Stress-related RNase PR-10c is post-translationally modified by glutathione in birch

The PR‐10c (previously termed as Bet v 1‐Sc3) protein of birch belongs to the family of intracellular pathogenesis‐related proteins. The high‐performance liquid chromatography electrospray ionization ion trap mass spectrometry (HPLC‐ESI‐MS) analysis of PR‐10c‐His fusion protein, produced in Escherichia coli, revealed three major peaks and masses. Enzymatic digestions and HPLC‐ESI‐MS and matrix assisted laser desorption/ionization – time of flight mass spectrometry (MALDI‐TOF‐MS) analyses of each fraction indicated that PR‐10c‐His protein is post‐translationally modified by carbamylation and S‐glutathiolation. Carbamylation was localized into the N‐terminal end of PR‐10c‐His and does not represent a biologically significant modification. The possible nuclease activity of PR‐10c was analysed with S‐glutathiolated and reduced fractions of PR‐10c‐His fusion protein. Both forms of PR‐10c‐His as well as the dimeric form of the protein possess RNase activity which is capable of digesting different RNA substrates. None of the fractions showed activity against single‐ or double‐stranded DNA. The MALDI‐TOF‐MS analysis of PR‐10c polypeptide extracted from zinc‐exposed birch roots showed that the protein is post‐translationally modified by glutathione (γ‐Glu‐Cys‐Gly) also in vivo. The S‐glutathiolated cysteine residue of PR‐10c is not conserved among Bet v 1 homologous proteins and is also unique in the PR‐10 family. As far as we know this is the first observation of S‐glutathiolation in plants, or any post‐translational modification in the PR‐10 family of proteins.     

Molecular Characterization of the Rice Pathogenesis-related Protein, OsPR-4b, and Its Antifungal Activity Against Rhizoctonia solani

We cloned and identified a new rice pathogenesis‐related (PR)‐4 gene, OsPR‐4b. OsPR‐4b encodes a 151 amino acid protein with a predicted molecular mass of 16.47 kDa and pI of 4.42. The putative OsPR‐4b shows high similarity to PR‐4 type proteins from various plant species and belongs to the Barwin family. Like other PR‐4s from monocot plants, OsPR‐4b contains a conserved Barwin domain and has a signal peptide at its N‐terminus. Recombinant OsPR‐4b protein expressed in Escherichia coli showed antifungal activity in vitro against the sheath blight fungus, Rhizoctonia solani. The results suggest that the OsPR‐4b may play a role in the disease resistance responses of rice against pathogen attacks through its antifungal activity.     

Proteomics in the liver of gilthead sea bream (Sparus aurata) to elucidate the cellular response induced by the intake of maslinic acid

Maslinic acid (MA) is a pentacyclic triterpene used as a feed additive to stimulate growth, protein‐turnover rates, and hyperplasia in fish. To further our understanding of cellular mechanisms underlying the action of MA, we have used 2‐DE coupled with MS to identify proteins differentially expressed in the livers of juvenile gilthead sea bream (Sparus aurata) grown under fish‐farm conditions and fed with a 100 mg/kg MA‐enriched diet (MA₁₀₀). After the comparison of the protein profiles from MA₁₀₀ fed fish and from control, 49 protein spots were found to be altered in abundance (≥2‐fold). Analysis by MALDI‐TOF/TOF allowed the unambiguous identification of 29 spots, corresponding to 19 different proteins. These proteins were: phosphoglucomutase, phosphoglucose isomerase, S‐adenosyl methionine‐dependent methyltransferase class I, aldehyde dehydrogenase, catalase, 6‐phosphogluconate dehydrogenase, fumarylacetoacetate hydrolase, 4‐hydroxyphenylpyruvic dioxygenase, methylmalonate‐semialdehyde dehydrogenase, lysozyme, urate oxidase, elongation factor 2, 60 kDa heat‐shock protein, 58 kDa glucose‐regulated protein, cytokeratin E7, type‐II keratin, intermediate filament proteins, 17‐β‐hydroxysteroid dehydrogenase type 4, and kinase suppressor of Ras1. Western blot analysis of kinase suppressor of Ras1, glucose 6‐phosphate dehydrogenase, elongation factor 2, 60 kDa heat‐shock protein, and catalase supported the proteome evidence. Based on the changes found in the protein‐expression levels of these proteins, we proposed a cellular‐signalling pathway to explain the hepatic‐cell response to the intake of a diet containing MA.     

