Abstract:Chen-Lu Tsou and Protein Folding Research in China

Abstract:Dr. Chen-Lu Tsou (1923-2006) obtained his doctorate from Cambridge in 1951, and became one of the founders of Chinese biochemistry. He spent most of his life on protein structure and function, making a number of original contributions. To commemorate his one hundred years of birth, two of his research achievements are elaborated in detail here, one of which is relation between the functional groups of proteins and their biological activity, and the other is on kinetics of irreversible modification of enzyme activity. This commemorative article intends to share with audiences how much can be achieved by the older generations of Chinese scientists with great minds but few resources, as epitomized in the two great pieces of work by Dr. TSOU.

Abstract:Transfer ribonucleic acid (tRNA) is the RNA molecule with the largest variety of post-transcriptional modifications. In particular its anticodon loop contains a large number of modifications. Mitochondria have a relatively independent protein synthesis system, mitochondrial tRNAs (mt-tRNAs) are all encoded by the mitochondrial genome. Studies have shown that 5-taurinomethyluridine modification (τm⁵U) only exists at position 34 of mitochondrial tRNAs of higher eukaryotes, regulating the fidelity of codon and anticodon interaction and contributing to translation speed and fidelity. Human GTPBP3 and MTO1 mediate mitochondrial τm⁵U modification, whose functional defects may cause mitochondrial encephalomyopathy. This review summarizes the biological properties of τm⁵U modification and its modifying enzymes, providing a new insight into the mechanism of τm⁵U modification and the pathogenesis of mitochondrial diseases caused by τm⁵U modification defects.

Abstract:Accumulation of unfolded proteins in the endoplasmic reticulum (ER) lumen causes ER stress, which triggers the unfolded protein response (UPR) to restore protein homeostasis. So far, three UPR sensors have been identified, including IRE1, PERK, and ATF6. All of them are ER transmembrane proteins, which become activated and initiate downstream UPR signals under ER stress. Though first discovered during the study of how cells respond to ER stress, it remains unclear how the ER stress signal is sensed by the three UPR sensors. Structural studies provide insight into how direct binding of ER-localized peptides to the lumenal domain of IRE1 and PERK facilitates their oligomerization and thus activation. In another model, dissociation of the ER chaperone BiP is the key to the UPR activation. In addition, further studies reveal that the UPR not only functions in maintaining protein homeostasis and UPR sersors is not solely activated in response to the accumulation of unfolded proteins in the ER. Lipid bilayer stress, cytosolic factors or intercellular signals may initiate the UPR. Despite the importance of the UPR in physiology and pathophysiology, how the UPR is activated under physiological or pathophysiological conditions are largely unknown. Developing novel strategy to monitor the unfolded and misfolded proteins in the ER lumen will advance our understanding of the relationship between ER stress and the UPR. This paper introduces the discovery and the canonical pathways of the UPR, with a focus on the current mechanistic understanding of the UPR activation, and discusses the relationship between the UPR and ER stress as well as related questions.

Abstract:Tardigrades, also known as water bears, possess an extraordinary ability to survive for extended periods under harsh conditions like extreme dryness, low temperatures, and low pressure. Under such condition, tardigrades enter into a state of cryptobiosis, where they undergo dehydration, body shrinkage, and metabolic halting, enabling them to endure extreme conditions for years. Once the environment improves, the cells or body in cryptobiosis can rehydrate and recover. Tardigrades owe this ability in part to some unique tardigrade disordered proteins (TDP), also called heat-soluble proteins, which safeguard their cells during dehydration by reshaping their structure to fix liquid water during desiccation. However, research on these proteins is still in its nascent stages, and the thorough mechanistic investigations are lacking. This article provides a concise overview of these unique proteins found in tardigrades, including their sequence, physicochemical properties, potential biological functions, and mechanisms. Currently, there are 3 major families of TDPs known as cytosolic-abundant heat-soluble proteins (CAHS), secretory-abundant heat-soluble proteins (SAHS), and mitochondrial-abundant heat-soluble proteins (MAHS). A large fraction of these proteins contain are intrinsic disorder regions that are critical to their function in desiccation tolerance. The CAHS proteins contain a long α-helix and two flanking intrinsic disordered regions, and play roles in the vitrification process during desiccation. The SAHS proteins contain a central β-barrel and a helix-turn-helix “cap” motif, and they may form a protective network outside the cells under extreme environment. The MAHS proteins contain a loosely organized “core” consist of 5 α-helix and the flanking region of disordered motifs, and their major roles is to protect the mitochondrial during desiccation. The unique structure and property of these TDPs may help to develop new technology in aiding higher animals to adapt to extreme environments, such as low temperatures and low oxygen. The ability of human cells to undergo cryptobiosis and reversible recovery in extreme environments has critical implications in fields like medicine, space exploration, and interstellar immigration.

