The intraviral protein-protein interaction of SARS-CoV-2 reveals the key role of N protein in virus-like particle assembly

Intraviral protein-protein interactions (PPIs) of SARS-CoV-2 in host cells may provide useful information for deep understanding of virology of SARS-CoV-2. In this study, 22 of 55 interactions of the structural and accessory proteins of SARS-CoV-2 were identified by biomolecular fluorescence complementation (BiFC) assay. The nucleocapsid (N) protein was found to have the most interactions among the structural and accessory proteins of SARS-CoV-2, and also specifically interacted with the putative packaging signal (PS) of SARS-CoV-2. We also demonstrated that the PS core containing PS576 RNA bears a functional PS, important for the assembly of the viral RNA into virus like particles (VLPs), and the packaging of SARS-CoV-2 RNA was N dependent.     

A combination of ultrasonic-assisted extraction with RRLC-QQQ method for the determination of artemisinin in the Chinese herb Artemisia annua L

Introduction – Artemisinin, the primary active ingredient of the Chinese herb Artemisia annua L., is known to have considerable anti‐malaria properties. However, rapid, sensitive and selective method for the determination of artemisinin in it is not currently available. Objective – To develop and validate an efficient method for extraction and analysis of artemisinin from the plant samples of Artemisia annua L. by rapid resolution liquid chromatography triple quadrupole mass spectrometry (RRLC‐QQQ). Methodology – Following ultrasound‐assisted extraction (USE), RRLC‐QQQ was utilised to separate and determine artemisinin from the plant sample of Artemisia annua L. The LC separation, QQQ‐MS detection and multiple reaction monitoring (MRM) mode were optimised, and the method validation concluding selectivity, calibration, accuracy and precision, and recovery were also evaluated. Results – LC separation was performed with an isocratic elution of 20% of methanol–water (10 mmol/L ammonium acetate, pH 4.0) on a C₁₈ column. The triple quadrupole MS detection was carried out under MRM mode of precursor ion [M + H]⁺ → fragment ions m/z 265.1 and m/z 247.2. The limits of detection and quantitation of artemisinin were 0.20 and 0.75 ng/mL, respectively. The intra‐ and inter‐day precisions did not exceed 3.71%, and the deviation of the intra‐ and inter‐day mean values did not exceed ±7.50. The average recoveries for artemisinin ranged from 92.45 to 103.8% with an RSD from 2.47 to 2.79%. Conclusion – The developed RRLC‐QQQ assay is an efficient method for separation and determination of artemisinin from the plant samples of Artemisia annua L. Copyright © 2011 John Wiley & Sons, Ltd.     

Crystal structure of YdaL, a stand-alone small MutS-related protein from Escherichia coli

Sequence homologs of the small MutS-related (Smr) domain, the C-terminal endonuclease domain of MutS2, also exist as stand-alone proteins. In this study, we report the crystal structure of a proteolyzed fragment of YdaL (YdaL??-???), a stand-alone Smr protein from Escherichia coli. In this structure, residues 86-170 assemble into a classical Smr core domain and are embraced by an N-terminal extension (residues 40-85) with an α/β/α fold. Sequence alignment indicates that the N-terminal extension is conserved among a number of stand-alone Smr proteins, suggesting structural diversity among Smr domains. We also discovered that the DNA binding affinity and endonuclease activity of the truncated YdaL??-??? protein were slightly lower than those of full-length YdaL?-???, suggesting that residues 1-38 may be involved in DNA binding.     

Quantitative proteomic analysis of tumor reversion in multiple myeloma cells

Tumor reversion is defined as the process by which cancer cells lose their malignant phenotype. However, relatively little is known about the cellular proteome changes that occur during the reversion process. A biological model of multiple myeloma (MM) reversion was established by using the H-1 parvovirus as a tool to select for revertant cells from MM cells. Isolated revertant cells displayed a strongly suppressed malignant phenotype both in vitro and in vivo. To explore possible mechanisms of MM reversion, the protein profiles of the revertant and parental MM cells were compared using a quantitative proteomic strategy termed SILAC-MS. Our results revealed that 379 proteins were either activated or inhibited during the reversion process, with a much greater proportion of the proteins, including STAT3, TCTP, CDC2, BAG2, and PCNA, being inhibited. Of these, STAT3, which is significantly down regulated, was selected for further functional studies. Inhibition of STAT3 expression by RNA interference resulted in suppression of the malignant phenotype and concomitant down regulation of TCTP expression, suggesting that myeloma reversion operates, at least in part, through inhibition of STAT3. Our results provide novel insights into the mechanisms of tumor reversion and suggest new alternative approaches for MM treatment.     

MutL associates with Escherichia coli RecA and inhibits its ATPase activity

Different DNA repair systems are known to cooperate to deal with DNA damage. However, the regulatory role of the cross-talk between these pathways is unclear. Here, we have shown that MutL, an essential component of mismatch repair, is a RecA-interacting protein, and that its highly conserved N-terminal domain is sufficient for this interaction. Surface plasmon resonance and capillary electrophoresis analyses revealed that MutL has little effect on RecA-ssDNA filament formation, but dose down-regulate the ATPase activity of RecA. Our findings identify a new role for MutL, and suggest its regulatory role in homologous recombination.     

