Advances of structure and mechanisms of bromodomain-containing protein 4 and its related research in tumors / 生物工程学报

The bromodomain and extraterminal domain (Bet) family are the regulators of the epigenome and also the pivotal driving factors for the expression of tumor related genes that tumor cells depend on for survival and proliferation. Bromodomain-containing protein 4 (Brd4) is a member of the Bet protein family. Generally, Brd4 identifies acetylated histones and binds to the promoter or enhancer region of target genes to initiate and maintain expression of tumor related genes. Brd4 is closely related to the regulation of multiple transcription factors and chromatin modification and is involved in DNA damage repair and maintenance of telomere function, thus maintaining the survival of tumor cells. This review summarizes the structure and function of Brd4 protein and the application of its inhibitors in tumor research.

Substitutability of metal-binding sites in an alcohol dehydrogenase / 生物工程学报

Covalently anchoring of a ligand/metal via polar amino acid side chain(s) is often observed in metalloenzyme, while the substitutability of metal-binding sites remains elusive. In this study, we utilized a zinc-dependent alcohol dehydrogenase from Thermoanaerobacter brockii (TbSADH) as a model enzyme, analyzed the sequence conservation of the three residues Cys37, His59, and Asp150 that bind the zinc ion, and constructed the mutant library. After experimental validation, three out of 224 clones, which showed comparative conversion and ee values as the wild-type enzyme in the asymmetric reduction of the model substrate tetrahydrofuran-3-one, were screened out. The results reveal that the metal-binding sites in TbSADH are substitutable without tradeoff in activity and stereoselectivity, which lay a foundation for designing ADH-catalyzed new reactions via metal ion replacement.

Research Advances in the Mechanisms of BRD4 and Its Inhibitors in Hematologic Malignancies--Review / 中国实验血液学杂志

Bromodomain-containing protein 4 (BRD4) is one of the most important members in the bromodomain and extra terminal domain(BET) family, it plays an important role in cellular physiology in human body, such as cell cycles, cell proliferation, and immune response. Recent studies have shown that BRD4 is associated with occurrence and development of acute myeloblastic leukemia, multiple myeloma and lymphoma. The mechanisms of BRD4 in hematologic malignancies including the regulation of c-Myc expression, and participation of the composition of super-enhancer, etc. At present, many kinds of inhibitors have been developed to target inhibit BRD4 for therapy in hematologic malignancies, and some of BRD4 inhibitors have entered phase Ⅱ clinical trials, which suggested that BRD4 inhibitors are expected to become new therapeutic agents for hematologic malignancies. In this review, the research advance of BRD4 and BRD4 inhibitors in hematologic malignancies was summarized briefly.

A study on KIF1A gene missense variant analysis and its protein expression and structure profiles of an autism spectrum disorder family trio / 中华医学遗传学杂志

OBJECTIVE@#To analyze the pathogenic variants of the KIF1A gene and its corresponding protein structure in an autism spectrum disorder (ASD) family trio carrying harmful missense variants in the KIF1A gene.@*METHODS@#The peripheral blood DNA of the patient and his parents was extracted and sequenced using whole exome sequencing (WES) technology and verified by Sanger sequencing. Bioinformatics software SIFT, PolyPhen-2, Mutation Taster, and CADD software were used to analyze the harmfulness and conservation of variants. The Human Brain Transcriptome (HBT) database was used to analyze the expression of the KIF1A gene in the brain. PredictProtein and SWISS-MODEL were further used to predict the secondary structure and tertiary structure of KIF1A wild-type protein and variant protein. PyMOL V2.4 was utilized to investigate the change of hydrogen bond connection after protein variant.@*RESULTS@#The WES sequencing revealed a missense variant c.664A>C (p.Asn222His) in the child's KIF1A gene, and this variant was a de novo variant. The harmfulness prediction results suggest that this variant is harmful. By analyzing expression level of KIF1A gene in the brain. It is found that KIF1A gene widely expressed in various brain regions during embryonic development. By analyzing the variant protein structure, the missense variant of KIF1A will cause many changes in the secondary structure of protein, such as alpha-helix, beta-strand, and protein binding domain. The connection of hydrogen bond and spatial structure will also change, thereby changing the original biological function.@*CONCLUSION@#The KIF1A gene may be a risk gene for ASD.

