Proteomic studies in biomedically and industrially relevant fungi

Historically, the proteomic investigation of filamentous fungi has been restrained by difficulties associated with efficient protein extraction and the lack of extensive fungal genome sequence databases. The advent of robust protein extraction and separation technologies, combined with protein mass spectrometry and emerging genome sequence data, is leading to the emergence of extensive new knowledge on the nature of these organisms. In this review, we discuss some recent technological advances and their role in exploring the proteome of Aspergillus spp., along with other biotechnologically relevant fungi.     

Proteomic approach to characterize mitochondrial complex I from plants

Mitochondrial NADH dehydrogenase complex (complex I) is by far the largest protein complex of the respiratory chain. It is best characterized for bovine mitochondria and known to consist of 45 different subunits in this species. Proteomic analyses recently allowed for the first time to systematically explore complex I from plants. The enzyme is especially large and includes numerous extra subunits. Upon subunit separation by various gel electrophoresis procedures and protein identifications by mass spectrometry, overall 47 distinct types of proteins were found to form part of Arabidopsis complex I. An additional subunit, ND4L, is present but could not be detected by the procedures employed due to its extreme biochemical properties. Seven of the 48 subunits occur in pairs of isoforms, six of which were experimentally proven. Fifteen subunits of complex I from Arabidopsis are specific for plants. Some of these resemble enzymes of known functions, e.g. carbonic anhydrases and l-galactono-1,4-lactone dehydrogenase (GLDH), which catalyzes the last step of ascorbate biosynthesis. This article aims to review proteomic data on the protein composition of complex I in plants. Furthermore, a proteomic re-evaluation on its protein constituents is presented.     

Proteomic changes in rice leaves during development of field-grown rice plants

Of the numerous factors affecting rice yield, how solar radiation is transformed into biomass through rice leaves is the most important. We have analyzed proteomic changes in rice leaves collected from six different developing stages (vegetative to ripening). We studied protein expression profiles of rice leaves by running two-dimensional gel electrophoresis. Differential protein expression among the six phases were analyzed by image analysis, which allowed the identification of 49 significantly different gel spots. The spots were further verified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry, in which 89.8% of them were confirmed to be rice proteins. Finally, we confirmed some of the interesting rice proteins by immunoblotting. Three major conclusions can be drawn from these experimental results. (i) Protein expression in rice leaves, at least for high or middle abundance proteins, is attenuated during growth (especially some chloroplast proteins). However, the change is slow and the expression profiles are relatively stable during rice development. (ii) Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), a major protein in rice leaves, is expressed at constant levels at different growth stages. Interestingly, a high ratio of degradation of the RuBisCO large subunit was found in all samples. This was confirmed by two approaches, mass spectrometry and immunoblotting. The degraded fragments are similar to other digested products of RuBisCO mediated by free radials. (iii) The expression of antioxidant proteins such as superoxide dismutase and peroxidase decline at the early ripening stage.     

Proteomic approach to characterize the supramolecular organization of photosystems in higher plants

A project to investigate the supramolecular structure of photosystems was initiated, which is based on protein solubilizations by digitonin, protein separations by Blue native (BN)-polyacrylamide gel electrophoresis (PAGE) and protein identifications by mass spectrometry (MS). Under the conditions applied, nine photosystem supercomplexes could be described for chloroplasts of Arabidopsis, which have apparent molecular masses between 600 and 3200 kDa on BN gels. Identities of the supercomplexes were determined on the basis of their subunit compositions as documented by 2D BN/SDS-PAGE and BN/BN-PAGE. Two supercomplexes of 1060 and approximately 1600 kDa represent dimeric and trimeric forms of photosystem I (PSI), which include tightly bound LHCI proteins. Compared to monomeric PSI, these protein complexes are of low abundance. In contrast, photosystem II mainly forms part of dominant supercomplexes of 850, 1000, 1050 and 1300 kDa. According to our interpretation, these supercomplexes contain dimeric PSII, 1-4 LHCII trimers and additionally monomeric LHCII proteins. The 1300-kDa PSII supercomplex (containing four LHCII trimers) is partially converted into the 1000-kDa PSII supercomplex (containing two LHCII trimers) in the presence of dodecylmaltoside on 2D BN/BN gels. Analyses of peptides of the trypsinated 1300-kDa PSII supercomplex by mass spectrometry allowed to identify known subunits of the PSII core complex and additionally LHCII proteins encoded by eight different genes in Arabidopsis. Further application of this experimental approach will allow new insights into the supermolecular organization of photosystems in plants.     

