Proline- and arginine-rich peptides constitute a novel class of allosteric inhibitors of proteasome activity

Substrate-specific inhibition of the proteasome has been unachievable despite great interest in proteasome inhibitors as drugs. Recent studies demonstrated that PR39, a natural proline- and arginine-rich antibacterial peptide, stimulates angiogenesis and inhibits inflammatory responses by specifically blocking degradation of IkappaBalpha and HIF-1alpha by the proteasome. However, molecular events involved in the PR39-proteasome interaction have not been elucidated. Here we show that PR39 is a noncompetitive and reversible inhibitor of the proteasome function. This effect is achieved by a unique allosteric mechanism allowing for specific inhibition of degradation of selected proteins without affecting total proteasome-dependent proteolysis. Atomic force microscopy (AFM) studies demonstrate that 20S and 26S proteasomes treated with PR39 or its derivatives exhibit serious perturbations in their structure and their normal allosteric movements. These effects are universal for proteasomes from yeast to human. The shortest functional sequence derived from PR39 still showing the allosteric inhibitory effect consists of eleven NH(2)-terminal residues containing essential three NH(2)-terminal arginines. The noncompetitive and reversible in vitro action of PR39 and its truncated derivatives is matched by the ability of the peptides to induce angiogenesis in vivo. We postulate that PR39 changes conformational dynamics of the proteasomes by interactions with the noncatalytic subunit alpha7 in a way that prevents the enzyme from cleaving the substrates of unique structural constraints.     

硝酸纤维膜和丙酮沉淀尿蛋白质保存方法的比较

硝酸纤维膜法是一种简单、快速、经济的尿液蛋白质保存方法,但其与传统尿液蛋白质丙酮沉淀方法的差异有待进一步研究。相同尿液分别经硝酸纤维膜法和丙酮沉淀法制备尿蛋白质,经液相串联质谱分析鉴定蛋白质,采用谱图数定量,研究两种不同方法的差别。结果显示硝酸纤维膜法和丙酮沉淀法鉴定蛋白质数目几乎相同,鉴定蛋白质在谱图数的分布上几乎相同,鉴定蛋白质在蛋白质变异系数的分布上也几乎相同。因此,硝酸纤维膜法处理尿蛋白质与丙酮沉淀法基本一致,可以应用于大规模临床尿液样本的保存。     

Comparative proteome analysis of breast cancer and normal breast

Breast cancer is a leading cause of death for women. The underlying molecular mechanism is still not well understood. In this study, two-dimensional gel electrophoresis combined with mass spectrometry was used to analyze changes in the proteome of infiltrating ductal carcinoma compared to normal breast tissue. Ten sets of two-dimensional gels per experimental condition were analyzed and more than 500 spots each were detected. This revealed 39 spots for which expression in breast cancer cells were reproducibly altered more than twofold compared to normal controls (p<0.01). These spots represented 25 different proteins after identification using the database search after mass spectrometry, comprising cell defense proteins, enzymes involved in glycolytic energy metabolism and homeostasis, protein folding and structural proteins, proteins involved in cytoskeleton and cell motility, and proteins involved in other functions. In addition, 28 nondifferentially expressed proteins with different functions were also mapped and identified, which might help to establish a two-dimensional gel electrophoresis reference map of human breast cancer. Our study shows that proteomics offers a powerful methodology to detect the proteins that show different expression patterns in breast cancer tissue and may provide an accurate molecular classification. The differentially expressed proteins may be used as potential candidate markers for diagnostic purposes or for determination of tumor sensitivity to therapy. The functional implications of the identified proteins are discussed.     