ADP-ribosylhydrolases: from DNA damage repair to COVID-19 / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Adenosine diphosphate (ADP)-ribosylation is a unique post-translational modification that regulates many biological processes, such as DNA damage repair. During DNA repair, ADP-ribosylation needs to be reversed by ADP-ribosylhydrolases. A group of ADP-ribosylhydrolases have a catalytic domain, namely the macrodomain, which is conserved in evolution from prokaryotes to humans. Not all macrodomains remove ADP-ribosylation. One set of macrodomains loses enzymatic activity and only binds to ADP-ribose (ADPR). Here, we summarize the biological functions of these macrodomains in DNA damage repair and compare the structure of enzymatically active and inactive macrodomains. Moreover, small molecular inhibitors have been developed that target macrodomains to suppress DNA damage repair and tumor growth. Macrodomain proteins are also expressed in pathogens, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, these domains may not be directly involved in DNA damage repair in the hosts or pathogens. Instead, they play key roles in pathogen replication. Thus, by targeting macrodomains it may be possible to treat pathogen-induced diseases, such as coronavirus disease 2019 (COVID-19).

Construction, expression and purification of a mammalian secretory recombinant fusion protein rPC / 生物工程学报

Drugs targeting immune checkpoint are used for cancer treatment, but resistance to single drug may occur. Combination therapy blocking multiple checkpoints simultaneously can improve clinical outcome. Therefore, we designed a recombinant protein rPC to block multiple targets, which consists of extracellular domains of programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). The coding sequence was inserted into expression vector and stably transfected into HEK293 cells. The culture supernatant was collected and rPC was affinity-purified. Real-time quantitative PCR was used to evaluate the expression levels of ligands for PD-1 and CTLA-4 in several human cancer cell lines. The binding of rPC with cancer cells was examined by immunofluorescence cell staining, the influence of rPC on cancer cell growth was assayed by CCK-8. The results showed that rPC could be expressed and secreted by stably transfected HEK293 cells, the purified rPC could bind to lung cancer NCI-H226 cells which have high levels of ligands for PD-1 and CTLA-4, no direct impact on cancer cell growth could be observed by rPC treatment. The recombinant protein rPC can be functionally assayed further for developing novel immunotherapeutic drugs for cancer.

Progress in shark single-domain antibody / 生物工程学报

Monoclonal antibody (mAb) is an important biological macromolecule and widely used in immune detection, in vitro diagnostics, and drug discovery. However, the inherent properties of mAb restrict its further development, such as high molecular weight and complex structure. Therefore, there is an urgent need to develop alternatives for mAb. Various types of miniaturized antibodies have been developed, among which the variable domain of immunoglobulin new antigen receptor (VNAR) is very attractive. The shark single-domain antibody, also known as shark VNAR, is an antigen-binding domain obtained by genetic engineering technology based on the immunoglobulin new antigen receptor (IgNAR) that naturally exists in selachimorpha. It has a molecular weight of 12 kDa, which is the smallest antigen-binding domain found in the known vertebrates at present. Compared with mAb, the shark VNAR exhibits various superiorities, such as low molecular weight, high affinity, tolerance to the harsh environment, good water solubility, strong tissue penetration, and recognition of the hidden epitopes. It has attracted wide attention in the fields of immunochemical reagents and drug discovery. In this review, various aspects of shark VNAR are elaborated, including the structural and functional characteristics, generating and humanization techniques, affinity maturation strategies, application fields, advantages and disadvantages, and prospects.

The 40-91 aa sequence of porcine epidemic diarrhea virus ORF3 protein is the key structural domain controlling its location in cytoplasm / 生物工程学报

ORF3 protein, the single accessory protein encoded by porcine epidemic diarrhea virus (PEDV), is related to viral pathogenicity. In order to determine the cytoplasmic location signal of PEDV ORF3, we constructed a series of recombinant plasmids carrying full-length or truncated segments of PEDV DR13 ORF3 protein. When the acquired plasmids were transfected into Vero cells, expression and distribution of the EGFP-fused full-length ORF3 protein and its truncated forms in the cells were observed by laser confocal microscopy. The results showed that ORF3 protein or their truncated forms containing 40-91 aa segment including two transmembrane domains were localized in the cytoplasm, whereas ORF3 truncated peptides without the 40-91 aa segment were distributed in the whole cell (in both cytoplasm and nucleus). This suggests that the 40-91 aa is the key structural domain determining cytoplasmic location of PEDV ORF3 protein. The discovery provides reference for further clarifying intracellular transport and biological function of PEDV ORF3 protein.

