Technical aspects of functional proteomics in plants

Since the completion of genome sequences of several organisms, attention has been focused to determine the function and functional network of proteins by proteome analysis. This analysis is achieved by separation and identification of proteins, determination of their function and functional network, and construction of an appropriate database. Many improvements in separation and identification of proteins, such as two-dimensional electrophoresis, nano-liquid chromatography and mass spectrometry, have rapidly been achieved. Some new techniques which include top-down mass spectrometry and tandem affinity purification have emerged. These techniques have provided the possibility of high-throughput analysis of function and functional network of proteins in plants. However, to cope with the huge information emerging from proteome analyses, more sophisticated techniques and software are essential. The development and adaptation of such techniques will ease analyses of protein profiling, identification of post-translational modifications and protein-protein interaction, which are vital for elucidation of the protein functions.     

Overcoming the dynamic range problem in mass spectrometry-based shotgun proteomics

Protein profiling using mass spectrometry technology has emerged as a powerful method for analyzing large-scale protein-expression patterns in cells and tissues. However, a number of challenges are present in proteomics research, one of the greatest being the high degree of protein complexity and huge dynamic range of proteins expressed in the complex biological mixtures, which exceeds six orders of magnitude in cells and ten orders of magnitude in body fluids. Since many important signaling proteins have low expression levels, methods to detect the low-abundance proteins in a complex sample are required. This review will focus on the fundamental fractionation and mass spectrometry techniques currently used for large-scale shotgun proteomics research.     

Overcoming the dynamic range problem in mass spectrometry-based shotgun proteomics

Protein profiling using mass spectrometry technology has emerged as a powerful method for analyzing large-scale protein-expression patterns in cells and tissues. However, a number of challenges are present in proteomics research, one of the greatest being the high degree of protein complexity and huge dynamic range of proteins expressed in the complex biological mixtures, which exceeds six orders of magnitude in cells and ten orders of magnitude in body fluids. Since many important signaling proteins have low expression levels, methods to detect the low-abundance proteins in a complex sample are required. This review will focus on the fundamental fractionation and mass spectrometry techniques currently used for large-scale shotgun proteomics research.     

Reverse genetics of essential genes in tissue-culture cells: 'dead cells talking'

In the 'post-genomic' era, cDNA and genomic sequences are now available that encode huge numbers of proteins. Assigning functions to these proteins is a daunting task. Cell biologists have traditionally approached this problem by disrupting protein function with dominant-negative or structural mutants. Here, we describe several alternative approaches whereby cells or cell lines lacking particular gene products can be generated from genomic sequences for use in functional studies. These include gene targeting in mouse, human and chicken DT40 cells, and recent advances in double-stranded RNA-mediated interference (RNAi).     

High-performance proteomics as a tool in biomarker discovery

Biomarkers allowing early detection of disease or therapy control have a huge influence in curing a disease. A wide variety of methods were applied to find new biomarkers. In contrast to methods focused on DNA or mRNA techniques, approaches considering proteins as potential biomarker candidates have the advantage that proteins are more diverse than DNA or RNA and are more reflective of a biological system. Here, we present an approach for the identification of new biomarkers relying on our experience from the past 10 years of proteomics, outlining a concept of "high-performance proteomics" This approach is based on quantitative proteome analysis using a sufficient number of clinical samples and statistical validation of proteomics data by independent methods, such as Western blot analysis or immunohistochemistry.     

Predicting mostly disordered proteins by using structure-unknown protein data

Background  

Predicting intrinsically disordered proteins is important in structural biology because they are thought to carry out various cellular functions even though they have no stable three-dimensional structure. We know the structures of far more ordered proteins than disordered proteins. The structural distribution of proteins in nature can therefore be inferred to differ from that of proteins whose structures have been determined experimentally. We know many more protein sequences than we do protein structures, and many of the known sequences can be expected to be those of disordered proteins. Thus it would be efficient to use the information of structure-unknown proteins in order to avoid training data sparseness. We propose a novel method for predicting which proteins are mostly disordered by using spectral graph transducer and training with a huge amount of structure-unknown sequences as well as structure-known sequences.     

