Application of proteomics to investigate stress-induced proteins for improvement in crop protection

Proteomics has contributed to defining the specific functions of genes and proteins involved in plant–pathogen interactions. Proteomic studies have led to the identification of many pathogenicity and defense-related genes and proteins expressed during phytopathogen infections, resulting in the collection of an enormous amount of data. However, the molecular basis of plant–pathogen interactions remains an intensely active area of investigation. In this review, the role of differential analysis of proteins expressed during fungal, bacterial, and viral infection is discussed, as well as the role of JA and SA in the production of stress related proteins. Resistance acquired upon induction of stress related proteins in intact plant leaves is mediated by potentiation of pathogens via signal elicitors. Stress related genes extensively used in biotechnology had been cited. Stress related proteins identified must be followed through for studying the molecular mechanism for plant defense against pathogens.     

From proteins to proteomics

During the second half of the 20th century, biochemistry and subsequently molecular biology blossomed into the core upon which all biological and biomedical sciences now depend. A major part of these closely related disciplines has been the study of the structure and function of proteins and the diverse biological functions that they perform. Early experimentation necessarily focused on individual entities, selected mainly for their activities, but as technology improved there developed a tendency to look at proteins as larger, interactive groups or clusters. Spurred by the recent exponential production of genomic sequence data for a rapidly increasing number of species, protein chemistry has now evolved into a new discipline, proteomics. In addition to embracing the methods and approaches that have served protein scientists well in the past, it includes, and is perhaps best defined by, high-throughput analyses based in large part on 2D gel electrophoresis, MALDI and ESI mass spectrometry and combinatorial arrays. Proteomic targets include the identification of all genome products and a mapping of their interactions and expression profiles. These hold great promise for the identification of disease markers and drug targets, but are not without their challenges and pitfalls.     

A proteomics approach to the identification of mammalian mitochondrial small subunit ribosomal proteins

Mammalian mitochondrial small subunit ribosomal proteins were separated by two-dimensional polyacrylamide gel electrophoresis. The proteins in six individual spots were subjected to in-gel tryptic digestion. Peptides were separated by capillary liquid chromatography, and the sequences of selected peptides were obtained by electrospray tandem mass spectrometry. The peptide sequences obtained were used to screen human expressed sequence tag data bases, and complete consensus cDNAs were assembled. Mammalian mitochondrial small subunit ribosomal proteins from six different classes of ribosomal proteins were identified. Only two of these proteins have significant sequence similarities to ribosomal proteins from prokaryotes. These proteins correspond to Escherichia coli S10 and S14. Homologs of two human mitochondrial proteins not found in prokaryotes were observed in the genomes of Drosophila melanogaster and Caenorhabditis elegans. A homolog of one of these proteins was observed in D. melanogaster but not in C. elegans, while a homolog of the other was present in C. elegans but not in D. melanogaster. A homolog of one of the ribosomal proteins not found in prokaryotes was tentatively identified in the yeast genome. This latter protein is the first reported example of a ribosomal protein that is shared by mitochondrial ribosomes from lower and higher eukaryotes that does not have a homolog in prokaryotes.     

Targeting of the mitochondrial membrane proteins to the cell surface for functional studies

Studying mitochondrial membrane proteins for ion or substrate transport is technically difficult, as the organelles are hidden within the cell interior and thus inaccessible to many conventional nondisruptive techniques. This technical barrier can potentially be overcome if the mitochondrial membrane proteins are targeted to the cell surface, where they can be more readily studied. We undertook experiments presented here to target two related mitochondrial membrane proteins, mitochondrial ATP-binding cassette-1 and -2 protein (mABC1 and mABC2, respectively) to the cell surface for functional studies. These two proteins have an N-terminal mitochondrial targeting signal (MTS), and we hypothesized that removal of this sequence or addition of a cell surface targeting signal would lead to cell membrane targeting of these proteins. When the MTS was removed from mABC1, it localized to intracellular secretory compartments as well as the plasma membrane. However, truncated mABC2 lacking the MTS aggregated inside the cell. Addition of a cell membrane signal sequence or the transmembrane domain from CD8 to the N-terminus of mABC1 or mABC2 resulted in similar subcellular localizations. We then performed patch clamp on cells expressing mABC1 on their surface. These cells exhibited nonselective transport of K(+) and Na(+) ions and resulted in the loss of membrane potential. Our findings open new ways to study mitochondrial membrane proteins in established cell culture systems by targeting them to the cell surface, where they can more reliably be studied using various molecular and cellular techniques.     