An overview of COVID-19 / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection emerged in Wuhan City, Hubei Province, China in December 2019. By Feb. 11, 2020, the World Health Organization (WHO) officially named the disease resulting from infection with SARS-CoV-2 as coronavirus disease 2019 (COVID-19). COVID-19 represents a spectrum of clinical manifestations that typically include fever, dry cough, and fatigue, often with pulmonary involvement. SARS-CoV-2 is highly contagious and most individuals within the population at large are susceptible to infection. Wild animal hosts and infected patients are currently the main sources of disease which is transmitted via respiratory droplets and direct contact. Since the outbreak, the Chinese government and scientific community have acted rapidly to identify the causative agent and promptly shared the viral gene sequence, and have carried out measures to contain the epidemic. Meanwhile, recent research has revealed critical aspects of SARS-CoV-2 biology and disease pathogenesis; other studies have focused on epidemiology, clinical features, diagnosis, management, as well as drug and vaccine development. This review aims to summarize the latest research findings and to provide expert consensus. We will also share ongoing efforts and experience in China, which may provide insight on how to contain the epidemic and improve our understanding of this emerging infectious disease, together with updated guidance for prevention, control, and critical management of this pandemic.

Extraction and purification of NUDT9 homology domain of human transient receptor potential melastatin 2 channel / 浙江大学学报·医学版

OBJECTIVE@#To develop methods of extraction and purification of Cterminal NUDT9 homology domain of human transient receptor potential melastatin 2 (TRPM2) channel.@*METHODS@#After sonication and centrifuge of strain Rosetta (DE3) which was induced by isopropylthio-β-D-galactoside, GST-NUDT9-H was collected after the binding of supernatant with GST beads and eluted with reduced glutathione. Then the elution buffer containing fusion protein was purified by size exclusion chromatography after concentration and centrifuge. Finally, with the cleavage of thrombin and binding with the GST beads, NUDT9-H with high purity in supernatant was collected.@*RESULTS@#The GST-NUDT9-H fusion protein was stabilized with lysis buffer containing 0.5% n-dodecyl -β-d-maltoside (DDM), and wash buffer containing 0.025% DDM in size-exclusion chromatography system, and finally the NUDT9-H with high purity was obtained after cleaved by thrombin (1 U/2 mg fusion protein) for 24 h.@*CONCLUSIONS@#Due to the poor stability of NUDT9-H, it is necessary to add DDM in extraction and purification buffer to stabilize the conformation of NUDT9-H, so as to increase its yields and purity.

Nucleus translocation of membrane/cytoplasm proteins in tumor cells / 浙江大学学报·医学版

Proteins are the physical basis of life and perform all kinds of life activities. Proteins have different orientations and function in different tissues. The same protein, located in different subcellular regions, can perform different and even opposite functions. Both functional and structural proteins are capable of undergoing re-localization which can directly or indirectly participate in signal transduction. Due to abnormal transduction of signals during carcinogenesis, the proteins originally expressed in the cytoplasm are translocated into the nucleus and lead to functional changes in the tumor tissue. The changes of protein localization are affected by many factors, including the interaction between proteins, expression level of proteins and the cleaved intracellular domain of transmembrane protein.