Roles for the two-hybrid system in exploration of the yeast protein interactome

Comprehensive analysis of protein-protein interactions is a challenging endeavor of functional proteomics and has been best explored in the budding yeast. The yeast protein interactome analysis was achieved first by using the yeast two-hybrid system in a proteome-wide scale and next by large-scale mass spectrometric analysis of affinity-purified protein complexes. While these interaction data have led to a number of novel findings and the emergence of a single huge network containing thousands of proteins, they suffer many false signals and fall short of grasping the entire interactome. Thus, continuous efforts are necessary in both bioinformatics and experimentation to fully exploit these data and to proceed another step forward to the goal. Computational tools to integrate existing biological knowledge buried in literature and various functional genomic data with the interactome data are required for biological interpretation of the huge protein interaction network. Novel experimental methods have to be developed to detect weak, transient interactions involving low abundance proteins as well as to obtain clues to the biological role for each interaction. Since the yeast two-hybrid system can be used for the mapping of the interaction domains and the isolation of interaction-defective mutants, it would serve as a technical basis for the latter purpose, thereby playing another important role in the next phase of protein interactome research.     

神经发育中的细胞周期时程

哺乳动物的神经发育经历一系列神经前体细胞的形态结构和机能改变,其细胞周期时程也呈现动态变化,从神经发生早期至后期,神经前体细胞的细胞周期时程逐渐延长,并与细胞发育命运转归有关,其调节因素包括周期蛋白复合体、Notch信号通路、原神经基因靶向蛋白、微管与分子马达蛋白等。细胞周期长度假说认为,细胞周期的长度影响到命运决定子的积累,因而决定细胞的命运。文章综述了相关的研究进展。     

Prediction of protein signal sequences and their cleavage sites

Protein signal sequences play a central role in the targeting and translocation of nearly all secreted proteins and many integral membrane proteins in both prokaryotes and eukaryotes. The knowledge of signal sequences has become a crucial tool for pharmaceutical scientists who genetically modify bacteria, plants, and animals to produce effective drugs. However, to effectively use such a tool, the first important thing is to find a fast and effective method to identify the "zipcode" entity; this is also evoked by both the huge amount of unprocessed data available and the industrial need to find more effective vehicles for the production of proteins in recombinant systems. In view of this, a sequence-encoded algorithm was developed to identify the signal sequences and predict their cleavage sites. The rate of correct prediction for 1,939 secretory proteins and 1,440 nonsecretory proteins by self-consistency test is 90.14% and that by jackknife test is 90.13%. The encouraging results indicate that the signal sequences share some common features although they lack similarity in sequence, length, and even composition and that they are predictable to a considerably accurate extent.     

Telomeres in cancer and ageing

Telomeres protect the chromosome ends from unscheduled DNA repair and degradation. Telomeres are heterochromatic domains composed of repetitive DNA (TTAGGG repeats) bound to an array of specialized proteins. The length of telomere repeats and the integrity of telomere-binding proteins are both important for telomere protection. Furthermore, telomere length and integrity are regulated by a number of epigenetic modifications, thus pointing to higher order control of telomere function. In this regard, we have recently discovered that telomeres are transcribed generating long, non-coding RNAs, which remain associated with the telomeric chromatin and are likely to have important roles in telomere regulation. In the past, we showed that telomere length and the catalytic component of telomerase, Tert, are critical determinants for the mobilization of stem cells. These effects of telomerase and telomere length on stem cell behaviour anticipate the premature ageing and cancer phenotypes of telomerase mutant mice. Recently, we have demonstrated the anti-ageing activity of telomerase by forcing telomerase expression in mice with augmented cancer resistance. Shelterin is the major protein complex bound to mammalian telomeres; however, its potential relevance for cancer and ageing remained unaddressed to date. To this end, we have generated mice conditionally deleted for the shelterin proteins TRF1, TPP1 and Rap1. The study of these mice demonstrates that telomere dysfunction, even if telomeres are of a normal length, is sufficient to produce premature tissue degeneration, acquisition of chromosomal aberrations and initiation of neoplastic lesions. These new mouse models, together with the telomerase-deficient mouse model, are valuable tools for understanding human pathologies produced by telomere dysfunction.     