Abstract:As a special member of the Rho family, RhoB exhibits distinct biological activities and plays a unique role in tumorigenesis, and its protein levels are tightly regulated compared with other family members. Acting as a tumor suppressor, RhoB has attracted increasing attention in targeted cancer therapy. However, RhoB has been reported to promote development of certain types of tumors, in which the underlying molecular mechanisms remain unclear. In this article we summarize the studies on post-translational modifications of RhoB, particularly its ubiquitination and sumoylation, and their roles in determining the fate of tumor cells. Under normal physiological conditions, RhoB is targeted to ubiquitination and degradation by E3 ubiquitin ligase Smurf1, therefore maintaining relatively low protein levels of RhoB for cell survival. In response to single strand DNA damage, activated Chk1 phosphorylates Smurf1 to induce its self-degradation, resulting an accumulation of RhoB. Meanwhile, RhoB is also phosphorylated by Chk1, leading to dissociation of RhoB from plasma membrane. Phosphorylated RhoB is further sumoylated by SUMO E3 PIAS1 in cytosol, which is required for translocation of TSC2 to lysosomes to inhibit mTORC1 activity and subsequent initiation of autophagy. Hence, RhoB plays a pivotal role in determining cell fate by switching between its ubiquitination and sumoylation. These multiple layers of regulation enable RhoB to execute appropriate cellular responses in different cellular states and environmental conditions. Further exploring the regulatory mechanisms of RhoB in tumor development may provide valuable clues and ideas for cancer treatment by targeting RhoB.

Abstract:Mitochondria are semi-autonomous cellular organelles responsible for oxidative phosphorylation (OXPHOS) and adenosine triphosphate (ATP) synthesis and are the powerhouses of cellular metabolism. Mitochondria are present in almost all eukaryotic organisms and are involved in apoptosis, calcium homeostasis, and regulation of the innate immune responses, which play a vital role in normal physiological processes. Mitochondria contain their own DNA that encodes 37 genes, including 2 rRNAs, 13 mRNA, and 22 tRNAs genes. Gene expression in mitochondria involves complex transcriptional and post-transcriptional processes, including cleavage of polycistronic RNA, RNA modification, and terminal processing of RNA. These processes require the coordinated spatiotemporal action of several enzymes, and many different factors are involved in the regulation and control of protein synthesis to maintain the stability and turnover of mitochondrial RNA. Disorders in mitochondrial RNA processing lead to changes in RNA expression profiles, interfere with protein translation, cause mitochondrial dysfunction, and result in a variety of mitochondria-related diseases. Although substantial progress has been made in the field of mitochondrial RNA processing and regulation, there are still many controversies and unknowns. This article reviews the latest research progress on mitochondrial DNA transcription, RNA post-transcriptional processing, and factors affecting RNA processing.

Abstract:As a kind of membrane-active lipopeptide antibiotics, polymyxins are the last-line therapy against multidrug-resistant Gram-negative pathogens. Through interacting with the lipopolysaccharide molecules, polymyxins disorganize the structure of bacterial outer membrane, and finally lead to the cell death. Nevertheless, the precise mechanisms of polymyxin pharmacology remain largely unknown, which is mainly due to the limited ability of current biochemical and structural approaches to characterize the interaction between cell membranes and drugs. This in turn significantly hinders the design and development of new-generation polymyxins. In recent years, molecular dynamics simulations have been successfully applied in the field of polymyxin pharmacology. In particular, a series of simulation models, including bacterial membranes-polymyxins, and human cell membrane-polymyxins, has been developed and tested. Previous studies have shown that polymyxin adopted a unique folded conformation in bacterial outer membrane, which played a key role in the antimicrobial activity of polymyxins. Further, various lipopolysaccharide modifications could change the structural and physical properties of bacterial outer membrane and thereby confer polymyxin resistance to bacteria. Moreover, recent studies revealed that polymyxins may disrupt the membrane of renal tubular cells, and also attenuate the function of different ion channels, which provide a clue to understand the detailed mechanism of polymyxin-induced nephrotoxicity. In this review, we summarized the applications of molecular dynamics simulations in the interaction of polymyxins with different biological membranes, with the aim to refresh our understanding of the link between polymyxin pharmacology and cell membranes and to provide mechanistic guides for the future design of novel antimicrobial drugs.

