Hydrophobicity of amino acid residues in globular proteins

During biosynthesis, a globular protein folds into a tight particle with an interior core that is shielded from the surrounding solvent. The hydrophobic effect is thought to play a key role in mediating this process: nonpolar residues expelled from water engender a molecular interior where they can be buried. Paradoxically, results of earlier quantitative analyses have suggested that the tendency for nonpolar residues to be buried within proteins is weak. However, such analyses merely classify residues as either "exposed" or "buried." In the experiment reported in this article proteins of known structure were used to measure the average area that each residue buries upon folding. This characteristic quantity, the average area buried, is correlated with residue hydrophobicity.     

Ubistatins inhibit proteasome-dependent degradation by binding the ubiquitin chain

To identify previously unknown small molecules that inhibit cell cycle machinery, we performed a chemical genetic screen in Xenopus extracts. One class of inhibitors, termed ubistatins, blocked cell cycle progression by inhibiting cyclin B proteolysis and inhibited degradation of ubiquitinated Sic1 by purified proteasomes. Ubistatins blocked the binding of ubiquitinated substrates to the proteasome by targeting the ubiquitin-ubiquitin interface of Lys(48)-linked chains. The same interface is recognized by ubiquitin-chain receptors of the proteasome, indicating that ubistatins act by disrupting a critical protein-protein interaction in the ubiquitin-proteasome system.     

Orchestration of the DNA-damage response by the RNF8 ubiquitin ligase

Cells respond to DNA double-strand breaks by recruiting factors such as the DNA-damage mediator protein MDC1, the p53-binding protein 1 (53BP1), and the breast cancer susceptibility protein BRCA1 to sites of damaged DNA. Here, we reveal that the ubiquitin ligase RNF8 mediates ubiquitin conjugation and 53BP1 and BRCA1 focal accumulation at sites of DNA lesions. Moreover, we establish that MDC1 recruits RNF8 through phosphodependent interactions between the RNF8 forkhead-associated domain and motifs in MDC1 that are phosphorylated by the DNA-damage activated protein kinase ataxia telangiectasia mutated (ATM). We also show that depletion of the E2 enzyme UBC13 impairs 53BP1 recruitment to sites of damage, which suggests that it cooperates with RNF8. Finally, we reveal that RNF8 promotes the G2/M DNA damage checkpoint and resistance to ionizing radiation. These results demonstrate how the DNA-damage response is orchestrated by ATM-dependent phosphorylation of MDC1 and RNF8-mediated ubiquitination.     

Phosphorylation-dependent ubiquitination of cyclin E by the SCFFbw7 ubiquitin ligase

Cyclin E binds and activates the cyclin-dependent kinase Cdk2 and catalyzes the transition from the G1 phase to the S phase of the cell cycle. The amount of cyclin E protein present in the cell is tightly controlled by ubiquitin-mediated proteolysis. Here we identify the ubiquitin ligase responsible for cyclin E ubiquitination as SCFFbw7 and demonstrate that it is functionally conserved in yeast, flies, and mammals. Fbw7 associates specifically with phosphorylated cyclin E, and SCFFbw7 catalyzes cyclin E ubiquitination in vitro. Depletion of Fbw7 leads to accumulation and stabilization of cyclin E in vivo in human and Drosophila melanogaster cells. Multiple F-box proteins contribute to cyclin E stability in yeast, suggesting an overlap in SCF E3 ligase specificity that allows combinatorial control of cyclin E degradation.     

Polarization of PAR proteins by advective triggering of a pattern-forming system

In the Caenorhabditis elegans zygote, a conserved network of partitioning-defective (PAR) polarity proteins segregates into an anterior and a posterior domain, facilitated by flows of the cortical actomyosin meshwork. The physical mechanisms by which stable asymmetric PAR distributions arise from transient cortical flows remain unclear. We present evidence that PAR polarity arises from coupling of advective transport by the flowing cell cortex to a multistable PAR reaction-diffusion system. By inducing transient PAR segregation, advection serves as a mechanical trigger for the formation of a PAR pattern within an otherwise stably unpolarized system. We suggest that passive advective transport in an active and flowing material may be a general mechanism for mechanochemical pattern formation in developmental systems.     

