Oncogenic KRAS G12C: Kinetic and redox characterization of covalent inhibition

The recent development of mutant-selective inhibitors for the oncogenic KRAS^G12C allele has generated considerable excitement. These inhibitors covalently engage the mutant C12 thiol located within the phosphoryl binding loop of RAS, locking the KRAS^G12C protein in an inactive state. While clinical trials of these inhibitors have been promising, mechanistic questions regarding the reactivity of this thiol remain. Here, we show by NMR and an independent biochemical assay that the pK_a of the C12 thiol is depressed (pK_a ∼7.6), consistent with susceptibility to chemical ligation. Using a validated fluorescent KRAS^Y137W variant amenable to stopped-flow spectroscopy, we characterized the kinetics of KRAS^G12C fluorescence changes upon addition of ARS-853 or AMG 510, noting that at low temperatures, ARS-853 addition elicited both a rapid first phase of fluorescence change (attributed to binding, K_d = 36.0 ± 0.7 μM) and a second, slower pH-dependent phase, taken to represent covalent ligation. Consistent with the lower pK_a of the C12 thiol, we found that reversible and irreversible oxidation of KRAS^G12C occurred readily both in vitro and in the cellular environment, preventing the covalent binding of ARS-853. Moreover, we found that oxidation of the KRAS^G12C Cys12 to a sulfinate altered RAS conformation and dynamics to be more similar to KRAS^G12D in comparison to the unmodified protein, as assessed by molecular dynamics simulations. Taken together, these findings provide insight for future KRAS^G12C drug discovery efforts, and identify the occurrence of G12C oxidation with currently unknown biological ramifications.     

高产青霉素酰化酶巨大芽胞杆菌的诱变选育及产酶条件优化

【目的】选育高产青霉素G酰化酶(PGA)工业菌株。【方法】采用LiCl-紫外线复合诱变以及常压室温等离子体(ARTP)诱变技术对巨大芽胞杆菌(Bacillus megaterium) ATCC 14945进行处理。处理菌体涂平板后,将长出的菌落接种到液体培养基中,向培养6 h后的二代菌液中添加终浓度为0.1%的苯乙酸,28 °C、250 r/min条件下诱导培养40 h。对离心后获得的上清(粗酶液)采用NIPAB法测定PGA酶活力。以PGA酶活力最高的菌株为材料,对苯乙酸最佳添加量和最佳诱导时间进行优化,采用NIPAB法测定PGA酶活力。采用SDS-PAGE检测诱变前后巨大芽胞杆菌粗酶液中PGA的蛋白特性。【结果】从诱变菌落中筛选到PGA酶活力为39.60 U/mL的菌株12-4,酶活力比出发菌株提高了8.5倍。该菌株在液体培养6 h后添加终浓度为0.2%的苯乙酸,继续培养50 h后,PGA酶活力可达78.45 U/mL,比出发菌株提高了16.8倍。诱变前后菌株培养液中的PGA蛋白均具α、β亚基;诱变后菌株PGA α亚基的量没有明显变化,β亚基的量明显增多;α、β亚基之间的蛋白条带明显增多。【结论】采用诱变技术可提高巨大芽胞杆菌PGA活性,获得的诱变菌株12-4及培养条件对PGA工业化生产具有重要价值。     

沙枣（Elaeagnus angustifolia）和孩儿拳头（Grewia.biloba G.Don var.parviflora）幼苗气体交换特征与保护酶对干旱胁迫的响应