Abstract:Phosphate (P_i) homeostasis is a tightly regulated process in all organisms. Dysfunction of P_i homeostasis leads to renal Fanconi syndrome in humans, severe growth retardation in plants, and lethality in microorganisms. To achieve a delicate balance between the biosynthetic requirements for P_i and the risks of excessive cytoplasmic P_i, unicellular organisms maintain important P_i stores in membrane-bound, acidocalcisome-like organelles in the form of inorganic polyphosphates (polyP). As the only known eukaryotic polyP synthetase, Saccharomyces cerevisiae vacuolar transporter chaperone (VTC) complex synthesizes polyP from adenosine triphosphate (ATP) and translocates polyP across the vacuolar membrane to maintain an intracellular P_i homeostasis. In this article, the latest progress on the structure and function of VTC complexes were reriewed from the aspects of structural characteristics, polyP synthesis, and polyP transport mechanism. The focus is on the recently published intact structural of VTC complexes and exploring the activation mechanism of VTC.

Abstract:The increasing prevalence of obesity and related diseases has made it imperative to address obesity as a pressing public health concern. Urgent attention is needed to develop innovative treatment options and strategies for obesity-related diseases. Fatty acid synthase (FAS) is a complex multiple enzyme that plays a critical role in the biosynthesis of long-chain fatty acids. Its dysregulation has been implicated in various human diseases, including obesity, type 2 diabetes, cancer, inflammation, and cardiovascular disease. Consequently, research on FAS inhibitors has received increasing attention over the past 30 years. In China, traditional Chinese medicines (TCMs) and functional foods are being recognized for their potential to alleviate disease status, particularly chronic diseases like obesity. Several TCMs have been found to have a strong inhibitory effect on FAS. This review aims to summarize the literature on the role of FAS as a biomarker and therapeutic target in obesity and related diseases while providing evidence to support the anti-obesity potential of TCMs and functional foods with FAS inhibitory activities.

Abstract:Tumors are a serious threat to human health. The current traditional methods of cancer treatment include surgery, chemotherapy, radiotherapy and targeted drug therapy. In recent years, tumor immunotherapy, especially chimeric antigen receptor (CAR) T cell immunotherapy has flourished in basic research and clinical application, and has achieved great success in the treatment of hematological malignancies. However, numerous studies have shown that various degrees of toxic and side effects may occur after cellular immunotherapy, and some patients relapse after remission. Therefore, it is of great significance to understand the challenges and limitations of cell therapy and find solutions to continue to exert the potential of cell immunotherapy. This article reviews the CAR structure, the selection of viral vectors, the challenges and prospects of cell therapy.

Abstract:β-Hydroxy-α-amino acids (HAAs) are a class of important chiral intermediates and have a wide range of uses in the pharmaceutical industry. Due to its adjacent chiral centers, it has attracted much attention to exploring strictly stereoselective biosynthesis methods. Threonine aldolase (TA) can catalyze the aldol reaction in one step for the synthesis of HAAs under mild conditions. TAs also exhibit a broad substrate spectrum and can catalyze the condensation of various aldehydes and amino acids, thereby constructing rich HAA libraries with immense potential for industrial applications. In this review, we have summarized the research progress of TA, including new enzyme mining, structure, catalytic mechanism analysis, high-throughput screening methods, protein engineering, and synthesis applications. Notably, we mainly focus on the research achievements of TA in structure-function relationship, mechanism analysis, and protein engineering. Currently, the catalytic process of TA has been elucidated, where it catalyzes the aldol reaction through the Schiff base exchange mechanism. Additionally, researchers have proposed diastereoselectivity regulating mechanisms of TA such as the “pathway hypothesis” and “dual conformation hypothesis”, which provide a foundation for unraveling the mystery of TA diastereoselectivity regulation. Moreover, significant progress has been made in TA molecular evolution, with multiple mutants obtained that showed strict diastereoselectivity synthesis for various chiral HAAs and their intermediates. Furthermore, we have discussed the current challenges and prospects of TA, which will guide for accelerating the industrial application of TAs as tool enzymes for synthesizing high-value HAAs compounds.

Abstract:With the aging population increasing worldwide, neurodegenerative diseases are becoming a major public health crisis. Neurodegenerative diseases are a group of neurologic disorders caused by the loss of the structure and function of neurons, mainly manifested by degenerative changes or death of neurons in specific regions. Neurodegenerative diseases are often classified into two categories: one affecting motor function, such as Parkinson’s disease (PD), Huntington’s disease (HD), and amyotrophic lateral sclerosis (ALS), and the other affecting cognitive function, such as Alzheimer’s disease (AD) and frontotemporal dementia (FTD). Mitochondrial impairment manifesting in the affected neurons is a common feature in these neurodegenerative diseases. The coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10) is a mitochondrial protein encoded by the nuclear genome, mainly located in the mitochondrial intermembrane space. CHCHD10 plays a critical role in the maintenance of structural integrity and function of mitochondria. Various CHCHD10 gene mutations have been identified in different neurodegenerative diseases, including FTD, ALS, PD, AD, etc. Mutations of the CHCHD10 gene or loss of its function can lead to the loss of mitochondrial cristae structure and abnormal mitochondrial function. However, the specific function of the CHCHD10 protein and the mechanism underlying mitochondrial damage caused by its gene mutations remain unclear. Here we review the recent advances in the structure and mitochondrial function of CHCHD10 and discuss the mechanism of mitochondrial dysfunction induced by gene mutations or functional loss of CHCHD10. Investigating the role of CHCHD10 in maintaining mitochondrial function will help us to understand the pathological mechanism of neurodegenerative diseases and explore potential therapeutic interventions for these devastating diseases.