Construction of a mutant of Actinoplanes sp. N902-109 that produces a new rapamycin analog / 中国天然药物

In the present study, we introduced point mutations into Ac_rapA which encodes a polyketide synthase responsible for rapamycin biosynthesis in Actinoplanes sp. N902-109, in order to construct a mutant with an inactivated enoylreductase (ER) domain, which was able to synthesize a new rapamycin analog. Based on the homologous recombination induced by double-strand breaks in chromosome mediated by endonuclease I-SceI, the site-directed mutation in the first ER domain of Ac_rapA was introduced using non-replicating plasmid pLYERIA combined with an I-SceI expression plasmid. Three amino acid residues of the active center, Ala-Gly-Gly, were converted to Ala-Ser-Pro. The broth of the mutant strain SIPI-027 was analyzed by HPLC and a new peak with the similar UV spectrum to that of rapamycin was found. The sample of the new peak was prepared by solvent extraction, column chromatography, and crystallization methods. The structure of new compound, named as SIPI-rapxin, was elucidated by determining and analyzing its MS and NMR spectra and its biological activity was assessed using mixed lymphocyte reaction (MLR). An ER domain-deficient mutant of Actinoplanes sp. N902-109, named as SIPI-027, was constructed, which produced a novel rapamycin analog SIPI-rapxin and its structure was elucidated to be 35, 36-didehydro-27-O-demethylrapamycin. The biological activity of SIPI-rapxin was better than that of rapamycin. In conclusion, inactivation of the first ER domain of rapA, one of the modular polyketide synthase responsible for macro-lactone synthesis of rapamycin, gave rise to a mutant capable of producing a novel rapamycin analog, 35, 36-didehydro-27-O-demethylrapamycin, demonstrating that the enoylreductase domain was responsible for the reduction of the double bond between C-35 and C-36 during rapamycin synthesis.

Construction of a mutant of Actinoplanes sp. N902-109 that produces a new rapamycin analog / 中国天然药物

In the present study, we introduced point mutations into Ac_rapA which encodes a polyketide synthase responsible for rapamycin biosynthesis in Actinoplanes sp. N902-109, in order to construct a mutant with an inactivated enoylreductase (ER) domain, which was able to synthesize a new rapamycin analog. Based on the homologous recombination induced by double-strand breaks in chromosome mediated by endonuclease I-SceI, the site-directed mutation in the first ER domain of Ac_rapA was introduced using non-replicating plasmid pLYERIA combined with an I-SceI expression plasmid. Three amino acid residues of the active center, Ala-Gly-Gly, were converted to Ala-Ser-Pro. The broth of the mutant strain SIPI-027 was analyzed by HPLC and a new peak with the similar UV spectrum to that of rapamycin was found. The sample of the new peak was prepared by solvent extraction, column chromatography, and crystallization methods. The structure of new compound, named as SIPI-rapxin, was elucidated by determining and analyzing its MS and NMR spectra and its biological activity was assessed using mixed lymphocyte reaction (MLR). An ER domain-deficient mutant of Actinoplanes sp. N902-109, named as SIPI-027, was constructed, which produced a novel rapamycin analog SIPI-rapxin and its structure was elucidated to be 35, 36-didehydro-27-O-demethylrapamycin. The biological activity of SIPI-rapxin was better than that of rapamycin. In conclusion, inactivation of the first ER domain of rapA, one of the modular polyketide synthase responsible for macro-lactone synthesis of rapamycin, gave rise to a mutant capable of producing a novel rapamycin analog, 35, 36-didehydro-27-O-demethylrapamycin, demonstrating that the enoylreductase domain was responsible for the reduction of the double bond between C-35 and C-36 during rapamycin synthesis.

YTH Domain: A Family of N-methyladenosine (mA) Readers / 基因组蛋白质组与生物信息学报·英文版

Like protein and DNA, different types of RNA molecules undergo various modifications. Accumulating evidence suggests that these RNA modifications serve as sophisticated codes to mediate RNA behaviors and many important biological functions. N-methyladenosine (mA) is the most abundant internal RNA modification found in a variety of eukaryotic RNAs, including but not limited to mRNAs, tRNAs, rRNAs, and long non-coding RNAs (lncRNAs). In mammalian cells, mA can be incorporated by a methyltransferase complex and removed by demethylases, which ensures that the mA modification is reversible and dynamic. Moreover, mA is recognized by the YT521-B homology (YTH) domain-containing proteins, which subsequently direct different complexes to regulate RNA signaling pathways, such as RNA metabolism, RNA splicing, RNA folding, and protein translation. Herein, we summarize the recent progresses made in understanding the molecular mechanisms underlying the mA recognition by YTH domain-containing proteins, which would shed new light on mA-specific recognition and provide clues to the future identification of reader proteins of many other RNA modifications.

