Understanding protein trafficking in plant cells through proteomics

The functions of approximately one-third of the proteins encoded by the Arabidopsis thaliana genome are completely unknown. Moreover, many annotations of the remainder of the genome supply tentative functions, at best. Knowing the ultimate localization of these proteins, as well as the pathways used for getting there, may provide clues as to their functions. The putative localization of most proteins currently relies on in silico-based bioinformatics approaches, which, unfortunately, often result in erroneous predictions. Emerging proteomics techniques coupled with other systems biology approaches now provide researchers with a plethora of methods for elucidating the final location of these proteins on a large scale, as well as the ability to dissect protein-sorting pathways in plants.     

抗增殖蛋白在肿瘤发生发展过程中扮演多种角色

抗增殖蛋白（prohibitins,PHBs）是一类进化保守的重要蛋白质。哺乳动物细胞中,抗增殖蛋白家族含有2个同源亚型PHB1和PHB2。PHBs涉及多种细胞功能,包括细胞增殖、细胞迁移和细胞凋亡。PHBs的亚细胞定位不同决定其行使不同的功能。细胞膜上的PHBs能够调节膜运输,并与细胞增殖迁移相关。细胞核内的PHBs参与调控转录和细胞周期。线粒体内膜上的PHBs参与维持线粒体基因组和线粒体形态的稳定,并参与线粒体内的凋亡途径。另外,PHBs可以在细胞核和线粒体之间“穿梭”,是细胞核与线粒体交流的重要媒介。近年来,PHBs的研究不断深入,发现PHBs与多种肿瘤的发生和发展密切相关。本文以PHBs在肿瘤发生发展过程中扮演的角色为切入点,从蛋白质的结构和定位,在肿瘤的发生、发展、迁移和凋亡中的作用及其靶向药物几方面进行综述。进一步揭示PHBs在不同类型肿瘤发生发展进程中的分子机制,为开发新的高效的药物靶点奠定了理论基础。     

Discovery of novel pathways for carbohydrate metabolism

Closing the gap between the increasing availability of complete genome sequences and the discovery of novel enzymes in novel metabolic pathways is a significant challenge. Here, we review recent examples of assignment of in vitro enzymatic activities and in vivo metabolic functions to uncharacterized proteins, with a focus on enzymes and metabolic pathways involved in the catabolism and biosynthesis of monosaccharides and polysaccharides. The most effective approaches are based on analyses of sequence-function space in protein families that provide clues for the predictions of the functions of the uncharacterized enzymes. As summarized in this Opinion, this approach allows the discovery of the catabolism of new molecules, new pathways for common molecules, and new enzymatic chemistries.     

真核蛋白质的亚细胞位点预测研究进展

研究真核蛋白质的亚细胞位点是了解真核蛋白质功能,深入研究蛋白质相关信号通路内在机制的基础。同时,可以为了解 疾病发病机制及为新药研发提供帮助。因此,研究真核蛋白质的亚细胞位点意义十分重大。随着基因组测序的完成,真核蛋白质 序列信息增长迅速,为真核蛋白质亚细胞位点的研究提出了更多的挑战。传统的实验法难以满足蛋白质信息量迅速增长的需求。 而采用生物信息学手段处理大规模数据的计算预测方法,可在较短时间内获得大量真核蛋白质亚细胞位点信息,弥补了实验法 的不足。因此,运用计算预测法预测真核蛋白质的亚细胞位点成为生物信息学领域的研究热点之一。本文主要从提取真核蛋白质 的特征信息、计算预测方法及预测效果的评价三个方面,介绍近年来真核蛋白质亚细胞位点预测的研究进展。     

The intracellular distribution and function of the high mobility group chromosomal proteins

This brief review provides a framework for discussing current approaches being used to determine the cellular localization and function of the high mobility group chromosomal (HMG) proteins. The four main constituents of this group (HMG 1, 2, 14, 17) are present in all four eukaryotic kingdoms, have a relatively well conserved primary sequence and contain several functional domains which enable them to interact with DNA, histones and other components of the genome. The evolutionary conservation in the primary and tertiary structure as well as the observed correlations between cell phenotype and quantitative changes in protein levels and in post-synthesis modifications suggests that these proteins are components obligatory for proper cellular function. Proteins HMG 1, 2 are DNA-binding proteins which can distinguish between various types of single-stranded regions of the genome. Proteins HMG 14, 17 may be involved in maintaining specific chromatin regions in particular conformations. The data available presently suggests that these proteins are important structural elements of chromatin and chromosomes.     

Proteomic approaches for generating comprehensive protein interaction maps

The availability of complete genome sequences of numerous model organisms has initiated the development of new approaches in biological research to complement conventional biochemistry and genetics. In this context, high-throughput methods for detecting protein interactions, such as mass spectrometry and yeast two-hybrid assays, have produced vast amounts of data that can be exploited to infer protein function and regulation. In this review, we explore different genome-wide protein interaction studies and comment on their extrapolation towards understanding protein functions. It is likely that improvements of these approaches, together with more sophisticated databases and the invention of novel technologies, will help to decipher the complex interactions among proteins and to integrate interacting proteins into existing and novel cellular pathways.     

Genetics and genomics of root symbiosis.

Model genetics and genomics have been developed as tools for studying the third largest family of flowering plants, the Leguminosae, which includes important crop plants. Functional genomics strategies for the global analysis of gene expression, the elucidation of pathways and reverse genetics are established. These approaches provide new possibilities for investigating rhizobial as well as mycorrhizal endosymbiosis. Plant genes with central functions in these mutualistic interactions have been identified by positional cloning and gene tagging. With progress in Lotus japonicus genome sequencing, which was recently initiated by Japanese researchers, comparative genomics will contribute to our understanding of symbiosis, pathogenesis and the evolution of plant genomes.     