以沙枣和孩儿拳头2年生盆栽苗为材料,采用称重控水的方法设置4个土壤含水量梯度(CK、T1、T2、T3),研究不同干旱胁迫对沙枣和孩儿拳头气体交换特征与保护酶的影响.结果显示:(1)干旱胁迫不仅引起两物种净光合速率、蒸腾速率、气孔导度、胞间 CO2 浓度的下降,而且使其日变化曲线在一定程度上发生改变;在轻度(T1)和中度胁迫(T2)下,两物种净光合速率下降主要是由气孔因素引起的,重度胁迫(T3)下,净光合速率下降主要是非气孔因素引起的.(2)随着干旱胁迫增加,沙枣瞬时水分利用效率呈现增加下降再增加趋势,孩儿拳头呈现下降趋势;两物种表观光能利用效率显著下降,重度胁迫下(T3),下降率达50%左右,孩儿拳头表观光能利用效率对干旱胁迫比较敏感;两物种表观CO2利用效率总体呈现下降趋势,沙枣表观CO2利用效率日进程经历了单峰(T1)、双峰(T2)、单峰(T3)的变化,孩儿拳头各处理的表观CO2利用效率日变化均呈现单峰曲线.(3)随着干旱胁迫加剧,两物种叶片的超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)活性先升高后降低,土壤含水量高于12.8%时,两物种SOD酶活性均高于CK,随着土壤含水量的降低,SOD酶活性低于CK;重度胁迫下(T3),沙枣POD酶活性虽然有所下降,但仍高于CK,而孩儿拳头则和CK无显著差异;两物种CAT酶活性在重度胁迫下(T3)显著低于CK;随着干旱胁迫程度的增加,两物种叶片中的丙二醛(MDA)含量均呈现升高趋势,孩儿拳头脂质过氧化程度受干旱胁迫的影响较大.     

Human C1orf27 protein interacts with α2A-adrenergic receptor and regulates its anterograde transport

The molecular mechanisms underlying the anterograde surface transport of G protein–coupled receptors (GPCRs) after their synthesis in the endoplasmic reticulum (ER) are not well defined. In C. elegans, odorant response abnormal 4 has been implicated in the delivery of olfactory GPCRs to the cilia of chemosensory neurons. However, the function and regulation of its human homolog, C1orf27, in GPCR transport or in general membrane trafficking remain unknown. Here, we demonstrate that siRNA-mediated knockdown of C1orf27 markedly impedes the ER-to-Golgi export kinetics of newly synthesized α_2A-adrenergic receptor (α_2A-AR), a prototypic GPCR, with the half-time being prolonged by more than 65%, in mammalian cells in retention using the selective hooks assays. Using modified bioluminescence resonance energy transfer assays and ELISAs, we also show that C1orf27 knockdown significantly inhibits the surface transport of α_2A-AR. Similarly, C1orf27 knockout by CRISPR-Cas9 markedly suppresses the ER–Golgi-surface transport of α_2A-AR. In addition, we demonstrate that C1orf27 depletion attenuates the export of β₂-AR and dopamine D2 receptor but not of epidermal growth factor receptor. We further show that C1orf27 physically associates with α_2A-AR, specifically via its third intracellular loop and C terminus. Taken together, these data demonstrate an important role of C1orf27 in the trafficking of nascent GPCRs from the ER to the cell surface through the Golgi and provide novel insights into the regulation of the biosynthesis and anterograde transport of the GPCR family members.     

G4-quadruplex-binding proteins: review and insights into selectivity

There are over 700,000 putative G4-quadruplexes (G4Qs) in the human genome, found largely in promoter regions, telomeres, and other regions of high regulation. Growing evidence links their presence to functionality in various cellular processes, where cellular proteins interact with them, either stabilizing and/or anchoring upon them, or unwinding them to allow a process to proceed. Interest in understanding and manipulating the plethora of processes regulated by these G4Qs has spawned a new area of small-molecule binder development, with attempts to mimic and block the associated G4-binding protein (G4BP). Despite the growing interest and focus on these G4Qs, there is limited data (in particular, high-resolution structural information), on the nature of these G4Q-G4BP interactions and what makes a G4BP selective to certain G4Qs, if in fact they are at all. This review summarizes the current literature on G4BPs with regards to their interactions with G4Qs, providing groupings for binding mode, drawing conclusions around commonalities and highlighting information on specific interactions where available.     

Battling for Ribosomes: Translational Control at the Forefront of the Antiviral Response

In the early stages of infection, gaining control of the cellular protein synthesis machinery including its ribosomes is the ultimate combat objective for a virus. To successfully replicate, viruses unequivocally need to usurp and redeploy this machinery for translation of their own mRNA. In response, the host triggers global shutdown of translation while paradoxically allowing swift synthesis of antiviral proteins as a strategy to limit collateral damage. This fundamental conflict at the level of translational control defines the outcome of infection. As part of this special issue on molecular mechanisms of early virus–host cell interactions, we review the current state of knowledge regarding translational control during viral infection with specific emphasis on protein kinase RNA-activated and mammalian target of rapamycin-mediated mechanisms. We also describe recent technological advances that will allow unprecedented insight into how viruses and host cells battle for ribosomes.     