Mutagenesis of mNeptune Red-Shifts Emission Spectrum to 681-685 nm

GFP-like fluorescent proteins with diverse emission wavelengths have been developed through mutagenesis, offering many possible choices in cellular and tissue imaging, such as multi-targets imaging, deep tissue imaging that require longer emission wavelength. Here, we utilized a combined approach of random mutation and structure-based rational design to develop new NIR fluorescent proteins on the basis of a far-red fluorescent protein, mNeptune (Ex/Em: 600/650 nm). We created a number of new monomeric NIR fluorescent proteins with the emission range of 681–685 nm, which exhibit the largest Stocks shifts (77–80 nm) compared to other fluorescent proteins. Among them, mNeptune681 and mNeptune684 exhibit more than 30 nm redshift in emission relative to mNeptune, owing to the major role of the extensive hydrogen-bond network around the chromophore and contributions of individual mutations to the observed redshift. Furthermore, the two variants still maintain monomeric state in solution, which is a trait crucial for their use as protein tags. In conclusion, our results suggest that there is untapped potential for developing fluorescent proteins with desired properties.     

Characterization of a monomeric heat-labile classical alkaline phosphatase from <Emphasis Type="Italic">Anabaena</Emphasis> sp. PCC7120

Alkaline phosphatases (APs), known inducible enzymes of the Pho regulon and poorly characterized in cyanobacteria, hydrolyze phosphomonoesters to produce inorganic phosphate (P_i) during P_i starvation. In this study, two predicted alkaline phosphatase genes in the genome of Anabaena sp. PCC 7120, all2843 and alr5291, were apparently induced during P_i starvation. Sequence analysis showed that alr5291 encodes a protein that is an atypical alkaline phosphatase like other cyanobacteria PhoAs, but the protein encoded by all2843 is very similar to the classical PhoAs, such as Escherichia coli alkaline phosphatase (EAP). To date, there have been no reports about classical phoA in cyanobacterial genomes. The alkaline phosphatase AP^A, coded by all2843, is characterized as a metalloenzyme containing Mg²⁺ and Zn²⁺ with molar ratio of 1: 2. Site-directed mutagenesis analysis indicated that, though the active center of AP^A is highly conserved in comparison with EAP, differences do exist between AP^A and EAP in metal ion coordination. Besides, biochemical analysis revealed that AP^A is a monomeric protein and inactivated rapidly at 50°C. These results suggest that AP^A is the first monomeric heat-labile classical PhoA found in cyanobacteria.     

麦冬根中总RNA的快速提取

目的:从富含多糖、多酚的麦冬根部组织中快速提取总RNA。方法:采用改进的苯酚法,提取液的配制为5%SDS、1mol/LNaAc(pH4.1)、20%HAC、0.1%PVP。结果:采用该方法提取的麦冬总RNA纯度高、完整性好,电泳条带清晰。通过琼脂糖凝胶电泳与紫外吸光度测定产量与纯度,麦冬根部组织总RNA的吸光值D260nm/D280nm值大于1.8,D260nm/D230nm值大于2.0,麦冬块根与不定根RNA的平均产量分别为79.716和76.144μg/g(鲜重)。结论:用本方法提取的RNA可用于后继的抑制消减杂交试验。     

金黄色葡萄球菌肠毒素 C2 的基因克隆、 表达及其生物学活性

利用 PCR 技术从金黄色葡萄球菌的基因组 DNA 中克隆 SEC2 全长基因, PCR 产物与 pGEM-T 载体连接,经测序证实后进行亚克隆,构建其表达载体 pET-28a-SEC2 ,在大肠杆菌 BL21(DE3) 中表达成熟重组蛋白 (rSEC2) , 纯化 rSEC2 蛋白并对其生物学活性进行研究 . 结果表明：成功克隆了 SEC2 全长基因,测序证实该基因共 717 bp ,编码 239 个氨基酸,与 GenBank 中收录的 SEC2 成熟蛋白质序列完全一致, SEC2 基因登录 GenBank(Accession number ： AY450554) ; 构建了 SEC2 的表达载体 pET-28a-SEC2 ,并在大肠杆菌 BL21(DE3) 中得到高效可溶性表达,可溶性的 rSEC2 经 Ni²⁺ 亲和层析纯化达到电泳纯,纯化的 rSEC2 蛋白经蛋白质印迹检测,并能有效刺激人外周血单个核细胞的增殖,被 rSEC2 刺激的外周血单个核细胞在体外对肿瘤细胞的生长有显著的抑制作用 .     

Live cell imaging of protein interactions in poliovirus RNA replication complex using fluorescence resonance energy transfer (FRET)

Poliovirus RNA replication is directed by a replication complex on the rosette-like arrangement of membranous vesicles. Proteins derived from the p3 region of the polioviral genome, such as 3D, 3AB, and 3B (VPg), play key roles in the formation and function of the replication complex. In the present study, by using an acceptor photobleaching protocol for fluorescence resonance energy transfer (FRET) imaging, we visualized the interactions of 3D, 3AB, and VPg in living cells. The interaction of 3AB-VPg was determined by live cell FRET analysis. Quantitative analyses showed that the FRET efficiencies of 3AB-3D, VPg-3D, and 3AB-VPg were 3.9 ± 0.4% (n = 36), 4.5 ± 0.4% (n = 39), and 8.3 ± 0.6% (n = 44), respectively, in the cell cytoplasm where viral replication complexes are formed and function. Poliovirus infection enhanced the protein interactions of VPg-3D and 3AB-3D, with FRET efficiencies in the virus-infected cells of 10.7 ± 1.1% (n = 39) and 9.0 ± 0.9% (n = 37), respectively. This method of live cell analysis of protein interactions in the poliovirus RNA replication complex lays the foundation for further understanding of the real-time process of poliovirus RNA replication.