氯氰菊酯的酶促降解

阴沟肠杆菌w 10 j15经过纯培养、超声波破碎和高速离心,提取到氯氰菊酯降解酶。在实验条件下测定了降解酶对氯氰菊酯的降解特性。结果表明,降解酶在40℃、pH 7.5时对氯氰菊酯显示最大的降解活性,对氯氰菊酯的降解率为78%;它在30~50℃或pH 6.5~8.0稳定性良好。L inew eaver-Burg法做图分析表明,其酶蛋白最大降解速率Vm ax为63.69 nm o.lmL-1.m in-1,米氏常数(Km)为377.35 nm o l/mL。     

Light chains of mouse myeloma proteins: partial amino acid sequence

Five kappa chains in the urinary proteins of the BALB/c mouse have the same carboxyl terminal amino acid sequence; this sequence resembles that of kappa light chains in human immunoglobulins. The five chains have amino acid sequence variations at the amino- terminal. The genetic basis for the amino- terminal variations is not understood but could be due either to a mecha nism for differently translating a single genetic message or to the presence of more than one kappa- type structural cistron in the BALB/c genome.     

Dual role of the Drosophila pattern gene tailless in embryonic termini

One of the first zygotically active genes required for formation of the terminal domains of the Drosophila embryo is tailless (tll). Expression of the tll gene is activated ectopically in gain-of-function mutants of the maternal terminal gene torso (tor); this suggests that tor normally activates the tll gene in the termini. Ectopic expression of tll under the control of an inducible promoter results in differentiation of ectopic terminal-specific structures, the Filzk?rper, and leads to the activation of at least one gene, hunchback, that is required to form these structures. Ectopic expression of the tll gene also represses segmentation by repressing the gap genes Krüppel and knirps and probably also pair rule genes.     

乳酸菌降解亚硝酸盐条件的优化

[目的]研究Lyoflora V_3乳杆菌对亚硝酸盐降解的影响,优化其降解条件。[方法]探讨了乳酸菌降解亚硝酸盐的影响因素,主要包括溶液pH、接种量、Na Cl含量和培养时间。采用L_9( 3~4)正交试验对整体工艺进行试验条件优化。[结果]影响乳酸菌降解亚硝酸盐的各因素次序为pH接种量Na Cl含量培养时间,最佳降解条件:pH为3.0,接种量为5%,Na Cl含量为9%,培养时间为72 h。在最佳降解条件下,乳酸菌降解亚硝酸盐的降解率可达98.50%。[结论]该研究可为乳酸菌发酵工艺中亚硝酸盐的控制提供参考。     

A general method for site-specific incorporation of unnatural amino acids into proteins

A new method has been developed that makes it possible to site-specifically incorporate unnatural amino acids into proteins. Synthetic amino acids were incorporated into the enzyme beta-lactamase by the use of a chemically acylated suppressor transfer RNA that inserted the amino acid in response to a stop codon substituted for the codon encoding residue of interest. Peptide mapping localized the inserted amino acid to a single peptide, and enough enzyme could be generated for purification to homogeneity. The catalytic properties of several mutants at the conserved Phe66 were characterized. The ability to selectively replace amino acids in a protein with a wide variety of structural and electronic variants should provide a more detailed understanding of protein structure and function.     

菠萝蛋白酶降解壳聚糖条件的研究

[目的]为菠萝蛋白酶降解壳聚糖制备壳寡糖提供工艺参数和科学依据。[方法]用DNS比色法测定还原糖的含量,探讨不同条件对菠萝蛋白酶降解壳聚糖的影响。用端基分析法测定壳寡糖的分子量,研究菠萝蛋白酶降解壳聚糖过程中壳聚糖分子量的变化。[结果]菠萝蛋白酶降解壳聚糖的最适pH为7.1,最适温度为53℃,最适酶浓度为0.30 g/L。在最适条件下,降解反应进行6 h以上,可以得到平均聚合度为8,平均分子量为1 306的壳寡糖。[结论]菠萝蛋白酶能有效降解壳聚糖,制备壳寡糖。     

有机磷降解剂对生菜农残降解效果分析

[目的]分析有机磷降解剂对生菜农残的降解效果.[方法]使用酶抑制分光光度法,以紫茎泽兰、秸秆为原料制备有机磷降解剂,检测其对生菜乐果的降解效果.[结果]不同pH的紫茎泽兰酶降解剂、秸秆酶降解剂对生菜中的有机磷有明显的降解作用,降解率高达79.34％～81.91％.[结论]该研究可为开发新型的有机磷降解剂提供理论依据.     

