Rapid determination of substrate specificity of Clostridium histolyticum beta-collagenase using an immobilized peptide library.

The molecular basis of the substrate specificity of Clostridium histolyticum beta-collagenase was investigated using a combinatorial method. An immobilized positional peptide library, which contains 24,000 sequences, was constructed with a 7-hydroxycoumarin-4-propanoyl (Cop) fluorescent group attached at the N terminus of each sequence. This immobilized peptide library was incubated with C. histolyticum beta-collagenase, releasing fluorogenic fragments in the solution phase. The relative substrate specificity (k(cat)/K(m)) for each member of the library was determined by measuring fluorescence intensity in the solution phase. Edman sequencing was used to assign structure to subsites of active substrate mixtures. Collectively, the substrate preference for subsites (P(3)-P(4)') of C. histolyticum beta-collagenase was determined. The last position on the C-terminal side in which the identity of the amino acids affects the activity of the enzyme is P(4)', and an aromatic side chain is preferred in this position. The optimal P(1)'-P(3)' extended substrate sequence is P(1)'-Gly/Ala, P(2)'-Pro/Xaa, and P(3)'-Lys/Arg/Pro/Thr/Ser. The Cop group in either the P(2) or P(3) position is required for a high substrate activity with C. histolyticum beta-collagenase. S(2) and S(3) sites of the protease play a dominant role in fixing the substrate specificity. The immobilized peptide library proved to be a powerful approach for assessing the substrate specificity of C. histolyticum beta-collagenase, so it may be applied to the study of other proteases of interest.     

Multiomic Metabolic Enrichment Network Analysis Reveals Metabolite–Protein Physical Interaction Subnetworks Altered in Cancer

Metabolism is recognized as an important driver of cancer progression and other complex diseases, but global metabolite profiling remains a challenge. Protein expression profiling is often a poor proxy since existing pathway enrichment models provide an incomplete mapping between the proteome and metabolism. To overcome these gaps, we introduce multiomic metabolic enrichment network analysis (MOMENTA), an integrative multiomic data analysis framework for more accurately deducing metabolic pathway changes from proteomics data alone in a gene set analysis context by leveraging protein interaction networks to extend annotated metabolic models. We apply MOMENTA to proteomic data from diverse cancer cell lines and human tumors to demonstrate its utility at revealing variation in metabolic pathway activity across cancer types, which we verify using independent metabolomics measurements. The novel metabolic networks we uncover in breast cancer and other tumors are linked to clinical outcomes, underscoring the pathophysiological relevance of the findings.     

环氧树脂固定化的Bacillus sp.DL-2胞外蛋白酶在拆分(±)-乙酸苏合香酯中的应用

环氧基是一个非常活跃的基团,它能与酶、蛋白质和核酸等生物分子发生反应形成共价键,有利于生物分子的固定化。经共价结合法固定化的酶其稳定性及重复使用性可得到显著提高。用环氧树脂ES-103B为载体采用共价结合法对海洋细菌Bacillus sp. DL-2的胞外蛋白酶进行固定化,经过单因素实验优化条件得出最优固定化条件为:p H 8. 0的胞外蛋白酶溶液,25 g/L的ES-103B,45℃下反应8h。采用此最优条件下的固定化酶拆分(±)-乙酸苏合香酯制备出了e. e. p=97. 5%的(R)-1-苯乙醇(产率为45. 0%)和e. e. s=99. 2%的(S)-乙酸苏合香酯(产率为83. 9%)。该固定化酶拆分(±)-乙酸苏合香酯在重复使用8次后制备出的(R)-1-苯乙醇的e. e. p仍大于90%,且固定化胞外蛋白酶在4℃下具有较好的储存稳定性。     

The 30-Base-Pair Deletion in Chinese Variants of the Epstein-Barr Virus LMP1 Gene Is Not the Major Effector of Functional Differences between Variant LMP1 Genes in Human Lymphocytes