The chloroplast‐localized small heat shock protein Hsp21 associates with the thylakoid membranes in heat‐stressed plants

The small heat shock protein (sHsp) chaperones are crucial for cell survival and can prevent aggregation of client proteins that partially unfold under destabilizing conditions. Most investigations on the chaperone activity of sHsps are based on a limited set of thermosensitive model substrate client proteins since the endogenous targets are often not known. There is a high diversity among sHsps with a single conserved β‐sandwich fold domain defining the family, the α‐crystallin domain, whereas the N‐terminal and C‐terminal regions are highly variable in length and sequence among various sHsps and conserved only within orthologues. The endogenous targets are probably also varying among various sHsps, cellular compartments, cell type and organism. Here we have investigated Hsp21, a non‐metazoan sHsp expressed in the chloroplasts in green plants which experience huge environmental fluctuations not least in temperature. We describe how Hsp21 can also interact with the chloroplast thylakoid membranes, both when isolated thylakoid membranes are incubated with Hsp21 protein and when plants are heat‐stressed. The amount of Hsp21 associated with the thylakoid membranes was precisely determined by quantitative mass spectrometry after metabolic ¹⁵N‐isotope labeling of either recombinantly expressed and purified Hsp21 protein or intact Arabidopsis thaliana plants. We found that Hsp21 is among few proteins that become associated with the thylakoid membranes in heat‐stressed plants, and that approximately two thirds of the pool of chloroplast Hsp21 is affected. We conclude that for a complete picture of the role of sHsps in plant stress resistance also their association with the membranes should be considered.     

Molecular farming in plants: host systems and expression technology

Plants provide an inexpensive and convenient system for the large-scale production of valuable recombinant proteins. This principle has been demonstrated by the commercial success of several first-generation products, and many others are currently under development. Over the past ten years, several efficient plant-based expression systems have emerged, and >100 recombinant proteins have now been produced in a range of different species. Plants have many advantages over other production systems, particularly in terms of practicality, economy and safety. However, several constraints that hinder the widespread use of plants as bioreactors remain to be addressed. Important factors include quality and homogeneity of the final product, the challenge of processing plant-derived pharmaceutical macromolecules under good manufacturing practice conditions and concerns about biosafety. Molecular farming in plants will only realize its huge potential if these constraints are removed through rigorous and detailed science-based studies.     

Mice with bad ends: mouse models for the study of telomeres and telomerase in cancer and aging

Telomeres are capping structures at the ends of eukaryotic chromosomes, which consist of repetitive DNA bound to an array of specialized proteins. Telomeres are part of the constitutive heterochromatin and are subjected to epigenetic modifications. The function of telomeres is to prevent chromosome ends from being detected as damaged DNA. Both the length of telomere repeats and the integrity of the telomere-binding proteins are important for telomere protection. Telomere length is regulated by telomerase, by the telomere-binding proteins, as well as by activities that modify the state of the chromatin. Various mouse models with altered levels of telomerase activity, or mutant for different telomere-binding proteins, have been recently generated. Here, I will discuss how these different mouse models have contributed to our understanding on the role of telomeres and telomerase in cancer and aging.     

大肠埃希氏杆菌UTI89分泌蛋白组的预测分析

目的：从大肠埃希氏杆菌UTI89基因组中筛选出全部潜在的分泌蛋白并进行初步研究。方法：使用SignalP3.0、TatP1.0、 SecretomeP2.0等蛋白分析软件对5211个ORF进行预测;对筛选出的信号肽及分泌蛋白的基本特征进行统计学分析;使用Blast 2 Sequences进行同源性分析。结果：共筛选出432个sec途径分泌蛋白,19个Tat途径分泌蛋白,386个非经典分泌蛋白;信号肽、分泌蛋白平均长度分别为25.5aa、282.8aa;信号肽中出现频率最高的3种氨基酸依次为L、A、S;仅有两个信号肽的氨基酸序列完全相同,相应的分泌蛋白高度同源。结论：大肠埃希氏杆菌UTI89基因组中有837个ORF可能编码分泌蛋白;分泌蛋白集中在500aa以下;组成信号肽的氨基酸相对保守,多数为疏水氨基酸;信号肽变异性较大,含相同信号肽的蛋白可能由同源基因编码。     

HUGE: a database for human large proteins identified in the Kazusa cDNA sequencing project

HUGE is a database for human large proteins newly identified in the Kazusa cDNA project, the aim of which is to predict the primary structure of proteins from the sequences of human large cDNAs (>4 kb). In particular, cDNA clones capable of coding for large proteins (>50 kDa) are the current targets of the project. HUGE contains >1100 cDNA sequences and detailed information obtained through analysis of the sequences of cDNAs and the predicted proteins. Besides an increase in the number of cDNA entries, the amount of experimental data for expression profiling has been largely increased and data on chromosomal locations have been newly added. All of the protein-coding regions were examined by GeneMark analysis, and the results of a motif/domain search of each predicted protein sequence against the Pfam database have been newly added. HUGE is available through the WWW at http://www.kazusa.or.jp/huge     

Protein strain in blue copper proteins studied by free energy perturbations.