The effect of simvastatin on the proteome of detergent‐resistant membrane domains: Decreases of specific proteins previously related to cytoskeleton regulation,calcium homeostasis and cell fate

Cell death induced by over‐activation of glutamate receptors occurs in different neuropathologies. Cholesterol depletors protect from neurotoxic over‐activation of glutamate receptors, and we have recently reported that this neuroprotection is associated with a reduction of the N‐methyl‐D ‐aspartate subtype of glutamate receptors in detergent‐resistant membrane domains (DRM). In the present study we used comparative proteomics to further identify which proteins, besides the N‐methyl‐D ‐aspartate receptor, change its percentage of association to DRM after treatment of neurons with simvastatin. We detected 338 spots in neuronal DRM subjected to 2‐DE; eleven of these spots changed its intensity after treatment with simvastatin. All 11 differential spots showed reduced intensity in simvastatin‐treated samples and were identified as adipocyte plasma membrane associated protein, enolase, calretinin, coronin 1a, f‐actin capping protein α1, f‐actin capping protein α2, heat shock cognate protein 71, malate dehydrogenase, n‐myc downregulated gene 1, prohibitin 2, Rab GDP dissociation inhibitor, translationally controlled tumor protein and voltage dependent anion selective channel protein 1. The proteins tested colocalized with the lipid raft marker caveolin‐1. Interestingly, the proteins we have identified in the present study had been previously reported to play a role in cell fate and, thus, they might represent novel targets for neuroprotection.     

An immunoinformatics approach to define T cell epitopes from polyketide and non‐ribosomal peptide synthesis proteins of Mycobacterium tuberculosis as potential vaccine candidates

The role of polyketide and non‐ribosomal proteins from the class of small molecule metabolism of Mycobacterium tuberculosis is well documented in envelope organization, virulence, and pathogenesis. Consequently, the identification of T cell epitopes from these proteins could serve to define potential antigens for the development of vaccines. Fourty‐one proteins from polyketide and non‐ribosomal peptide synthesis of small molecule metabolism proteins of M tuberculosis H37Rv were analyzed computationally for the presence of HLA class I binding nanomeric peptides. All possible overlapping nanomeric peptide sequences from 41 small molecule metabolic proteins were generated through in silico and analyzed for their ability to bind to 33 alleles belonging to A, B, and C loci of HLA class I molecule. Polyketide and non‐ribosomal protein analyses revealed that 20% of generated peptides were predicted to bind HLA with halftime of dissociation T_1/2 ≥ 100 minutes, and 77% of them were mono‐allelic in their binding. The structural bases for recognition of nanomers by different HLA molecules were studied by structural modeling of HLA class I‐peptide complexes. Pathogen peptides that could mimic as self‐peptides or partially self‐peptides in the host were excluded using a comparative study with the human proteome; thus, subunit or DNA vaccines will have more chance of success.     