Proteomic studies of the intrinsically unstructured mammalian proteome

Intrinsically unstructured proteins (IUPs) represent an important class of proteins primarily involved in cellular signaling and regulation. The aim of this study was to develop methodology for the enrichment and identification of IUPs. We show that heat treatment of NIH3T3 mouse fibroblast cell extracts at 98 degrees C selects for IUPs. The majority of these IUPs were cytosolic or nuclear proteins involved in cell signaling or regulation. These studies represent the first large-scale experimental investigation of the intrinsically unstructured mammalian proteome.     

Proteomic studies of lipopolysaccharide-induced polypeptides in the silkworm, Bombyx mori

Silkworm larvae at the 5th instar were injected with lipopolysaccharide from Escherichia coli and inducible polypeptides were examined within a pI range of 3-10 and a size range of 14-97 kDa by proteomics, including peptide mass fingerprinting. No polypeptides were induced in the midgut. FB1 and H1-4 polypeptides were significantly induced in fat body and hemolymph, respectively. FB1 and H1 were estimated to be antitrypsin and serpin-2 proteinase inhibitors respectively. H2 and H3 were novel polypeptides. H4 was estimated to be attacin antibacterial polypeptide with high coverage of sequence. The amounts of all the induced polypeptides decreased at 48 h after the injection.     

Proteomic studies on the white matter of human brain

Limited information on the protein expression profiles of the different components of mammalian brain is available to date. In the present study, proteomic analysis was performed on 32 white matter samples obtained from 8 different regions of brains of four post mortem cases. Proteins were separated by 2D gel electrophoresis and identified by mass spectrometry. Most of the protein spots (98%) are reproducibly present in all the samples analyzed. A total of 64 different proteins were identified and divided into seven functional groups. These include metabolic proteins (33%), structural proteins (9%), proteins involved in signal transduction (9%), blood proteins (8%), stress related proteins (23%), and proteins involved in the ubiquitin mediated proteolysis (6%). This protein database obtained from the white matter of human brain contributes to deepen our knowledge on the molecular mechanisms that control several pathologies affecting this key component of the brain.     

Proteomic studies of human and other vertebrate muscle proteins

This review summarizes results of some systemic studies of muscle proteins of humans and some other vertebrates. The studies, started after introduction of two-dimensional gel electrophoresis of OFarrell, were significantly extended during development of proteomics, a special branch of functional genomics. Special attention is paid to analysis of characteristic features of strategy for practical realization of the systemic approach during three main stages of these studies: pre-genomic, genomic (with organizational registration of proteomics), and post-genomic characterized by active use of structural genomics data. Proteomic technologies play an important role in detection of changes in isoforms of various muscle proteins (myosins, troponins, etc.). These changes possibly reflecting tissue specificity of gene expression may underline functional state of muscle tissues under normal and pathological conditions, and such proteomic analysis is now used in various fields of medicine.Translated from Biokhimiya, Vol. 69, No. 11, 2004, pp. 1574–1591.Original Russian Text Copyright © 2004 by Shishkin, Kovalyov, Kovalyova     

Combining proteomic and genetic studies in plants

Plant proteomics is still in its infancy, although numerous experiments have been undertaken since the end of the 1970s. In this review we focus on the interactions between proteomics and genetics. A given genome can express various proteomes according to differentiation, development, tissues, cells and subcellular compartments, and proteomes are modified in function of biotic and abiotic environment. These different proteomes and the way they respond to environment can be compared between genotypes, allowing the characterization of mutants or lines, the study of mutation pleiotropic effects, the genetic mapping of expressed genes. These comparisons also permit to hypothesize for "candidate proteins" that might be involved in the genetic variation of traits of economic or agronomic interest.     

食虫植物研究的现状和趋势

综述了食虫植物的研究概况 ,包括研究历史、食虫植物的捕虫与消化机制 ,食虫的效果以及食虫植物与环境的生态关系 ,旨在促进我国关于食虫植物的研究     

Proteomics studies of post-translational modifications in plants

Post-translational modifications of proteins greatly increase protein complexity and dynamics, co-ordinating the intricate regulation of biological events. The global identification of post-translational modifications is a difficult task that is currently accelerated by advances in proteomics techniques. There has been significant development in sample preparation methods and mass spectrometry instrumentation. To reduce the complexity and to increase the amount of modified proteins available for analysis, proteins are usually subjected to prefractionation such as chromatographic purification and affinity enrichment. In this review, the post-translational modification studies in plants are summarized. The sample preparation strategies applied to each study are also described. These include affinity-based enrichment methods, immobilized metal affinity chromatography and immunoprecipitation used for phosphorylation and ubiquitination studies, respectively, and the phase partitioning approach for glycosylphosphatidylinositol modification studies.     