Localization of DNA polymerases eta and iota to the replication machinery is tightly co-ordinated in human cells

Y-family DNA polymerases can replicate past a variety of damaged bases in vitro but, with the exception of DNA polymerase eta (poleta), which is defective in xeroderma pigmentosum variants, there is little information on the functions of these polymerases in vivo. Here, we show that DNA polymerase iota (poliota), like poleta, associates with the replication machinery and accumulates at stalled replication forks following DNA-damaging treatment. We show that poleta and poliota foci form with identical kinetics and spatial distributions, suggesting that localization of these two polymerases is tightly co-ordinated within the nucleus. Furthermore, localization of poliota in replication foci is largely dependent on the presence of poleta. Using several different approaches, we demonstrate that poleta and poliota interact with each other physically and that the C-terminal 224 amino acids of poliota are sufficient for both the interaction with poleta and accumulation in replication foci. Our results provide strong evidence that poleta targets poliota to the replication machinery, where it may play a general role in maintaining genome integrity as well as participating in translesion DNA synthesis.     

Proteomic and functional analysis of the mitotic Drosophila centrosome

Regulation of centrosome structure, duplication and segregation is integrated into cellular pathways that control cell cycle progression and growth. As part of these pathways, numerous proteins with well‐established non‐centrosomal localization and function associate with the centrosome to fulfill regulatory functions. In turn, classical centrosomal components take up functional and structural roles as part of other cellular organelles and compartments. Thus, although a comprehensive inventory of centrosome components is missing, emerging evidence indicates that its molecular composition reflects the complexity of its functions. We analysed the Drosophila embryonic centrosomal proteome using immunoisolation in combination with mass spectrometry. The 251 identified components were functionally characterized by RNA interference. Among those, a core group of 11 proteins was critical for centrosome structure maintenance. Depletion of any of these proteins in Drosophila SL2 cells resulted in centrosome disintegration, revealing a molecular dependency of centrosome structure on components of the protein translation machinery, actin‐ and RNA‐binding proteins. In total, we assigned novel centrosome‐related functions to 24 proteins and confirmed 13 of these in human cells.     

Localization of DNA polymerases eta and iota to the replication machinery is tightly co-ordinated in human cells

Y-family DNA polymerases can replicate past a variety of damaged bases in vitro but, with the exception of DNA polymerase eta (poleta), which is defective in xeroderma pigmentosum variants, there is little information on the functions of these polymerases in vivo. Here, we show that DNA polymerase iota (poliota), like poleta, associates with the replication machinery and accumulates at stalled replication forks following DNA-damaging treatment. We show that poleta and poliota foci form with identical kinetics and spatial distributions, suggesting that localization of these two polymerases is tightly co-ordinated within the nucleus. Furthermore, localization of poliota in replication foci is largely dependent on the presence of poleta. Using several different approaches, we demonstrate that poleta and poliota interact with each other physically and that the C-terminal 224 amino acids of poliota are sufficient for both the interaction with poleta and accumulation in replication foci. Our results provide strong evidence that poleta targets poliota to the replication machinery, where it may play a general role in maintaining genome integrity as well as participating in translesion DNA synthesis.     

Chloroplast proteomics: potentials and challenges

With the available Arabidopsis genome and near-completion of the rice genome sequencing project, large-scale analysis of plant proteins with mass spectrometry has now become possible. Determining the proteome of a cell is a challenging task, which is complicated by proteome dynamics and complexity. The biochemical heterogeneity of proteins constrains the use of standardized analytical procedures and requires demanding techniques for proteome analysis. Several proteome studies of plant cell organelles have been reported, including chloroplasts and mitochondria. Chloroplasts are of particular interest for plant biologists because of their complex biochemical pathways for essential metabolic functions. Information from the chloroplast proteome will therefore provide new insights into pathway compartmentalization and protein sorting. Some approaches for the analysis of the chloroplast proteome and future prospects of plastid proteome research are discussed here.     

Arrayed adenoviral expression libraries for functional screening

With the publication of the sequence of the human genome, we are challenged to identify the functions of an estimated 70,000 human genes and the much larger number of proteins encoded by these genes. Of particular interest is the identification of gene products that play a role in human disease pathways, as these proteins include potential new targets that may lead to improved therapeutic strategies. This requires the direct measurement of gene function on a genomic scale in cell-based, functional assays. We have constructed and validated an individually arrayed, replication-defective adenoviral library harboring human cDNAs, termed PhenoSelect library. The adenoviral vector guarantees efficient transduction of diverse cell types, including primary cells. The arrayed format allows screening of this library in a variety of cellular assays in search for gene(s) that, by overexpression, induce a particular disease-related phenotype. The great majority of phenotypic assays, including morphological assays, can be screened with arrayed libraries. In contrast, pooled-library approaches often rely on phenotype-based isolation or selection of single cells by employing a flow cytometer or screening for cell survival. An arrayed placental PhenoSelect library was screened in cellular assays aimed at identifying regulators of osteogenesis, metastasis, and angiogenesis. This resulted in the identification of known regulators, as well as novel sequences that encode proteins hitherto not known to play a role in these pathways. These results establish the value of the PhenoSelect platform, in combination with cellular screens, for gene function discovery.