The role of two superoxide dismutase mRNAs in rye aluminium tolerance

Aluminium (Al) is the main factor that limits crop production in acidic soils. There is evidence that antioxidant enzymes such as superoxide dismutase (SOD) play a key role against Al‐induced oxidative stress in several plant species. Rye is one of the most Al‐tolerant cereals and exudes both citrate and malate from the roots in response to Al. The role of SOD against Al‐induced oxidative stress has not been studied in rye. Al accumulation, lipid peroxidation, H₂O₂ production and cell death were significantly higher in sensitive than in tolerant rye cultivars. Also, we characterised two genes for rye SOD: ScCu/ZnSOD and ScMnSOD. These genes were located on the chromosome arms of 2RS and 3RL, respectively, and their corresponding hypothetical proteins were putatively classified as cytosolic and mitochondrial, respectively. The phylogenetic relationships indicate that the two rye genes are orthologous to the corresponding genes of other Poaceae species. In addition, we studied Al‐induced changes in the expression profiles of mRNAs from ScCu/ZnSOD and ScMnSOD in the roots and leaves of tolerant Petkus and sensitive Riodeva rye. These genes are mainly expressed in roots in both ryes, their repression being induced by Al. The tolerant cultivar has more of both mRNAs than the sensitive line, indicating that they are probably involved in Al tolerance.     

Plant and animal PR1 family members inhibit programmed cell death and suppress bacterial pathogens in plant tissues

A role for programmed cell death (PCD) has been established as the basis for plant–microbe interactions. A functional plant‐based cDNA library screen identified possible anti‐PCD genes, including one member of the PR1 family, designated P14a, from tomato. Members of the PR1 family have been subject to extensive research in view of their possible role in resistance against pathogens. The PR1 family is represented in every plant species studied to date and homologues have been found in animals, fungi and insects. However, the biological function of the PR1 protein from plants has remained elusive in spite of extensive research regarding a role in the response of plants to disease. Constitutive expression of P14a in transgenic tomato roots protected the roots against PCD triggered by Fumonisin B1, as did the human orthologue GLIPR1, indicating a kingdom crossing function for PR1. Tobacco plants transformed with a P14a‐GFP fusion construct and inoculated with Pseudomonas syringae pv. tabaci revealed that the mRNA was abundant throughout the leaves, but the fusion protein was restricted to the lesion margins, where cell death and bacterial spread were arrested. Vitus vinifera grapes expressing the PR1 homologue P14a as a transgene were protected against the cell death symptoms of Pierce's disease. A pull‐down assay identified putative PR1‐interacting proteins, including members of the Rac1 immune complex, known to function in innate immunity in rice and animal systems. The findings herein are consistent with a role of PR1 in the suppression of cell death‐dependent disease symptoms and a possible mode of action.     

Proteomic analysis of phosphoproteins regulated by abscisic acid in rice leaves

Abscisic acid (ABA) is a hormone that regulates plant development and adaptation to environmental stresses. Protein phosphorylation has been recognized as an important mechanism for ABA signaling. However, the target phosphoproteins regulated by ABA are still largely unknown. Here, we report the identification of ABA-regulated phosphoproteins in rice using proteomic approaches. Six ABA-regulated phosphoproteins were identified as G protein beta subunit-like protein, ascorbate peroxidase, manganese superoxide dismutase, triosephosphate isomerase, putative Ca²⁺/H⁺ antiporter regulator protein, and glyoxysomal malate dehydrogenase. These results provide new insight into the regulatory mechanism for some ABA signaling proteins and implicate several previously unrecognized proteins in ABA action.     

Differential proteomic response to heat stress in thermal Agrostis scabra and heat‐sensitive Agrostis stolonifera