Abstract:The high specificity and sustainability of enzymes make them widely used as green catalysts, and their stability and catalytic activity are vital for their practical applicability. Recently, enzymes have been endowed with desired physical and catalytic properties via using protein structural modification. From the protein structural point of view, enzyme thermal stability has been improved by modulation of non-covalent/covalent interactions (hydrophobic interaction, hydrogen bonding, salt bridges, aromatic interaction and disulfide bonds), loop truncation, C-/N-terminal engineering, introduction of proline with highest conformational rigidity in the flexible region, and substitution of glycine with highest conformational entropy. Meanwhile, the catalytic function has been enhanced or altered by various methods, including reducing steric hindrance, widening the binding pocket, moderating substrate binding affinity and active site flexibility. While, the generation of new features or improvement of the existing features often comes at the expense of the other ones. Thus, strategies include screening suitable mutation sites, co-selection for stability and activity, and using highly stable proteins as the parental backbones are also discussed to overcome the stability-activity trade-off. This review summarized recent advances in structural modification to improve the stability or/and catalytic activity of enzymes and further provided a brief prospect in the future developments.

Abstract:In eukaryotic cells, deadenylation is achieved by deadenylases, which are 3"-5" exonuclease that specifically degrade poly(A) or oligo(A) at the 3"-end of RNAs. Most eukaryotic cells contain more than a dozen of deadenylase isoenzymes. Among them, the CCR4-NOT complex and the PAN2-PAN3 complex are the main contributors of non-specific deadenylation of mRNAs, while PARN and PNLDC1 are involved in highly regulated deadenylation of mRNAs and the biogenesis of non-coding RNAs. Besides their roles in RNA metabolism, deadenylases are also regulators of transcription, translation efficiency, stress response, immunological response, genome integrity, and self-renewal and differentiation of stem cells. In vitro and in vivo studies have discovered that deadenylase activity can be modulated by low-molecular-weight compounds, intramolecular interactions between catalytic and non-catalytic/structural domains, post-translational modifications, and binding partners. By regulating the 3"-tail length of poly(A) or oligo(A) of RNAs, deadenylases have been found to participate in diverse cellular, physiological and pathological processes by modulating RNA homeostasis. Particularly, deadenylases are key players of development by regulating the clearance of maternal mRNAs, the expression of tissue-specific genes and the cross-talk with developmental signaling pathways. Recently, inherited mutations or aberrant expression of deadenylases has been associated with many diseases including telomere diseases, cardiovascular diseases, neurodevelopmental diseases, cancers, and metabolic diseases. The precise regulation of deadenylases in their diverse intracellular functions may be achieved by a complicated network composing of various cis-acting elements in the targeted RNAs, thousands of trans-acting RNA-binding proteins, and numerous post-translational modifications. In this network, RNA-binding proteins may act as hubs to bind with targeted RNAs with specific cis-acting elements and to recruit a distinct deadenylase via protein-protein interactions, and thereby to modulate RNA fate by modifying the poly(A) length or trimming the oligo(A) at the 3"-end. The changes in the expression profile of RNA-binding proteins and in the post-translational modifications of deadenylase-binding partners provide a dynamic and responsive network to achieve the spatiotemporal regulation of gene expression. This complicated regulating network facilitates the cells to maintain RNA homeostasis or switch transcriptome to meet the demands of cell growth, proliferation, cell differentiation, stress response and cell death. The regulating network of deadenylases may also cross-talk with the other cellular pathways such as signaling transduction, autophagy, and anabolism of various biomacromolecules. In this review, we will discuss the regulators of deadenylases, the mechanisms of RNA homeostasis regulated by deadenylation, and the emerging roles of deadenylases in health and diseases.