Transmembrane domain dependent inhibitory function of FcγRIIB

FcγRIIB, the only inhibitory IgG Fc receptor, functions to suppress the hyper-activation of immune cells. Numerous studies have illustrated its inhibitory function through the ITIM motif in the cytoplasmic tail of FcγRIIB. However, later studies revealed that in addition to the ITIM, the transmembrane (TM) domain of FcγRIIB is also indispensable for its inhibitory function. Indeed, recent epidemiological studies revealed that a non-synonymous single nucleotide polymorphism (rs1050501) within the TM domain of FcγRIIB, responsible for the I232T substitution, is associated with the susceptibility to systemic lupus erythematosus (SLE). In this review, we will summarize these epidemiological and functional studies of FcγRIIB-I232T in the past few years, and will further discuss the mechanisms accounting for the functional loss of FcγRIIB-I232T. Our review will help the reader gain a deeper understanding of the importance of the TM domain in mediating the inhibitory function of FcγRIIB and may provide insights to a new therapeutic target for the associated diseases.

Carboxylesterases in lipid metabolism: from mouse to human

Mammalian carboxylesterases hydrolyze a wide range of xenobiotic and endogenous compounds, including lipid esters. Physiological functions of carboxylesterases in lipid metabolism and energy homeostasis in vivo have been demonstrated by genetic manipulations and chemical inhibition in mice, and in vitro through (over)expression, knockdown of expression, and chemical inhibition in a variety of cells. Recent research advances have revealed the relevance of carboxylesterases to metabolic diseases such as obesity and fatty liver disease, suggesting these enzymes might be potential targets for treatment of metabolic disorders. In order to translate pre-clinical studies in cellular and mouse models to humans, differences and similarities of carboxylesterases between mice and human need to be elucidated. This review presents and discusses the research progress in structure and function of mouse and human carboxylesterases, and the role of these enzymes in lipid metabolism and metabolic disorders.

Functional characterization of human equilibrative nucleoside transporter 1

Equilibrative nucleoside transporters (ENTs), which facilitate cross-membrane transport of nucleosides and nucleoside-derived drugs, play an important role in the salvage pathways of nucleotide synthesis, cancer chemotherapy, and treatment for virus infections. Functional characterization of ENTs at the molecular level remains technically challenging and hence scant. In this study, we report successful purification and biochemical characterization of human equilibrative nucleoside transporter 1 (hENT1) in vitro. The HEK293F-derived, recombinant hENT1 is homogenous and functionally active in proteoliposome-based counter flow assays. hENT1 transports the substrate adenosine with a K of 215 ± 34 µmol/L and a V of 578 ± 23.4 nmol mg min. Adenosine uptake by hENT1 is competitively inhibited by nitrobenzylmercaptopurine ribonucleoside (NBMPR), nucleosides, deoxynucleosides, and nucleoside-derived anti-cancer and anti-viral drugs. Binding of hENT1 to adenosine, deoxyadenosine, and adenine by isothermal titration calorimetry is in general agreement with results of the competitive inhibition assays. These results validate hENT1 as a bona fide target for potential drug target and serve as a useful basis for future biophysical and structural studies.

Structure-based assessment of disease-related mutations in human voltage-gated sodium channels

Voltage-gated sodium (Na) channels are essential for the rapid upstroke of action potentials and the propagation of electrical signals in nerves and muscles. Defects of Na channels are associated with a variety of channelopathies. More than 1000 disease-related mutations have been identified in Na channels, with Na1.1 and Na1.5 each harboring more than 400 mutations. Na channels represent major targets for a wide array of neurotoxins and drugs. Atomic structures of Na channels are required to understand their function and disease mechanisms. The recently determined atomic structure of the rabbit voltage-gated calcium (Ca) channel Ca1.1 provides a template for homology-based structural modeling of the evolutionarily related Na channels. In this Resource article, we summarized all the reported disease-related mutations in human Na channels, generated a homologous model of human Na1.7, and structurally mapped disease-associated mutations. Before the determination of structures of human Na channels, the analysis presented here serves as the base framework for mechanistic investigation of Na channelopathies and for potential structure-based drug discovery.

