an electron gun, commonly fitted with a tungsten filament cathode as the electron source. The electron beam is accelerated by an anode, focused by electrostatic and electromagnetic lenses, and transmitted through the specimen that is in part transparent to electrons and in part scatters them out of the beam. When it emerges from the specimen, the electron beam carries information about the structure of the specimen that is magnified by the objective lens system of the microscope. The spatial variation in this information (the “image”) may be viewed by projecting the magnified electron image onto a fluorescent viewing screen, a photographic film or the sensor of a digital camera.

Sulfonamide antibiotics and cephalosporins

ZHANG Yu, 12 May 2022

1. Sulfonamide antibiotics

Sulfonamide drugs were the first broadly used antibiotics and paved the way for the antibiotic revolution in medicine. Sulfonamide is a functional group act as competitive inhibitors of the enzyme dihydropteroate synthase (DHPS) which is an enzyme involved in folate synthesis. Folate is vital to the growth and development of the livings. Compared with the bacteria, human can acquire folate though the diet. Therefore, cutting of the folate supply by using the sulfonamide antibiotics can help us inhibit bacterial infection specifically without hurting ourselves. However, as a competitive inhibitor, the sulfonamide antibiotics can only inhibit the growth and multiplication of bacteria, but not kill them.^[1]

Prontosil, as Bayer named the new drug, was the first medicine ever discovered that could effectively treat a range of bacterial infections inside the body. It had a strong protective action against infections caused by streptococci, including blood infections, childbed fever, and erysipelas, and a lesser effect on infections caused by other cocci. However, it had no effect at all in the test tube, exerting its antibacterial action only in live animals. Later, Daniel Bovet, Federico Nitti, and Jacques and Thérèse Tréfouël found that the drug was metabolized into two parts inside the body, releasing a smaller, colorless, active compound called sulfonamide. ^[3]

The discovery of Prontosil paved the road for the development of sulfonamide antibiotics and filled the blank of the bacteria infection treatment before Penicillin. With the discovery of Penicillin, the sulfonamide antibiotics slowly faded out from the antibiotic market because of more side effects. However, sulfonamide drugs still work as diuretics or COX-2 inhibitors in pharmaceutical field.

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

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

PROTAC

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

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

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.

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.).^[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.^[4]

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]

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).

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.

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 [1].

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.

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 [1].

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 [1].

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 [1].

One of the cheapest methods uses calcium phosphate, originally discovered by F. L. Graham and A. J. van der Eb in 1973 [2]. 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 [1].

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 [3].

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 [4]. Lipofection generally uses a positively charged (cationic) lipid (cationic liposomes or mixtures) to form an aggregate with the negatively charged (anionic) genetic material [5]. This transfection technology performs the same tasks as other biochemical procedures utilizing polymers, DEAE-dextran, PEI and, calcium phosphate [6]. 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 [5].

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 [8] 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. [11]

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.

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).

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.

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.

T3SS

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.

Bioorthogonal reactions

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^[1]. 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) ^[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) ^[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) ^[3].

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.