Abstract:Green polymer synthesis routes especially for enzymatic polymerization, have become a hot spot in the preparation of biomedical polymers owing to its characteristics of mild reaction conditions, low dispersity index, no residue of metal catalysts, and high enantio- and regio-selectivity. Oxidoreductases, hydrolases and transferases have been successfully applied in the polymer synthesis. Among them, lipase-catalyzed polycondensation and ring-opening polymerization were most widely studied, which dramatically facilitated the preparation of polymeric materials with high molecular mass and narrow distribution. Meanwhile, enzymatic reversible deactivation radical polymerization including reversible addition-fragmentation chain transfer polymerization and atom transfer radical polymerization has been rapidly developed because of its unique advantages of mild reaction, high efficiency and oxygen resistance. In addition, to solve the limitations of structure, property and application of polymers, enzymatic polymerization has been combined with chemical routes including atom transfer radical polymerization and ring-opening metathesis polymerization, and the polymers have been applied in the field of drug/gene delivery. Based on the existing literatures, this paper reviewed the progress of lipase-catalyzed polymerization, enzyme-catalyzed reversible radical polymerization and chemoenzymatic polymerization, and the limitations of current research and the future directions were also discussed.

Abstract:The problem of protein folding has been one of the frontiers of biological research. Disruption of proteostasis is closely related to ageing and the pathogenesis of many neurodegenerative diseases. The correct folding of proteins and proteostasis largely depend on the complex network including molecular chaperones. Many studies have shown that antibodies can act as molecular chaperones to promote proper protein folding and prevent abnormal protein aggregation. The strict substrate specificity gives them the potential to be used to treat specific protein-misfolding diseases and to help refolding of inclusion bodies. This paper briefly introduces the progress of research on molecular chaperones, elaborates the research progress of antibodies and single-chain fragment variable antibodies with chaperone function, and discusses on the recent research status of antibodies which can inhibit protein aggregation.

Abstract:Nucleic acid metabolism processes such as the synthesis and degradation of DNA and RNA are the basic metabolic units to maintain the growth and development, metabolism, genetic variation and aging, and widely participate in the whole process of the body’s life activity. The enzyme activity related to nucleic acid metabolism is crucial for maintaining the stability of the intracellular environment, and the change of the activity may cause the occurrence and development of many diseases. The enzymes related to nucleic acid metabolism have become important targets for the study of various diseases and are indispensable tools in the field of biotechnology and bioengineering, such as polymerase chain reaction, site-directed mutagenesis, molecular cloning and DNA sequencing. Therefore, nucleic acid metabolism is the basis of all nucleic acid studies and related life science researches. In this paper, we introduce the common methods of enzymatic analysis of nucleic acid metabolism, and focus on the simple and fast real-time fluorescence method, classify and compare them according to their principles, development history and applications, and also prospect the future study and development of tools for enzymatic analysis of nucleic acid.

Abstract:Pancreatic cancer is one of the most difficult malignant tumors to be diagnosed and treated, with insidious onset, rapid progression and poor prognosis. Presently, surgery is still the preferred method for the treatment of pancreatic cancer. However, due to lack of early symptoms, approximately 70% of patients are diagnosed with local spread or distant metastasis, making it impossible to undergo surgical treatment. Development of effective approaches for better administration of the disease will be unmet and effective way for reducing the mortality and the morbidity. Unfortunately, detection of pancreatic cancer, especially at early stage, is challenged by the lack of highly sensitive and specific biomarkers. Imaging methods (CT, MRI, EUS, etc.) often fail to detect early lesions and is easily influenced by operator. Routine clinical markers such as CA19-9, CA125, CA242 and CEA were limited with unsatisfactory sensitivity or specificity. In recent years, extensive studies on biomarkers mainly focused on genetics, transcriptomics, and proteomics. Especially, non-protein coding RNA (ncRNA) consisting of microRNA (miRNA), long non-coding RNA (lncRNA) and circular RNA (circRNA) have proposed many new ideas about early detection of pancreatic cancer. However, the majority of them remain in the laboratory research stage. Few of them, to our knowledge, have gone into clinical practice. A mature study on biomarker may integrate data from genomics, transcriptomics, proteomics, or metabolomics, and combine with individual characteristics of patients (such as body mass index, history of diabetes, smoking, drinking and other risk factors) through large-scale, prospective and validation studies.

Abstract:Autoradiography generally involves in metallic silver stain formation through irradiation to photosensitive materials (such as X-ray film) with further development and fixation steps using a radiolabeled probe, and the stain density indicates the relative amount of its target molecules and its distribution in tissue slices. This traditional method has a wide range of applications both in biological studies and preclinical drug researches. Phosphor film imaging has significantly shortened the autoradiography’s experimental intervals, and the positron nuclides with short-half-lives can also be performed. The purpose of this paper focuses on the frontier of some experimental works that can’t be completely replaced by non-radioactive labeled methods, such as some enzymatic activity assays, protein or peptide phosphorylation site analyses, nucleic acid assays with low concentrations, and the target (such as receptors) molecule distributions in tissue slices by radiolabeled selective ligands. Thus the technological platform should be reformulated to adapt to the current trends of experimental works.