An integrated machine learning system to computationally screen protein databases for protein binding peptide ligands

A fairly large set of protein interactions is mediated by families of peptide binding domains, such as Src homology 2 (SH2), SH3, PDZ, major histocompatibility complex, etc. To identify their ligands by experimental screening is not only labor-intensive but almost futile in screening low abundance species due to the suppression by high abundance species. An ideal way of studying protein-protein interactions is to use high throughput computational approaches to screen protein sequence databases to direct the validating experiments toward the most promising peptides. Predictors with only good cross-validation were not good enough to screen protein databases. In the current study we built integrated machine learning systems using three novel coding methods and screened the Swiss-Prot and GenBank protein databases for potential ligands of 10 SH3 and three PDZ domains. A large fraction of predictions has already been experimentally confirmed by other independent research groups, indicating a satisfying generalization capability for future applications in identifying protein interactions.     

A method for generation of arbitrary peptide libraries using genomic DNA

Random peptide libraries can be constructed either by in vitro synthesis of random peptides, or through translation of DNA sequences from synthetic random oligonucleotides. Here we describe an alternative way of making arbitrary peptide libraries with high diversity that can be used in screening as random peptide libraries. Genomic DNA digested with a frequent-cutting restriction enzyme recognizing four nucleotides will theoretically consist of small DNA pieces with average length of 256 nucleotides, and on average around 10⁷ fragments can be generated from a genome of 3 × 10⁹ bases. A peptide library translated from these fragments will have sufficient diversity for some protein interaction screening experiments. Moreover, the same genome digested with a different four-cutter enzyme or ligated into different reading frames will result in different nonoverlapping libraries. A series of such libraries could be generated with genomic DNAs from different species. In this study, human genomic DNA was digested with four-cutter restriction enzymes DpnII and Tsp509I, respectively, and cloned into yeast expression vector pGADT7 to generate arbitrary peptide libraries. These libraries were used in yeast two-hybrid assays to screen for binding motifs of the PDZ domain containing protein synectin. Our results showed that in addition to various native carboxy-terminal tails, synectin could also bind to many artificial ones, some of which contained a consensus sequence—(S/T)XC-COOH.     

A tool for biomarker discovery in the urinary proteome: a manually curated human and animal urine protein biomarker database

Urine is an important source of biomarkers. A single proteomics assay can identify hundreds of differentially expressed proteins between disease and control samples; however, the ability to select biomarker candidates with the most promise for further validation study remains difficult. A bioinformatics tool that allows accurate and convenient comparison of all of the existing related studies can markedly aid the development of this area. In this study, we constructed the Urinary Protein Biomarker (UPB) database to collect existing studies of urinary protein biomarkers from published literature. To ensure the quality of data collection, all literature was manually curated. The website (http://122.70.220.102/biomarker) allows users to browse the database by disease categories and search by protein IDs in bulk. Researchers can easily determine whether a biomarker candidate has already been identified by another group for the same disease or for other diseases, which allows for the confidence and disease specificity of their biomarker candidate to be evaluated. Additionally, the pathophysiological processes of the diseases can be studied using our database with the hypothesis that diseases that share biomarkers may have the same pathophysiological processes. Because of the natural relationship between urinary proteins and the urinary system, this database may be especially suitable for studying the pathogenesis of urological diseases. Currently, the database contains 553 and 275 records compiled from 174 and 31 publications of human and animal studies, respectively. We found that biomarkers identified by different proteomic methods had a poor overlap with each other. The differences between sample preparation and separation methods, mass spectrometers, and data analysis algorithms may be influencing factors. Biomarkers identified from animal models also overlapped poorly with those from human samples, but the overlap rate was not lower than that of human proteomics studies. Therefore, it is not clear how well the animal models mimic human diseases.     

