海葵毒素多肽的分离和初步表征

经冻融法提取,丙酮沉淀,SP Sephadex C 25强离子交换和HPLC ODS C₁₈反相色谱层析,从海葵Actinia cari中提纯到两个新的毒素多肽,命名为RSAPⅠ和RSAPⅡ,经SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）和基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）鉴定RSAPⅠ和RSAPⅡ均为单一成分,相对分子质量分别为5 008和4 992.初步钠通道药理学实验表明,RSAPⅠ和RSAPⅡ对豚鼠心室肌细胞钠通道表现出活性,RSAPⅠ使钠离子内流增加,起开放作用;RSAPⅡ使钠离子内流减少,起阻滞作用.二者的圆二色谱均表现为100% β转角.     

转基因植物中Bt杀虫蛋白的重组噬菌体辅助检测

以LRP和棉花总蛋白为本底蛋白,纯化的Bt杀虫蛋白为目标蛋白,利用噬菌体展示技术从噬菌体的随机七肽库中筛选与Bt蛋白特异性结合的七肽.ELISA检测表明经过三轮淘筛过程,特异性多肽得到了高度富集,其中PH5可与Bt蛋白特异性结合.将筛选出的PH5作为Bt蛋白的“类抗体”用于抗虫转基因植物的检测,由此建立了一种新的检测方法,讨论了噬菌体展示技术在植物基因工程中的潜在应用价值.     

含霍乱肠毒素B亚单位基因植物表达载体的构建

构建含CTB基因的植物双元表达载体,采用高保真PCR方法调出CTB基因,经测序证实核酸序列正确后,再亚克隆到含植物表达调控原件的载体上,采用冻融法和电击法,将含CTB的植物表达载体转入根癌农杆菌中,通过一系列分子克隆的方法获得含CTB基因的植物双元表达载体pBI-CTB和pBI-CTBK,并经酶切证实。     

Phage display screening identifies a prostate specific antigen (PSA)–/lo prostate cancer cell specific peptide to retard castration resistance of prostate cancer

Patients with prostate cancer (PCa) will eventually progress to castrate-resistant prostate cancer (CRPC) after androgen deprivation therapy (ADT) treatment. Prostate-specific antigen (PSA)^−/lo cells which harbor self-renewing long-term tumor-propagating cells that can be enriched using ALDH+CD44+α2β1+ and can initiate tumor development may represent a critical source of CRPC cells. Our purpose was to find a peptide that specifically targets PSA^−/lo PCa cells to retard the development of CRPC. PSA⁺ and PSA^−/lo cells were successfully separated from LNCaP xenograft tumors after prostate- PSAP-GFP vector infection and FACS. A variety of PSA^−/lo cells specifically targeting peptide (named as “TAP1” targeted affinity peptide 1) was identified by using phage display library screening. The highest binding rate in TAP1 binding cell subpopulations are identified to be among ALDH⁺CD44⁺CXCR4⁺CD24⁺ cells. TAP1 significantly inhibited PCa growth both in vitro and in vivo. TAP1 significantly improved the anti-proliferation effect of the anti-androgens (Charcoal dextran-stripped serum (CDSS)+Bicalutamide, Enzalutamide) and chemotherapeutic agents (Abiraterone, Docetaxel, Etoposide) in vitro. TAP1 treatment shortens the length of telomeres in ALDH⁺CD44⁺CXCR4⁺CD24⁺ cells and significantly reduces the expression of Homeobox B9 (HOXB9) and TGF-β2. In conclusion, PSA^−/lo PCa cell-specific targeting peptide (TAP1) that suppressed PCa cell growth both in vitro and in vivo and improved the drug sensitivities of anti-androgens and chemotherapeutic agents at least through shortening the length of telomere and reducing the expression of HOXB9 and TGF-β2. Therapeutic peptides that specifically target prostate cancer stem cell might be a very valuable and promising approach to overcome chemoresistance and prevent recurrence in patients with PCa.     

Modulation of DNA and RNA by PNA

Peptide nucleic acid (PNA), a synthetic DNA mimic that is devoid of the (deoxy)ribose-phosphate backbone yet still perfectly retains the ability to recognize natural nucleic acids in a sequence-specific fashion, can be employed as a tool to modulate gene expressions via several different mechanisms. The unique strength of PNA compared to other oligonucleotide analogs is its ability to bind to nucleic acid targets with secondary structures such as double-stranded and quadruplex DNA as well as RNA. This digest aims to introduce general readers to the advancement in the area of modulation of DNA/RNA functions by PNA, its current status and future research opportunities, with emphasis on recent progress in new targeting modes of structured DNA/RNA by PNA and PNA-mediated gene editing.     