Abstract:Acetylation is a reversible post-translational modification of proteins mediated by acetyltransferases and deacetylase. Acetyltransferase engages in transferring the acetyl moiety of acetyl-CoA to the amino acid residue of the substrate protein, and the acetyl moiety of protein is removed by deacetylase. Acetylation is involved in the regulation of many basic biological processes, acting on histones and non-histone proteins, thus affecting a series of cellular processes such as gene transcription, regulating mRNA stability, mediating protein localization and degradation, etc. More and more studies have shown that protein acetylation plays an important role in the pathogenic process of pathogenic bacteria. Legionella pneumophila (L. pneumophila) is an intravacuolar pathogen that can cause acute pneumonia in susceptible human hosts. L. pneumophila can be airborne to infect macrophages in human lung tissue. Successful intracellular survival in hosts depends on the formation of the specialized Legionella-containing vacuolar (LCV). Upon invading host cells by phagocytosis, more than 300 different effectors are delivered into host cells via the Icm/Dot type IV system (T4SS) into host cells. These effectors hijack a variety of host cellular processes by diverse mechanisms, redirecting components of the host cell secretory pathway to remodel and maturate the LCV, and play an extremely important role in L. pneumophila survival and replication in host. Chief among them are mediated the post-translational modification that perturb host signal pathway. Among L. pneumophila effectors are quite a bit proteins that show primary amino acid sequence homology to eukaryotic GNAT family acetyltransferases or prokaryotic serine/threonine acetyltransferases. This article mainly reviews acetylation modification, the pathogenic mechanism of L. pneumophila, and the role of acetylation in the pathogenic process of the pathogen and highlights the known virulence as acetyltransferase and their role in host interaction, providing a reference for understanding the mechanism of acetylation modification in the pathogenic process of L. pneumophila.

Abstract:R-loops are formed during transcription when the nascent RNA generated by RNA polymerases hybridizes with its complementary DNA template, giving rise to a region of DNA∶RNA hybrid and a displaced single-stranded DNA. R-loops are stable structures that have important beneficial physiological functions, but also could pose a threat to genomic stability. Unscheduled R-loops induce cell cycle checkpoint activation, DNA damage, and chromosome rearrangement in mammalian cells. R-loops expose unstable single-stranded DNA, which is prone to transcription-related mutations and recombination. R-loops may also directly block DNA replication, leading to DNA double strand breaks. Abnormal accumulations of R-loops have been found in some syndromes, human neurological disorders, and cancers. On the other hand, R-loops also play positive roles in physiological processes, such as epigenetic modification, DNA repair, gene regulation and mitochondrial stability. R-loops forming on transcription-termination regions, promote RNA polymerase pausing before termination. R-loops are regulated delicately in cells. Collisions between replication and transcription cause accumulation of R-loops. Replication stress, DNA damage and RNA Pol II pausing also induce R-loop formation. To resolve R-loops when they form, cell evolve numerous dissolution mechanisms. Ribonuclease RNase H1 and RNase H2 bind to R-loops and then catalyze the cleavage of RNA. Helicases, such as SETX, DHX9, DDX21, unwind the RNA from the R-loops. Defects in RNA processing factors, chromatin modulators, DNA repair proteins, cause accumulation of R-loops, suggesting they are involved in R-loop regulations. To detect R-loops, several methods have been developed and are mainly based on the S9.6 antibody and the HBD domain of RNase H1, however, both of them possess some issues. Understanding the regulatory mechanisms of R-loop formation and clearance could help us better know how cells maintain genomic stability and prevent disease development. In this review article, we summarized functions and regulations of R-loops. We also discussed methodologies used to detect R-loops. Finally, we proposed some future perspectives of R-loop research.

Abstract:With the development of biopharmacology, many therapeutic enzymes have been developed for treatment of various diseases, including metabolic diseases, thrombotic cardiovascular diseases and cancers. Most of the approved therapeutic enzymes are hydrolases, which are used to clean the toxic organic compounds and biomacromolecules in vivo, such as saccharides, lipids, proteins and their aggregates. Due to the high catalytic activity, affinity and specificity of enzyme towards substrate, enzyme therapy has a shorter time frame and fewer side reactions compared to other therapeutic approaches. However, there are several critical bottlenecks that limit the effectiveness of therapeutic enzymes, including immunogenicity, short circulation time, and lack of tissue specificity. Many approaches have been used to overcome these challenges. Several second generation therapeutic enzymes with significantly improved effectiveness have been developed using molecular engineering technologies such as glycan modification and pegylation. In addition, enzyme gene therapy becomes an emerging approach for treatment of diseases caused by enzymes deficiencies. Here, we reviewed the current enzyme-based therapeutics, and discussed its advantages, challenges and future perspectives.