Targeting of proteins to mitochondria

A clear picture has emerged over the past years on how a 'classic' mitochondrial protein, like subunit IV of cytochrome c oxidase, might be targeted to mitochondria. The targeting and subsequent import process involves the commitment of the TOM (translocase in the outer mitochondrial membrane) receptor complex on the mitochondrial surface, a TIM (translocase in the inner mitochondrial membrane) translocation complex in the mitochondrial inner membrane, and assorted chaperones and processing enzymes within the organelle. Recent work suggests that while very many mitochondrial precursor proteins might follow this basic targeting pathway, a large number have further requirements if they are to be successfully imported. These include ribosome-associated factors and soluble factors in the cytosol, soluble factors in the mitochondrial intermembrane space, an additional TIM translocase in the inner membrane and a range of narrow specificity assembly factors in the inner membrane. This review is focused on the targeting of proteins up to the stage at which they enter the TOM complex in the outer membrane.     

Finding the right organelle. Targeting signals in mitochondrial outer-membrane proteins

The mitochondrial outer membrane contains a diverse set of proteins that includes enzymes, components of the preprotein translocation machinery, pore-forming proteins, regulators of programmed cell death, and those that control the morphology of the organelle. All these proteins, like the vast majority of mitochondrial proteins, are encoded in the nucleus, so they are synthesized in the cytosol and contain signals that are essential for their subsequent import into mitochondria. This review summarizes our current knowledge of the signals that target mitochondrial outer-membrane proteins to their correct intracellular location. In addition, the mechanisms by which these signals are decoded by the mitochondria are discussed.     

线粒体蛋白质组学

线粒体作为真核细胞内一种重要的细胞器,在许多生理病理过程中发挥重要作用。蛋白质组学研究技术的不断发展推动了线粒体蛋白质组的研究。本文对近年来线粒体蛋白质组学的研究现状、存在的影响因素及发展前景进行了综述。     

Targeting of proteins to the Golgi apparatus

The Golgi apparatus maintains a highly organized structure in spite of the intense membrane traffic which flows into and out of this organelle. Resident Golgi proteins must have localization signals to ensure that they are targeted to the correct Golgi compartment and not swept further along the secretory pathway. There are a number of distinct groups of Golgi membrane proteins, including glycosyltransferases, recyclingtrans-Golgi network proteins, peripheral membrane proteins, receptors and viral glycoproteins. Recent studies indicate that there are a number of different Golgi localization signals and mechanisms for retaining proteins to the Golgi apparatus. This review focuses on the current knowledge in this field.     

Targeting of proteins to the Golgi apparatus

 The proteins that reside in the Golgi carry out functions associated with post-translational modifications, including glycosylation and proteolytic processing, membrane transport, recycling of endoplasmic reticulum proteins and maintenance of the structural organisation of the organelle itself. The latter includes Golgi stacking, interconnections between stacks and the microtubule-dependent positioning of the organelle within the cell. There are a number of distinct groups of Golgi membrane proteins, including glycosyltransferases, recycling trans-Golgi network (TGN) proteins, peripheral membrane proteins and receptors. Considerable effort has been directed at understanding the basis of the localisation of Golgi glycosyltransferases and recycling TGN proteins; in both cases there is increasing evidence that multiple signals may be involved in their specific localisation. A number of models for the Golgi retention of glycosyltransferases have been proposed including oligomerisation, lipid-mediated sorting and intra-Golgi retrograde transport. More information is required to determine the contribution of each of these potential mechanisms in the targeting of different glycosyltransferases. Future work is also likely to focus on the relationship between the localisation of resident Golgi proteins and the maintenance of Golgi structure. Accepted: 15 October 1997     

Ternary solvents to investigate proteins at zub-zero temperatures.

Mixtures of water, ethylene glycol and methanol in different volume ratios have been selected to carry out kinetics of enzyme reactions at sub-zero temperatures with the intention to reduce maximally the viscosity. Density, viscosity and dielectric constant values of these mixtures as a function of temperature are reported, as well as values of the protonic activity of several buffers under such conditions. A procedure to avoid or delay the eventual damaging effect of methanol on proteins is described.     