Knowledge of heat‐responsive proteins is critical for further understanding of the molecular mechanisms of heat tolerance. The objective of this study was to compare proteins differentially expressed in two C₃ grass species contrasting in heat tolerance, heat‐tolerant thermal Agrostis scabra and heat‐sensitive Agrostis stolonifera L., and to identify heat‐responsive proteins for short‐ and long‐term responses. Plants were exposed to 20/15°C (day/night, control) or 40/35°C (day/night, heat stress) in growth chambers. Leaves were harvested at 2 and 10 days after temperature treatment. Proteins were extracted and separated by fluorescence difference gel electrophoresis (DIGE). Thermal A. scabra had superior heat tolerance than A. stolonifera, as indicated by the maintenance of higher chlorophyll content and photochemical efficiency under heat stress. The two‐dimensional difference electrophoresis detected 68 heat‐responsive proteins in the two species. Thermal A. scabra had more protein spots either down‐ or up‐regulated at 2 days of heat stress, but fewer protein spots were altered at 10 days of heat stress compared with A. stolonifera. Many protein spots exhibited transient down‐regulation in thermal A. scabra (only at 2 days of heat treatment), whereas down‐regulation of many proteins was also found at 10 days of heat treatment in A. stolonifera, which suggested that protein metabolism in thermal A. scabra might acclimate to heat stress more rapidly than those in A. stolonifera. The sequences of 56 differentially expressed protein spots were identified using mass spectrometry. The results suggest that the maintenance or less severe down‐regulation of proteins during long‐term (10 days) heat stress may contribute to the superior heat tolerance in thermal A. scabra, including those involved in photosynthesis [RuBisCo, RuBisCo activase, chloroplastic glyceraldehydes‐3‐phosphate dehydrogenase (GAPDH), chloroplastic aldolase, oxygen‐evolving complex, photosystem I subunits], dark respiration (cytosolic GAPDH, cytoplasmic aldolase, malate dehydrogenase, hydroxypyruvate reductase, sedoheptulose‐1,7‐bisphosphatase), photorespiration [(hydroxypyruvate reductase, alanine aminotransferase (AlaAT), hydroxymethyltransferase (SHMT), glycine decarboxylase (GDC)], as well as heat and oxidative stress protection [heat shock cognate (HSC) 70 and FtsH‐like protein].     

Proteome profiling of aging in mouse models: Differential expression of proteins involved in metabolism,transport, and stress response in kidney

Aging is a time‐dependent complex biological phenomenon observed in various organs and organelles of all living organisms. To understand the molecular mechanism of age‐associated functional loss in aging kidneys, we have analyzed the expression of proteins in the kidneys of young (19–22 wk) and old (24 months) C57/BL6 male mice using 2‐DE followed by LC‐MS/MS. We found that expression levels of 49 proteins were upregulated (p ≤ 0.05), while that of only ten proteins were downregulated (p ≤ 0.05) due to aging. The proteins identified belong to three broad functional categories: (i) metabolism (e.g., aldehyde dehydrogenase family, ATP synthase β‐subunit, malate dehydrogenase, NADH dehydrogenase (ubiquinone), hydroxy acid oxidase 2), (ii) transport (e.g., transferrin), and (iii) chaperone/stress response (e.g., Ig‐binding protein, low density lipoprotein receptor‐related protein associated protein 1, selenium‐binding proteins (SBPs)). Some proteins with unknown functions were also identified as being differentially expressed. ATP synthase β subunit, transferrin, fumarate hydratase, SBPs, and albumin are present in multiple forms, possibly arising due to proteolysis or PTMs. The above functional categories suggest specific mechanisms and pathways for age‐related kidney degeneration.     

DIFFERENTIAL PROTEOME ANALYSIS OF THE MALE AND FEMALE ANTENNAE FROM Holotrichia parallela

To understand the olfactory mechanisms of Holotrichia parallela antennae in detecting volatile compounds in the environment, protein profiles of H. parallela antennae were analyzed using two‐dimensional electrophoresis followed by mass spectrometry and bioinformatics analyses. Approximately 1,100 protein spots in silver staining gel were detected. Quantitative image analysis revealed that in total 47 protein spots showed significant changes in different genders of adult antennae. Thirty‐five differentially expressed proteins were identified by Matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI‐TOF/TOF) tandem mass spectrometer, among which 65.7% are involved in carbohydrate and energy metabolism, antioxidant system, transport, and amino acid/nucleotide metabolism. Some proteins identified here have not been reported previously in insect antennae. Identified male‐biased proteins included odorant‐binding protein 4, pheromone‐binding protein‐related protein 2, odorant‐binding protein 14, prophenoloxidase‐I, acyl‐CoA dehydrogenase, aldo‐keto reductase‐like, carbamoyl phosphate synthetase, etc. whereas some proteins are female biased, such as antennae‐rich cytochrome P450, aldehyde dehydrogenase, and putative glutamine synthetase. Alterations in the levels of some proteins were further confirmed by real time polymerase chain reaction (RT‐PCR). The proteomic resources displayed here are valuable for the discovery of proteins from H. parallela antennae.     