Free energy perturbations have been performed on two blue copper proteins, plastocyanin and nitrite reductase. By changing the copper coordination geometry, force constants, and charges, we have estimated the maximum energy with which the proteins may distort the copper coordination sphere. By comparing this energy with the quantum chemical energy cost for the same perturbation on the isolated copper complex, various hypotheses about protein strain have been tested. The calculations show that the protein can only modify the copper-methionine bond length by a modest amount of energy-<5 kJ/mol-and they lend no support to the suggestion that the quite appreciable difference in the copper coordination geometry encountered in the two proteins is a result of the proteins enforcing different Cu-methionine bond lengths. On the contrary, this bond is very flexible, and neither the geometry nor the electronic structure change appreciably when the bond length is changed. Moreover, the proteins are rather indifferent to the length of this bond. Instead, the Cu(II) coordination geometries in the two proteins represent two distinct minima on the potential surface of the copper ligand sphere, characterized by different electronic structures, a tetragonal, mainly sigma-bonded, structure in nitrite reductase and a trigonal, pi-bonded, structure in plastocyanin. In vacuum, the structures have almost the same energy, and they are stabilized in the proteins by a combination of geometric and electrostatic interactions. Plastocyanin favors the bond lengths and electrostatics of the trigonal structure, whereas in nitrite reductase, the angles are the main discriminating factor. Proteins 1999;36:157-174.     

Finding function through structural genomics

The recent availability of whole-genome sequences and large numbers of protein-coding regions from high-throughput cDNA analysis has fundamentally changed experimental biology. These efforts have provided huge databases of protein sequences, many of which are of unknown function. Deciphering the functions of these myriad proteins presents a major intellectual challenge.     

微生物tRNA与密码子系统应用研究进展*

tRNA作为生命中心法则中翻译过程的重要参与分子,其种类、丰度都会对蛋白质的正常合成产生巨大影响。近年来通过对微生物tRNA的结构功能以及合成修饰过程的解析获得诸多启发,开展密码子扩展的研究,实现将非天然氨基酸引入特定位置从而获得新功能蛋白。同时,通过化学合成微生物基因组开展的密码子重编码工作将释放更多的密码子与tRNA用于更加广泛的密码子扩展研究。对微生物tRNA与密码子系统在合成生物学中的最新应用研究进展进行了综述,并讨论其未来的发展趋势。     

Nuclear pore structure and function

Nuclear pores are huge macromolecular assemblies, approximately 120 nm in diameter, that perforate the nuclear membrane and mediate nucleocytoplasmic transport. Nuclear pores are constructed from a cylindrical spoke-plug complex sandwiched between nucleoplasmic and cytoplasmic rings. The spoke-plug complex has pronounced 8-fold rotational symmetry, which is also present in the rings. Nucleocytoplasmic transport is an energy-requiring process that takes place through the centre of the pores and can accommodate particles up to about 25 nm diameter. Translocation is preceded by a separate binding step which does not require energy. Several nuclear pore proteins have been isolated and characterized. Many of these proteins contain O-linked N-acetyl glucosamine residues and may have similar modular domain structures.     

Kinetics of different processes in human insulin amyloid formation

Human insulin has long been known to form amyloid fibrils under given conditions. The molecular basis of insulin aggregation is relevant for modeling the amyloidogenesis process, which is involved in many pathologies, as well as for improving delivery systems, used for diabetes treatments. Insulin aggregation displays a wide variety of morphologies, from small oligomeric filaments to huge floccules, and therefore different specific processes are likely to be intertwined in the overall aggregation. In the present work, we studied the aggregation kinetics of human insulin at low pH and different temperatures and concentrations. The structure and the morphogenesis of aggregates on a wide range of length scales (from monomeric proteins to elongated fibrils and larger aggregates networks) have been monitored by using different experimental techniques: time-lapse atomic force microscopy (AFM), quasi-elastic light-scattering (QLS), small and large angle static light-scattering, thioflavin T fluorescence, and optical microscopy. Our experiments, along with the analysis of scattered intensity distribution, show that fibrillar aggregates grow following a thermally activated heterogeneous coagulation mechanism, which includes both tip-to-tip elongation and lateral thickening. Also, the association of fibrils into bundles and larger clusters (up to tens of microns) occurs simultaneously and is responsible for an effective lag-time.