线粒体蛋白质组学研究进展

线粒体是真核生物中重要的细胞器,其包含的全部蛋白质称为线粒体蛋白质组。人类线粒体大约包含1500多种蛋白质,由核基因和线粒体基因共同编码。线粒体是细胞能量合成和物质代谢的中心,其功能障碍将直接或问接引起许多疾病。目前线粒体蛋白质组学正是系统性地研究线粒体在生理、病理过程中的功能变化以及研究疾病发生机制的重要方法。将线粒体蛋白质组的研究方法、研究进展、线粒体蛋白质组的性质及其在相关疾病研究中的作用进行综述,并对线粒体蛋白质组学在疾病发生机制和诊断治疗中的发展前景进行展望。     

Functional proteomics approach to investigate the biological activities of cDNAs implicated in breast cancer

Functional proteomics approaches that comprehensively evaluate the biological activities of human cDNAs may provide novel insights into disease pathogenesis. To systematically investigate the functional activity of cDNAs that have been implicated in breast carcinogenesis, we generated a collection of cDNAs relevant to breast cancer, the Breast Cancer 1000 (BC1000), and conducted screens to identify proteins that induce phenotypic changes that resemble events which occur during tumor initiation and progression. Genes were selected for this set using bioinformatics and data mining tools that identify genes associated with breast cancer. Greater than 1000 cDNAs were assembled and sequence verified with high-throughput recombination-based cloning. To our knowledge, the BC1000 represents the first publicly available sequence-validated human disease gene collection. The functional activity of a subset of the BC1000 collection was evaluated in cell-based assays that monitor changes in cell proliferation, migration, and morphogenesis in MCF-10A mammary epithelial cells expressing a variant of ErbB2 that can be inducibly activated through dimerization. Using this approach, we identified many cDNAs, encoding diverse classes of cellular proteins, that displayed activity in one or more of the assays, thus providing insights into a large set of cellular proteins capable of inducing functional alterations associated with breast cancer development.     

Use of functional proteomics to investigate PKC epsilon-mediated cardioprotection: the signaling module hypothesis

The characterization of biological processes on the basis of alterations in the cellular proteins, or "proteomic" analysis, is a powerful approach that may be adopted to decipher the signaling mechanisms that underlie various pathophysiological conditions, such as ischemic heart disease. This review represents a prospectus for the implementation of proteomic analyses to delineate the myocardial intracellular signaling events that evoke cardioprotection against ischemic injury. In concert with this, the manifestation of a protective phenotype has recently been shown to involve dynamic modulation of protein kinase C-epsilon (PKC epsilon) signaling complexes (Ping P, Zhang J, Pierce WM Jr, and Bolli R. Circ Res 88: 59--62, 2001). Accordingly, "the signaling module hypothesis" is formulated as a plausible mechanism by which multipurpose stress-activated proteins and signaling kinases may function collectively to facilitate the genesis of cardioprotection.     

Identification of proteins binding to decursinol by chemical proteomics

Decursinol, found in the roots of Angelica gigas Nakai, has been traditionally used to treat anemia and other various diseases. Recently, numerous biological activities such as cytotoxic effect on leukemia cells, and antitumor, neuroprotection, and antibacterial activities have been reported for this compound. Although a number of proteins including protein kinase C, androgen receptor, and acetylcholinesterase were proposed as molecular targets responsible for the activities of decursinol, they are not enough to explain such a diverse biological activity mentioned above. In this study, we employed a chemical proteomic approach, leading to identification of seven proteins as potential proteins interacting with decursinol. Most of the proteins contain a defined ATP or nucleic acid binding domain and have been implied to be involved in the pathogenesis and progression of various human diseases including cancer, autoimmune disorders, or neurodegenerative diseases. The present results may provide clues to understand the molecular mechanism of the biological activities shown by decursinol, an anticancer natural product.     

Membrane proteins and membrane proteomics

Biological membranes form an essential barrier between living cells and their external environments, as well as serve to compartmentalize intracellular organelles within eukaryotes. The latter includes membranes that envelope the nucleus, the outer and inner membranes of the mitochondria, membrane cisternae complex of the ER, Golgi apparatus, as well as lysosomes and secretory vesicles. Depending on their localizations in the whole organism and also within the cell, these membranes have different, highly specialized functions. Although 30% of naturally occurring proteins are predicted to be embedded in biological membranes, membrane proteomics is traditionally understudied due to difficulties in solubilizing, separating, and identifying membrane proteins. Given the importance of membrane proteins in the various cellular processes listed in this review, as well as the roles they play in diseases and their potential as drug targets, it is imperative that this class of proteins be better studied. With the recent advancement in technology, it is expected that some of the difficulties in membrane proteomics will be overcome, yielding new data on membrane proteins.