Changes in proteins and isoenzymes in leaves of wheat when infected by stem rust

Summary Biuret assay, gel electrophoresis and immunochemistry were used to study concentrations, forms and activities of proteins of uredospores of Puccinia graminis Pers. f. sp. tritici, in healthy wheat leaves, wheat leaves that had been inoculated with incompatible races of stem rust and leaves which had become rusted.The soluble proteins of primary leaves increased by 25–117% following infection by compatible races of stem rust. There was a corresponding decrease of proteins in uninfected younger leaves. Infection by an incompatible strain of rust led to a temporary 29% increase in soluble proteins.Immunoelectrophoresis and gel electrophoresis of infected leaves showed the presence in them of the forms of malate dehydrogenase, glucose-6-phosphate dehydrogenase, catalase and -amylase characteristic of the rust fungus. In the infected leaves, the activity of certain bands of host glucose-6-phosphate dehydrogenase and catalase changed with the development of the pathogen; the malate dehydrogenase and -amylase of the host were unaffected. In leaves inoculated with an incompatible race there were no obvious changes of any of these enzymes.     

Proteome Analysis of Aniline-Induced Proteins in Acinetobacter lwoffii K24

Acinetobacter lwoffii K24 is a soil bacterium that can use aniline as a sole carbon and nitrogen source (by β-ketoadipate pathway genes (cat genes)) and has two copies of catABC gene separately located on the chromosome. In order to identify aniline-induced proteins, two-dimensional electrophoresis (2-DE) was applied to soluble protein fractions of A. lwoffii K24 cultured in aniline and succinate media. In the range of pH3–10, more than 370 spots were detected on the silver stained gels. Interestingly, more than 20 spots were selectively induced on aniline-cultured bacteria. Twenty-three protein spots of A. lwoffii K24 were analyzed by N-terminal microsequencing and internal microsequencing with in-gel digestion. Of 20 aniline induced protein spots, we identified six β-ketoadipate pathway genes, one subunit of amino group transfer (putative subunit of aniline oxygenase), malate dehydrogenase, putative ABC transporter, putative hydrolase, HHDD isomerase, and five unknown proteins. Especially in case of two catechol 1,2-dioxygenases (CDI₁ and CDI₂), more than three isotypes were detected on the 2D gel. This study showed that the proteome analysis of A. lwoffii K24 may be helpful for identification of genes induced by aniline and understanding of their function in the cell. Received: 2 April 2001 / Accepted: 14 May 2001     

On the interaction of ribosomal protein RPL22e with microtubules

Microtubule (MT) protein preparations often contain components of the translation machinery, including ribosome proteins. To understand the biological meaning of it we studied the interaction of ribosomal protein RPL22e with the MT. We found that bacteria expressed purified RPL22e‐GFP‐6His did co‐sediment with brain tubulin MTs with 1.3 µM dissociation coefficient. Such a K_D is comparable to some specific MT‐associated proteins. Distinct in vitro interaction of RPL22e‐GFP with MTs was also observed by TIRF microscopy. In real‐time assay, RPL22e‐GFP molecules stayed bound to MTs for several seconds, and 15% of them demonstrated random‐walk along MTs with diffusion coefficient 0.03 µ²/s. Deletion of basic areas of RPL22e did not have an impact on K_D, and deletion of acidic tail slightly increased association with MTs. Interestingly, the deletion of acidic tail increased diffusion coefficient as well. The interaction of RPL22e with MTs is hardly noticeable in vivo in cultured cells, probably since a significant part of the protein is incorporated into the ribosomes. The mobility of ribosomal protein on the MTs probably prevents its interfering with MT‐dependent transport and could ameliorate its transport to the nucleus.