Crystal structure of a Vip3B family insecticidal protein reveals a new fold and a unique tetrameric assembly

Vegetatively expressed insecticidal proteins (VIPs) produced by Bacillus thuringiensis fall into several classes of which the third, VIP3, is known for their activity against several key Lepidopteran pests of commercial broad acre crops and because their mode of action does not overlap with that of crystalline insecticidal proteins. The details of the VIP3 structure and mode of action have remained obscure for the quarter century that has passed since their discovery. In the present article, we report the first crystal structure of a full‐length VIP3 protein. Crystallization of this target required multiple rounds of construct optimization and screening—over 200 individual sequences were expressed and tested. This protein adopts a novel global fold that combines domains with hitherto unreported topology and containing elements seemingly borrowed from carbohydrate‐binding domains, lectins, or from other insecticidal proteins.     

A chemically stable peptide agonist to neuromedin U receptor type 2

Neuromedin U (NMU) is a peptide with appetite suppressive activity and other physiological activities via activation of the NMU receptors NMUR1 and NMUR2. In 2014, we reported the first NMUR2 selective agonist, 3-cyclohexylpropionyl-Leu-Leu-Dap-Pro-Arg-Asn-NH₂ (CPN-116). However, we found that CPN-116 in phosphate buffer is unstable because of Nα-to-N^β acyl migration at the Dap residue. In this study, the chemical stability of CPN-116 was evaluated under various conditions, and it was found to be relatively stable in buffers such as HEPES and MES. We also performed a structure-activity relationship study to obtain an NMUR2-selective agonist with improved chemical stability. Consequently, CPN-219 bearing a Dab residue in place of Dap emerged as a next-generation hexapeptidic NMUR2 agonist.     

Cytotoxic T-lymphocyte elicited therapeutic vaccine candidate targeting cancer against MAGE-A11 carcinogenic protein

Immunotherapy is a breakthrough approach for cancer treatment and prevention. By exploiting the fact that cancer cells have overexpression of tumor antigens responsible for its growth and progression, which can be identified and removed by boosting the immune system. In silico techniques have provided efficient ways for developing preventive measures to ward off cancer. Herein, we have designed a potent cytotoxic T-lymphocyte epitope to elicit a desirable immune response against carcinogenic melanoma-associated antigen-A11. Potent epitope was predicted using reliable algorithms and characterized by advanced computational avenue CABS molecular dynamics simulation, for full flexible binding with HLA-A*0201 and androgen receptor to large-scale rearrangements of the complex system. Results showed the potent immunogenic construct (KIIDLVHLL), from top epitopes using five algorithms. Molecular docking analyses showed the strong binding of epitope with HLA-A*0201 and androgen receptor with docking score of −780.6 and −641.06 kcal/mol, respectively. Molecular dynamics simulation analysis revealed strong binding of lead epitope with androgen receptor by involvement of 127 elements through atomic-model study. Full flexibility study showed stable binding of epitope with an average root mean square deviation (RMSD) 2.21 Å and maximum RMSD value of 6.48 Å in optimal cluster density area. The epitope also showed remarkable results with radius of gyration 23.0777 Å, world population coverage of 39.08% by immune epitope database, and transporter associated with antigen processing (TAP) affinity IC₅₀ value of 2039.65 nm. Moreover, in silico cloning approach confirmed the expression and translation capacity of the construct within a suitable expression vector. The present study paves way for a potential immunogenic construct for prevention of cancer.     

Fluorescent probes for investigating peptidoglycan biosynthesis in mycobacteria

Tuberculosis killed 1.5 million people in 2018. Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is the most deadly infectious bacteria in the world. A strength of mycobacterial pathogens — their formidable cell wall — could also be one of their greatest molecular vulnerabilities. As in other bacteria, peptidoglycan (PG) maintenance and integrity is essential to mycobacterial survival. But Mtb PG is unique, and a better understanding of its biosynthetic machinery could lead to new drugs or more effective treatment regimens. Such investigations are being accelerated by the application of fluorescent probes, including those based on vancomycin, β-lactams, PG stem mimics, d-amino acids, and reactive glycans. This review will describe how fluorescent probes are being used to uncover new information on the regulation and drug susceptibility of two classes of enzymes that fortify the Mtb PG: the penicillin-binding proteins and the L,D-transpeptidases.     

Revealing the functional roles of tyrosine sulfation using synthetic sulfopeptides and sulfoproteins

The decoration of proteins with post-translational modifications (PTMs) serves as a mechanism to expand the functional repertoire of the proteome. Tyrosine sulfation is a PTM that has been shown to be a key regulator of extracellular protein–protein interactions in a select number of examples. However, the challenges associated with identifying and characterising the functional consequences of tyrosine sulfation have hindered our ability to understand the full scope of its role in the wider proteome when compared with that of other PTMs, for example, phosphorylation and glycosylation. In this account, we highlight recent advances in the prediction and detection of tyrosine sulfation and outline the need for continued innovation in this area. We also discuss the utility of emerging solid-phase synthesis and peptide ligation strategies for accessing libraries of homogeneously sulfated peptides and proteins to help reveal functional aspects of the sulfoproteome.