甲氨蝶呤对大鼠尿液蛋白质组的影响

甲氨蝶呤是常见的免疫抑制剂,曾被大剂量用于治疗癌症,近年来其被小剂量用于治疗类风湿性关节炎。文中尝试研究甲氨蝶呤对大鼠尿蛋白质组的影响。大鼠口服甲氨蝶呤构造用药模型,再收集大鼠10 h内的尿液,并且采用液相色谱串联质谱(liquid chromatography tandem mass spectrometry,LC-MS/MS)分析大鼠的尿蛋白。总共鉴定到31个差异蛋白,其中7个蛋白与甲氨蝶呤药物作用和类风湿性关节炎症状有关。部分大鼠的生物学过程反映了甲氨蝶呤对机体谷胱甘肽代谢以及对JAK/STAT信号通路的影响。结果显示,尿蛋白具有反映甲氨蝶呤对大鼠机体影响的能力。对单个大鼠个体差异蛋白的分析体现出不同个体对该药物的反应有较大差异。     

Novel nonphosphorylated peptides with conserved sequences selectively bind to Grb7 SH2 domain with affinity comparable to its phosphorylated ligand

The Grb7 (growth factor receptor-bound 7) protein, a member of the Grb7 protein family, is found to be highly expressed in such metastatic tumors as breast cancer, esophageal cancer, liver cancer, etc. The src-homology 2 (SH2) domain in the C-terminus is reported to be mainly involved in Grb7 signaling pathways. Using the random peptide library, we identified a series of Grb7 SH2 domain-binding nonphosphorylated peptides in the yeast two-hybrid system. These peptides have a conserved GIPT/K/N sequence at the N-terminus and G/WD/IP at the C-terminus, and the region between the N-and C-terminus contains fifteen amino acids enriched with serines, threonines and prolines. The association between the nonphosphorylated peptides and the Grb7 SH2 domain occurred in vitro and ex vivo. When competing for binding to the Grb7 SH2 domain in a complex, one synthesized nonphosphorylated ligand, containing the twenty-two amino acid-motif sequence, showed at least comparable affinity to the phosphorylated ligand of ErbB3 in vitro, and its overexpression inhibited the proliferation of SK-BR-3 cells. Such nonphosphorylated peptides may be useful for rational design of drugs targeted against cancers that express high levels of Grb7 protein.     

加热洗脱硝酸纤维膜上保存的尿蛋白质

用膜保存尿蛋白质对于生物标志物的研发意义重大,从保存尿蛋白质的硝酸纤维膜上洗脱蛋白质的有效性,决定着保存方法被接受的程度和应用的范围。加热洗脱蛋白质的方法,通过提高硝酸纤维膜溶解时的温度等方式;并运用SDS-PAGE和LC-MS/MS分析加热洗脱方法所制备的尿蛋白质样品,将其与强烈涡旋洗脱方法和直接丙酮沉淀方法所制备的尿蛋白质样品比较。结果显示,加热洗脱方法比强烈涡旋洗脱方法获得更多的蛋白质(P0.05),且蛋白质无降解;加热洗脱方法和直接丙酮沉淀方法制备的蛋白质样品,质谱所鉴定到蛋白质重叠率无明显差异(分别是92.6%、96.8%),蛋白质丰度CV值20%的蛋白质占总蛋白质的比例均很高(分别是85.2%、94.4%)。加热洗脱法具有很好的技术重复性,操作更加高效、简单,有利于用膜保存尿蛋白质方法的推广应用。     

大鼠急性低氧模型尿液蛋白质组的变化

本研究旨在探究急性低氧对大鼠尿液蛋白质组造成的影响。在该项研究中,大鼠被放置于模拟海拔5 000 m高原环境的低氧舱内24 h。在低氧后0、12、24 h收集尿液样本,并使用液相色谱-串联质谱技术（LC-MS/MS）对尿蛋白进行分析。与低氧0 h相比,低氧12 h组共鉴定到144个差异蛋白,低氧24 h组共鉴定到129个差异蛋白。功能分析显示,差异蛋白参与了一系列与低氧应激有关的生物学通路,如抗氧化应激、糖酵解、补体和凝血级联反应等。研究结果表明,尿液蛋白质组可以反映急性低氧刺激后的显著变化。这些发现可能提供了一种判断机体缺氧状态的方法,有助于辅助检测缺氧状态。