One group of sequence variants of Epstein-Barr virus is characterized by a 10-amino-acid deletion within the CTAR-2 functional domain of the latent membrane protein, LMP1. A role for this deletion in enhancing the tumorigenicity of the viral oncogene in rodent fibroblasts was recently demonstrated. We examined the effect of this deletion upon LMP1 function in four human lymphoid cell lines by using three natural variants of LMP1: the prototype B95.8 gene and the CAO and AG876 genes, both of which have codons 343 to 352 of the B95.8-LMP1 deleted. These experiments revealed that LMP1-mediated upregulation of CD40 and CD54 was markedly impaired (by 60 to 90%) with CAO-LMP1 compared with B95.8-LMP1. In contrast, the function of AG876-LMP1 was indistinguishable from that of B95.8-LMP1 in two lines and was only slightly impaired in the other two lines. Activation of NF-κB by CAO-LMP1 was not impaired in any of the lines; rather, activation of an NF-κB reporter by CAO-LMP1 was consistently about twofold greater than the activation with B95.8- or AG876-LMP1. Therefore, while the CAO-LMP1 is functionally distinct from the prototype B95.8-LMP1 in human lymphocytes, the 10-amino-acid deletion appears not to be directly responsible. This conclusion was confirmed by using a B95.8-LMP1 mutant with codons 343 to 352 deleted and chimerae of CAO- and B95.8-LMP1 in which the CTAR-2 domains of these genes were exchanged. Sequences outside the CTAR-2 domain were implicated in the distinct functional characteristics of CAO-LMP1 in human lymphoid cells.     

Solution structure of the N‐terminal domain of DC‐UbP/UBTD2 and its interaction with ubiquitin

DC‐UbP/UBTD2 is a ubiquitin (Ub) domain‐containing protein first identified from dendritic cells, and is implicated in ubiquitination pathway. The solution structure and backbone dynamics of the C‐terminal Ub‐like (UbL) domain were elucidated in our previous work. To further understand the biological function of DC‐UbP, we then solved the solution structure of the N‐terminal domain of DC‐UbP (DC‐UbP_N) and studied its Ub binding properties by NMR techniques. The results show that DC‐UbP_N holds a novel structural fold and acts as a Ub‐binding domain (UBD) but with low affinity. This implies that the DC‐UbP protein, composing of a combination of both UbL and UBD domains, might play an important role in regulating protein ubiquitination and delivery of ubiquitinated substrates in eukaryotic cells.     

格网尺度下典型旅游城市生态服务价值估算和时空分异特征——以三亚为例

以典型旅游城市三亚市为案例地，利用2006-2018年4期Landsat遥感影像数据，借助ENVI、ArcGIS平台定量识别土地利用演变特征，在1km×1km格网尺度下估算旅游地生态系统服务价值，并结合空间探索性数据分析揭示生态系统服务价值时空分异特征及其与旅游地发展的时空耦合关系。结果表明：（1）2006-2018年间，三亚市生态系统服务价值总量呈逐年下降趋势，由6.73×10⁹元降至5.76×10⁹元，累计减少9.78×10⁸元；（2）空间格局上，三亚市呈"南低北高"空间分异格局，2006-2018年增值区域连片分布于崖州区、天涯区、吉阳区南部区域，且呈逐年减少趋势，减值区域集聚于天涯区东北部、海棠区；（3）空间集聚上，生态系统服务价值截面各年份均呈显著空间正相关且相关性先降后增。高高集聚区位于天涯区北部区域，低低集聚区分布于沿海、海湾地区；（4）旅游发展与生态系统服务价值时空演化特征关联性较强。三亚市天涯区北部林地生态环境良好，生态系统服务价值略有下降但绝对数值稳定，是生态系统服务价值主要来源；旅游发展较为迅速的三亚湾、崖州湾以及海棠湾，相对增值区域较多，但绝对生态系统服务价值损失显著，严重滞后于其他区域。     

Repeated pre-training sleep restriction in adolescent rats impaired spatial performance

Sleep and Biological Rhythms - Chronic sleep deprivation (SD) is an overwhelming problem in young students. Firstly, we investigated whether different levels of pre-training SD had effects on...