不同降解方法下β-胡萝卜素降解产物的分析

为了给卷烟加香提供一种新思路,比较分析了不同降解方法下β-胡萝卜素的降解产物,利用双氧水氧化、硫酸铜催化-双氧水氧化、硝酸银催化-双氧水氧化和高锰酸钾催化氧化-双氧水氧化等4种不同的降解方法对β-胡萝卜素进行降解,并对降解产物进行GC/MS分析。所鉴定出的降解产物均为烟叶中的重要香味成分,其中二氢猕猴桃内酯和β-紫罗兰酮相对含量最高;硝酸银催化-双氧水氧化和高锰酸钾催化氧化-双氧水氧化法所得到的降解产物中,二氢猕猴桃内酯相对含量高于另外2种方法;双氧水氧化和高锰酸钾催化氧化-双氧水氧化法所得到的降解产物中,β-紫罗兰酮相对含量高于另外2种方法。β-胡萝卜素经过降解,可以产生二氢猕猴桃内酯、β-紫罗兰酮、异佛尔酮、氧化异佛尔酮等致香物质;不同的降解方法,得到的降解产物和比例不同。     

Identification of SCF ubiquitin ligase substrates by global protein stability profiling

Ubiquitin-mediated proteolysis regulates all aspects of cellular function, and defects in this process are associated with human diseases. The limited number of identified ubiquitin ligase-substrate pairs is a major bottleneck in the ubiquitin field. We established and applied genetic technologies that combine global protein stability (GPS) profiling and genetic perturbation of E3 activity to screen for substrates of the Skp1-cullin-F-box (SCF) ubiquitin ligase in mammalian cells. Among the >350 potential substrates identified, we found most known SCF targets and many previously unknown substrates involved in cell cycle, apoptosis, and signaling pathways. Exploring cell cycle-stage stability, we found that several substrates used the SCF and other E3s in different cell cycle stages. Our results demonstrate the potential of these technologies as general platforms for the global discovery of E3-substrate regulatory networks.     

酵母双杂交技术筛选与绵羊微管解聚蛋白相互作用的蛋白

采用TRIzol法提取绵羊卵巢组织总RNA,运用RT–PCR技术扩增绵羊微管解聚蛋白基因(STMN1)的编码区序列;通过同源重组技术构建重组诱饵表达载体p GBKT7–STMN1,鉴定该诱饵蛋白在酵母双杂交系统中的自激活活性、细胞毒性和表达情况;利用诱饵质粒p GBKT7–STMN1从绵羊卵巢组织细胞的酵母双杂交cDNA文库筛选互作的宿主蛋白。结果显示：成功构建重组诱饵质粒p GBKT7–STMN1,并对酵母细胞无自激活活性和细胞毒性作用,且能在酵母细胞中正常表达;筛选获得12个与STMN1相互作用的克隆,经测序比对分析和点对点验证,得到CREB和FOXM1共2个互作的宿主蛋白,这2个蛋白可能参与调控细胞的增殖、周期、凋亡等过程。     

Structure of the GDP domain of EF-Tu and location of the amino acids homologous to ras oncogene proteins

A 2.7 angstrom resolution x-ray diffraction analysis of a trypsin-modified form of the Escherichia coli elongation factor Tu reveals that the GDP-binding domain has a structure similar to that of other nucleotide-binding proteins. The GDP ligand is located at the COOH-terminal end of the beta sheet and is linked to the protein via a Mg2+ ion salt bridge. The location of the guanine ring is unusual; the purine ring is located on the outer edge of the domain, not deep within a hydrophobic pocket. The amino acids from Pro10 to Arg44 and from Gly59 to Glu190 have been assigned to the electron density with computer graphic techniques, and the resulting model is consistent with all known biochemical data. An analysis of the structure reveals that four regions of the amino acid sequence that are homologous with the family of ras oncogene proteins, termed p21, are located in the vicinity of the GDP-binding site, and most of the invariant amino acids shared by the proteins interact directly with the GDP ligand.     

过氧化氢降解有机磷农药的研究II.降解动力学及降解液的毒性

研究了H2O2光降解动力学机理及其降解液的毒性. 结果表明,光照对H2O2降解农药有显著影响,农药降解率随反应时间延长而增加. 有机磷农药的H2O2光催化降解符合零级反应动力学,其反应速率常数(k)随农药浓度的增加而增大,半衰期(t0.5)延长. 用生测法测定降解液残留物的毒性发现,H2O2光催化降解农药是降毒降解的. 10 μg/mL甲胺磷、10 μg/mL毒死蜱、100 μg/mL久效磷的H2O2光催化降解初始反应液处理家蚕Bombyx mori 2龄幼虫的24 h死亡率分别为60%、100%、100%,而12 h降解液的死亡率均小于5%. 黄曲条跳甲Phyllotreta striolata生物测定所得结果与家蚕结果一致.