Analysis of phenotypes and genetic mutations in two pedigrees affected with hereditary protein C deficiency / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the pathogenesis of protein C deficiency in two pedigrees through mutation detection and model analysis.</p><p><b>METHODS</b>Chromogenic substrate method and enzyme linked immunosorbent assay (ELISA) were used to determine the plasma protein C activity (PC: A) and protein C antigen (PC: Ag) in the two probands and their family members. All of the 9 exons and intron-exon boundaries of the PROC gene were amplified by PCR and analyzed with Sanger sequencing after purification. Corresponding mutate sites of the family members were also amplified and sequenced. The PolyPhen-2 software was used to analyze the perniciousness of the mutations and Clustal X was to analyze the conservatism. The protein model and amino acids interaction of the mutations were analyzed by Swiss-PdbViewer software.</p><p><b>RESULTS</b>The PC: A and PC: Ag of proband 1 was 30% and 35%, while PC:A of his father, mother and aunt were all slightly under the reference range. Two heterozygous missense mutations were found in exons 7 and 5 of the PROC gene, namely c.565 C>T (p.Arg147Trp) and c.383 G>A (p.Gly86Asp). His father and aunt were carriers for c.565 C>T, while his mother had carried c.383 G>A. The PC: A of proband 2 and his son were 50% and 64%, respectively. And they were both positive for p.Arg147Trp. Analysis of PolyPhen-2 indicated that p.Arg147Trp was benign, while p.Gly86Asp was damaging. Clustal X analysis indicated that the p.Arg147Trp was non-conservative, while the p.Gly86Asp was highly conservative. Modeling for the mutant proteins revealed that the simple aromatic ring of Trp147 in p.Arg147Trp destroyed the two hydrogen bonds between Arg147-Lys146 and Arg147-Lys151, and steric hindranted with Arg178. The side chain of Asp86 extended and generated steric clash with Gln90 with the occurrence of p.Gly86Asp. The change of hydrogen bonds and steric effects has altered the spatial configuration of amino acids, which led to unstable mutate proteins and interfered with the secretion.</p><p><b>CONCLUSION</b>Both probands had hereditary protein C deficiencies, for which their parents were all carriers. The heterozygous mutations p.Arg147Trp and p.Gly86Asp were the main cause for PC: A activity decrease. Among these, p.Gly86Asp was discovered for the first time.</p>

Molecular cloning, characterization and expression analysis of woodchuck retinoic acid-inducible gene I / 华中科技大学学报（医学）（英德文版）

Cytosolic retinoic acid-inducible gene I (RIG-I) is an important innate immune RNA sensor and can induce antiviral cytokines, e.g., interferon-β (IFN-β). Innate immune response to hepatitis B virus (HBV) plays a pivotal role in viral clearance and persistence. However, knowledge of the role that RIG-I plays in HBV infection is limited. The woodchuck is a valuable model for studying HBV infection. To characterize the molecular basis of woodchuck RIG-I (wRIG-I), we analyzed the complete coding sequences (CDSs) of wRIG-I, containing 2778 base pairs that encode 925 amino acids. The deduced wRIG-I protein was 106.847 kD with a theoretical isoelectric point (pI) of 6.07, and contained three important functional structures [caspase activation and recruitment domains (CARDs), DExD/H-box helicases, and a repressor domain (RD)]. In woodchuck fibroblastoma cell line (WH12/6), wRIG-I-targeted small interfering RNA (siRNA) down-regulated RIG-I and its downstrean effector-IFN-β transcripts under RIG-I' ligand, 5'-ppp double stranded RNA (dsRNA) stimulation. We also measured mRNA levels of wRIG-I in different tissues from healthy woodchucks and in the livers from woodchuck hepatitis virus (WHV)-infected woodchucks. The basal expression levels of wRIG-I were abundant in the kidney and liver. Importantly, wRIG-I was significantly up-regulated in acutely infected woodchuck livers, suggesting that RIG-I might be involved in WHV infection. These results may characterize RIG-I in the woodchuck model, providing a strong basis for further study on RIG-I-mediated innate immunity in HBV infection.