Abstract:Adenylate kinases (AK) are widely existing in various organisms, which play critical roles in maintaining the normal content of nucleotides and regulation of energy metabolism in cells. Among AK family members, AK6, also known as human coilin-interacting nuclear ATPase protein (hCINAP), is an atypical adenylate kinase that possesses both activities of adenylate kinase and ATPase. We have been carried out long-term research on the structure, enzymatic activity and functions of this enzyme, and demonstrated that AK6/hCINAP plays critical roles in many biological processes, including gene transcription, ribosome quality control, embryonic development, senescence, cell metabolism, cell proliferation and apoptosis, DNA damage responses, inflammatory response, and tumor development. In this review, we summarize the structural features, biological roles and transcriptional regulators of AK6/hCINAP, which provides important insights into its activity and functions, and contributes to the screening of specific inhibitors of AK6/hCINAP and its application in clinical therapy in the future.

Abstract:The canonical functions of aminoacyl-tRNA synthetases (aaRS) are charging amino acids to their cognate tRNAs to ensure the precise protein synthesis. Over the course of evolution, aaRS progressively incorporated domains and motifs that have no essential connections to tRNA charging but play roles in cell signaling. These include mediating protein-protein interaction, protein subcellular localization and sensing and transmitting metabolites signals. Deregulated noncanonical aaRS functions are associated with array of human diseases. These all suggest that aaRS play roles beyond their tRNA charging activity, however, the underlying biochemical mechanisms remain to be elucidated. Recent studies revealed that aaRS have aminoacyl transferase activities. An amino acid can be specifically recognized and activated into aminoacyl-AMP by its cognate aaRS, and the formed aminoacyl-AMP in aaRS can modify lysines in proteins that physically interact with this aaRS. This aminoacylation senses and transmits amino acids abundance and side chain information into cell signaling network, provides opportunities to understand why additional domains are acquired by aaRS during evolution and how mutations in an aaRS causes specific human diseases. This review summarizes the noncanonical functions of aaRS and discusses how aaRS mutations may be linked to diseases.

Abstract:Aging is a major risk factor for neurodegenerative diseases such as Alzheimer?s disease (AD). Oxidative stress and free radicals have important biological functions. However, redox imbalance results in oxidative stress that has been implicated in the pathology of many human diseases including AD. The author reviews the involvement of reactive oxygen species (ROS) in the pathogenesis of neurodegenerative diseases, particularly, the interaction of oxidative stress with other critical mechanisms of AD, especially summarized the results about protect effects of tea polyphenols, L-theanine, astaxanthin, EGb761,soy isoflavones and nicotine on AD in cell, animal models and clinical treatments. Hopefully, this review can provide insights into novel preventive and therapeutic strategies for AD.

Abstract:Polyglutamine (polyQ) diseases are a kind of neurodegenerative disorders caused by unstable repeat expansion of CAG trinucleotide in the specific gene sequences. Nine types of polyQ diseases have been discovered, and most of the pathogenic proteins play an important role in transcriptional regulation in disease pathology. The abnormal repeat expansion of glutamine in polyQ protein will cause protein misfolding and thereby form aggregates accumulated in cells. The protein aggregates can sequester transcription factors, ubiquitin (Ub) adapters or receptors, molecular chaperones and other cellular factors into aggregates or inclusions through specific interactions mediated by their own domains, RNAs or Ub conjugates. Decrease of the soluble fractions and available amounts of these essential factors will impair the cellular function of transcriptional regulation and cause pathogenic transcriptional disorders. Therefore, studying polyQ-expanded protein aggregation that may sequester cellular transcription factors and other components will be beneficial to elucidating the pathogenesis of polyQ diseases at the molecular level, and provide potential therapeutic strategy for clinical application.

Abstract:Antibody drug conjugate (ADC) is typically composed of a monoclonal antibody conjugated with a cytotoxic small molecule drug via a linker. It is an emerging and promising class of targeted cancer therapeutics, combining both the highly cytotoxic activity of chemical drugs and highly targeting ability and specificity of monoclonal antibody. Fourteen ADCs have been approved for marketing so far worldwide, and more than 140 ADC drug candidates have been investigated in clinical studies. Various ADC technologies have been well developed to manufacture these ADC drugs in commercial scale as well as clinical scale. In this review, we describe the molecular structure, mechanisms of action and development history of ADCs. We then provide an overview of the current landscape and recent advances in each key element of ADCs, including antibody, linker, payload and conjugation, and their advantages and disadvantages. Future directions in ADC development may encompass smaller sized forms of antibodies such as antibody fragments and nanobodies to improve the penetration and accumulation of ADCs in the solid tumors. Novel linkers are also being tested to enhance the stability in circulation systems and reduce off-target toxicities. Emerging payloads of new functional mechanisms are also explored in the construction of ADCs to overcome the drug resistance resulted from currently used payloads of marketed ADCs. Various site-specific conjugation technologies have been adopted to reduce the heterogeneity of drug-load species and optimize the pharmacokinetic properties of ADCs. This review article aims to enhance systemic understanding and careful considerations in designing an ADC drug with improved efficacy and safety.