Eco-physiological studies on desert plants

Summary The O₂ uptake and RQ of germinating negatively photoblastic Zygophyllum coccineum seeds were studied during the first fourty hours after soaking in water under dark or light conditions. Five phases could be distinguished in the course of O₂ uptake in water in dark. The respiration course in light did not differ from that in dark till the 12th hour after soaking, then it deviated showing a low level, The RQ values in dark and light are nearly identical; both had sharp decline between the 8th and the 16th hour after soaking. Light, by blocking germination, affected the O₂ uptake.Moisture stress simulated by mannitol solutions caused a decrease in the O₂ uptake of seeds and seedlings. Na₂SO₄ caused a reduction and a lag in the time of maximum O₂ uptake. NaCl showed a particular effect on respiration of germinating seeds causing earlier rise and decrease in O₂ uptake than in water. NaCl was effective in rising the O₂ uptake of seedlings specially those supplied with glucose. Its effect was not pronounced on starved seedlings. O₂ uptake of seedlings germinated in NaCl was considerably high when compared with that of seeds germinating in the same medium 40 hours after soaking.The decreasing effect of moisture stress caused by mannitol on O₂ uptake was reversed by diluting the medium or replacing it with water. Values of O₂ uptake on number of seedlings basis are different from those calculated on fresh weight basis due to the difference in water content of seedlings germinated under various conditions.     

Eco-physiological studies on desert plants

Summary The Egyptian Zygophyllum species, though limited in number, contribute in considerable measure to the desert vegetation. They represent a group of succulent plants which are drought resistant or salt tolerant living under severe dry climatic conditions. Their abundance is attributed to these characteristics in addition to their unpalatibility.The distribution of the studied species is deduced from the data collected by the present authors and others working in the Egyptian desert. The growth and distribution of some Zygophyllum species are dependant on the chemical nature of the substratum. Z. occineum is a widespread species in the limestone territories of the Eastern desert. Z. decumbens is confined to a limited area within the borders of the area inhabited by Z. coccineum. Z. album is a salt tolerant plant with wide geographical range growing in dry littoral or inland salt marshes. Z. simplex, the only ephemeral species, has wide geographical and ecological ranges. The soil carbonate content does not exercise any significant effect on its distribution though it is more ecologically related to Z. occineum than the other species. Z. decumbens is ecologically related to Z. coccineum, while Z. album has its own ecological amplitude.     

Further studies of drought resistance in woody plants

This publication is an extension of the 1956 review of drought resistance in woody plants. The more important recently used terms are listed and defined. Summaries of reports of drought effects on forest trees are presented in tabular form. Frost-drought is discussed as an important factor in the limitation of woody plants’ geographical ranges. The role that droughts play in the initiation of tree diseases is considered, especially in the light of new evidence from studies of diebacks-declines of forest trees. Interactions between soil moisture and transpiration are discussed as well as ways to control transpiration in large stands. Also considered are drought experiments with tree seedlings and symptomatology of drought injury. The means by which woody plants survive drought (excluding ephemerals) are discussed.     

Phylogeographic studies in plants: problems and prospects

Genetic structuring of plant populations is strongly influenced by both common ancestry and current patterns of interpopulation genetic exchange. The interaction of these two forces is particularly confounding and hence interesting in plants. This complexity of plant genetic structures is due in part to a diversity of reproductive ecologies affecting genetic exchange and the fact that reproductive barriers are often weak between otherwise morphologically well-defined species. Phylogeographic methods provide a means of examining the history of genetic exchange among populations, with the potential to distinguish biogeographic patterns of genetic variation caused by gene flow from those caused by common ancestry. With regard to plants, phylogeography will be most useful when applied broadly across the entire spectrum of potential genetic exchange. Although current phylogeographic studies of plants show promise, widespread application of this approach has been hindered by a lack of appropriate molecular variation; this problem is discussed and possible solutions considered.