嗜盐菌群对酸性金黄G的脱色特性和机理

【目的】为了获得能够在高盐环境下脱色偶氮染料的嗜盐菌群及其降解机理。【方法】采用富集驯化的方法获得一个嗜盐菌群,采用Illumina HiSeq2500测序平台对其群落结构进行测定;采用分光光度法测定了其降解特性;采用GC-MS和红外图谱分析了其降解机理;采用微核实验的方法比较了偶氮染料降解前后的毒性。【结果】该菌群在10%的盐度下,使100mg/L的酸性金黄G在8h内脱色。菌群主要由Zobellella、Rheinheimera、Exiguobacterium和Marinobacterium组成。最适宜的脱色条件是:pH=6,酵母粉为碳源,蛋白胨或硝酸钾作为氮源,盐度为1%–10%。酸性金黄G降解产物的毒性比降解前降低。酸性金黄G主要的降解产物是对氨基二苯胺和二苯胺。此外,该菌群还能使酸性大红GR和直接湖蓝5B等多种偶氮染料脱色,具有较好的脱色广谱性。【结论】获得了快速降解偶氮染料的嗜盐菌群及降解机理,为该嗜盐菌群应用于高盐印染废水的处理提供菌种资源和理论支持。     

Is CD36 gene polymorphism in region encoding lipid-binding domain associated with early onset CAD?

CD36 is a fatty acid translocase in striated muscle cells and cardiomyocytes. Some study suggested that alterations in CD36 gene may be associated with coronary artery disease (CAD) risk. The aim of the current study was to compare the frequency of CD36 variants in region encoding lipid-binding domain in Caucasian patients with early-onset CAD, no-CAD adult controls and neonates. The study group comprised 100 patients with early onset CAD. The genetic control groups were 306 infants and 40 no-CAD adults aged over 70 years. Exons 4, 5 and 6 including fragments of flanking introns were studied using the denaturing high-performance liquid chromatography technique and direct sequencing. Changes detected in analyzed fragment of CD36: IVS3-6 T/C (rs3173798), IVS4-10 G/A (rs3211892), C311T (Thr104Ile, not described so far) in exon 5, G550A (Asp184Asn, rs138897347), C572T (Pro191Leu, rs143150225), G573A (Pro191Pro, rs5956) and A591T (Thr197Thr, rs141680676) in exon 6. No significant differences in the CD36 genotype, allele and haplotype frequencies were found between the three groups. Only borderline differences (p = 0.066) were found between early onset CAD patients and newborns in the frequencies of 591T allele (2.00% vs 0.50%) and CGCGCGT haplotype (2.00% vs 0.50%) with both IVS3-6C and 591T variant alleles. In conclusion, CD36 variants: rs3173798, rs3211892, rs138897347, rs5956, rs143150225 rs141680676 and C311T do not seem to be involved in the risk of early-onset CAD in Caucasian population.     

一株碱性淀粉酶产生菌Bacillus flexus XJU-3的分离鉴定及酶学特性分析

[目的]从新疆石河子市一处碱性工业污水(pH11.0)分离﹑鉴定高产碱性淀粉酶菌株,并对其所产酶酶学特性进行研究.[方法]在碱性淀粉酶分离培养基上对所分离菌株进行筛选,分离到一株高产碱性淀粉酶菌株,并将其编号XJU-3.应用生理生化试验,脂肪酸含量,16S rDNA序列以及(G C) mol%含量等方法对菌株进行鉴定,同时对XJU-3所产碱性淀粉酶的生物学特性进行研究.[结果] XJU-3可在pH4.0～12.5的LB培养基上生长,最适生长温度37℃.16S rDNA序列构建的系统进化树表明XJU-3与Bacillus flexus类聚在一起,且序列同源性为99%.该菌产生的淀粉酶最适pH10.0,最适温度40℃,且在pH9.0～13.0内有较高活性和稳定性.Co2 和Mg2 能明显提高酶的活性.[结论] XJU-3被鉴定为Bacillus flexus,由于XJU-3与B. flexus DSM 1320T在尿素水解和优势脂肪酸含量上有差异,且具有宽范围pH耐受性,因此XJU-3被认为是B.flexus的一个新菌株.XJU-3所产的碱性淀粉酶酶学特性良好,具有极大的工业应用潜力.