Abstract:The structure of protein is determined by the sequence, and the function of protein is determined by its structure. The advent of accurate protein structure prediction tools has created new opportunities and challenges in the fields of structural biology, structural bioinformatics, drug discovery and many other fields of life sciences. The accuracy of single-chain protein structure prediction has reached a level comparable to that of experimental methods. In this review, we provide an overview of the theoretical basis, development history, and recent advances in the field of protein structure prediction. Additionally, we discuss how the large number of predicted protein structures and artificial intelligence-based methods affect experimental structural biology. Open questions and future research directions in the field of protein structure prediction are analyzed.

Abstract:Objective Prion diseases are infectious, lethal neurodegenerative disorders principally caused by the conformational conversion of prion protein (PrP) from its cellular form (PrP^C) into a protease-resistant, aggregated form (PrP^Sc) in humans and various vertebrate species. We have recently reported a cryo-EM structure of an amyloid fibril formed by full-length human PrP, which features a parallel in-register intermolecular β sheet architecture. However, it is unclear whether amyloid fibrils from full-length human PrP^C are cytotoxic and transmissible.Methods Sarkosyl-insoluble Western blotting and cell viability assays were used to detect PrP aggregation and cell viability, respectively. Oxidative stress detection and annexin V-FITC apoptosis detection assays were also used for the determination of ROS production and cell apoptosis, respectively.Results Human PrP fibrils are cytotoxic, and transmissible to induce the misfolding of endogenous PrP^C not only in cells but also in the frontal cortices of infant mice. The PrP fibrils also induce severe mitochondrial damage in cells stably expressing PrP^C. Importantly, the PrP fibrils elevate ROS production via aggravating mitochondrial stress resulting from PrP aggregation and induce severe late apoptosis in cells stably expressing PrP^C.Conclusion We demonstrate that PrP fibrils prepared in vitro are cytotoxicity and have pathogenic potential.

Abstract:Objective To study the trained immunity effect of Drosophila melanogaster at the individual and molecular level, and provide a basis for the follow-up in-depth study of the molecular mechanism of trained immunity using genetic tools available to Drosophila.Methods Firstly, a germ-free Drosophila culture model was constructed. Subsequently, the Drosophila adult and cross-developmental trained immunity models were constructed. Two Gram-negative bacteria, Erwinia carotovora carotovora 15 and Pseudomonas aeruginosa, were respectively used to infect Drosophila orally. With a repeated infection elicited after the first infection completely subsided, the effects of potential trained immunity is demonstrated by comparing the survival rate and bacterial load of Drosophila melanogaster during the two infection phases. The induction of immune deficiency (IMD) pathway by Gram-negative bacteria as the expression level of corresponding innate immunity-related genes was detected by real-time quantitative PCR.Results The primary infection of during either adults or larva developmental stage can significantly improve the survival rate of secondary challenge. A higher bacterial clearance efficiency and maximum bacterial load of death is consistently observed after a second infection. The basal expression of immune response genes in IMD signaling pathway is boosted prior to secondary infection than naive animal, explaining the molecular basis of gained infection resistance. Midgut is examined to be primary anatomic site of immune response, and the effects of secondary immunization were faster and more intense than those of primary infection. The numbers of intestinal stem cells in the midgut were significantly higher during the second infection compared with the first one.Conclusion A robust trained immunity in Drosophila melanogaster intestine can be triggered by oral infection of either homologous or heterologous Gram-negative bacteria, and the immunological memory can persist across developmental stages. It may act on chromatin and store immunologic memory at relevant gene loci through chromatin modifications. A potential way for the passage of immunologic memory across developmental stages is through JNK/STAT activation of intestinal stem cells, which may carry on the immune imprint from larval to adult developmental stages in the gut.

Abstract:Evolution is the natural history where biological diversity is generated and retained. To expedite the evolution process in the lab to achieve specific functional optimization by artificial selection, targeted mutagenesis boosts are introduced into fast-proliferating prokaryotes and simple eukaryotes. If tolerated, the functional diversity a fast-evolving system generated offers unique opportunities to obtain artificially engineered bio-molecules best suited for bioengineering applications. In this perspective, we discuss the current state of art in vivo continuous evolution platforms, focusing on advances made in both phage and yeast artificial evolution. Their successful applications in biotechnology are presented, followed by a brief outlook of near-future developments in this burgeoning field.

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