Agrobacterium tumefaciens VirB proteins are essential for gene transfer from bacteria to plants. These proteins are postulated to form a transport pore to allow transfer of the T-strand DNA intermediate. To study the function of the VirB proteins in DNA transfer, we developed an expression system in A. tumefaciens. Analysis of one VirB protein, VirB9, by Western blot assays showed that under nonreducing conditions VirB9, when expressed alone, migrates as a -31-kDa band but that it migrates as a -36-kDa band when expressed with all other VirB proteins. The 36-kDa band is converted to the 31-kDa band by the reducing agent 2-mercaptoethanol. Using strains that contain a deletion in a defined virB gene and strains that express specific VirB proteins, we demonstrate that the 36-kDa band is composed ofVirB9 and VirB7 that are linked to each other by a disulfide bond. Mutational studies demonstrate that cysteine residues at positions 24 of VirB7 and 262 of VirB9 participate in the formation of this complex.The transformation of a susceptible plant cell by the soil bacterium Agrobacterium tumefaciens results from the transfer and integration of a segment of the tumor-inducing (T,) plasmid DNA into the plant nuclear genome. The virulence (vir) genes of the Ti plasmid play an essential role in the DNA transfer and integration processes. The portion of the T1 plasmid that is transferred to plant cells (T-DNA) is defined by a 24-bp direct repeat sequence known as the border sequence. Two proteins of the virD operon, VirDl and VirD2, initiate the processing of the T-DNA by introducing a site-and strandspecific nick at the T-DNA borders, leading to the formation of a single-stranded T-strand DNA composed of the bottom strand of the T-DNA. The T-strand DNA contains a VirD2 molecule covalently attached to its 5'-end and is believed to be an intermediate in DNA transfer to plants (for reviews, see refs. 1 and 2).Little is known about the mechanism of DNA transfer from bacteria to plants. It is postulated that DNA transfer occurs through a transport pore primarily composed of the VirB proteins. Of the 11 VirB proteins, 10 (VirB2-VirB11) are essential for DNA transfer. The other protein (VirBl) is required for a high efficiency of DNA transfer (3). The observations that the VirB proteins have no role in T-strand DNA synthesis and that most of the VirB proteins are membrane associated led to their proposed role in DNA transfer (4-10). Subsequently, DNA sequence analysis of the trb genes of the conjugative plasmid pRP4 and that of the ptl operon of the pathogenic bacteria Bordetella pertussis revealed that these operons encode homologs of the VirB proteins (11,12). The requirement of the trb genes in conjugal transfer of plasmid DNA, that of the ptl genes in the secretion of the Bordetella toxin protein, and that of the virB genes in T-DNA transfer suggests the existence of a common transport pathway for macromolecule export. The requirement of the virB genes in tumor formation, in DNA transfer to plants in a transient DN...

No abstract

, and ؉18 daltons. The masses of the modifications suggest that the tryptophan is modified to kynurenine (؉4), a keto-͞ amino-͞hydroxy-(؉16) derivative, and a dihydro-hydroxy-(؉18) derivative of the indole side chain. Peptide synthesis and MS͞MS confirmed the kynurenine assignment. The ؉16 and ؉18 tryptophan modifications may be intermediates formed during the oxidative cleavage of the indole ring to give kynurenine. The sitedirected mutations, W352C, W352L, and W352A, exhibit an increased rate of photoinhibition relative to wild type. We hypothesize that Trp-352 oxidative modifications are a byproduct of PSII water-splitting or electron transfer reactions and that these modifications target PSII for turnover. As a step toward understanding the tertiary structure of this CP43 peptide, structural modeling was performed by using molecular dynamics.mass spectrometry ͉ collision-induced dissociation ͉ tryptophan ͉ kynurenine ͉ photoinhibition P hotosystem II (PSII) is a protein-pigment complex located in thylakoid membranes of plants, eukaryotic algae, and cyanobacteria. PSII catalyzes the light-driven oxidation of water to O 2 , and the reduction of plastoquinone. PSII contains both intrinsic and extrinsic polypeptides. The intrinsic polypeptides include chlorophyll-binding proteins, CP47, CP43, and the D1 and D2 polypeptides (reviewed in ref. 1). The D2͞D1 heterodimer binds P 680 , pheophytin, and the quinone receptors, Q A and Q B (2). Three extrinsic subunits, the manganese stabilizing, 24-kDa, and 18-kDa proteins, are required for maximum oxygen evolution in plants (3, 4). Recently, a 3.8-Å structure of the cyanobacterial PSII reaction center has been reported (5, 6).The intrinsic PSII subunits, CP43 and CP47, function as light-harvesting proteins and play a role in PSII assembly and activity (7-11). CP47 and CP43 have similar tertiary and secondary structures (5). Each polypeptide has six membranespanning regions and a large luminal, hydrophilic loop (E) between helix V and VI (5, 7). In Synechocystis sp. PCC 6803, loop E of the CP43 subunit extends from residue Asn-280 to . Mutations or deletions in this loop inactivate or impair PSII activity in Synechocystis (9, 11-13).Posttranslational modifications can play important roles in the assembly, degradation, structure, and function of proteins. However, little is known about the roles of such modifications in membrane proteins. For example, in cytochrome c oxidase, a crosslinked tyrosine-histidine cofactor has been identified at the binuclear metal site (14); the function of this cofactor has not yet been definitively established. Recently, it has been suggested that posttranslationally modified amino acids, containing carbonyl groups, covalently bind hydrazines and amines at the catalytic site of PSII (10, 15). Because amines and hydrazines are inhibitors of photosynthetic water oxidation, it was suggested that these carbonyl-containing amino acids play roles in the structure, function, or assembly of PSII.To obtain more information about posttranslational mod...

Peripheral tissue injury is associated with changes in protein expression in sensory neurons that may contribute to abnormal nociceptive processing. We used cultured dorsal root ganglion (DRG) neurons as a model of axotomized neurons to investigate early changes in protein expression after nerve injury. Comparing protein levels immediately after DRG dissociation and 24 h later by proteomic differential expression analysis, we found a substantial increase in the levels of the neurotrophin-inducible protein VGF (nonacronymic), a putative neuropeptide precursor. In a rodent model of nerve injury, VGF levels were increased within 24 h in both injured and uninjured DRG neurons, and the increase persisted for at least 7 d. VGF was also upregulated 24 h after hindpaw inflammation. To determine whether peptides derived from proteolytic processing of VGF participate in nociceptive signaling, we examined the spinal effects of AQEE-30 and LQEQ-19, potential proteolytic products shown previously to be bioactive. Each peptide evoked dose-dependent thermal hyperalgesia that required activation of the mitogen-activated protein kinase p38. In addition, LQEQ-19 induced p38 phosphorylation in spinal microglia when injected intrathecally and in the BV-2 microglial cell line when applied in vitro. In summary, our results demonstrate rapid upregulation of VGF in sensory neurons after nerve injury and inflammation and activation of microglial p38 by VGF peptides. Therefore, VGF peptides released from sensory neurons may participate in activation of spinal microglia after peripheral tissue injury.

Despite successful revascularization of hibernating myocardium, regional function and blood flow remained depressed during catecholamine stress. Electron transport chain proteins known to be downregulated during adaptive process within hibernating myocardium did not normalize after revascularization. These data demonstrate a potential bioenergetic cause of persistent dysfunction and heart failure within successfully revascularized hibernating myocardium.

Ovarian cancer is the fifth leading cause of cancer death for women in the U.S., yet survival rates are over 90% when it is diagnosed at an early stage, highlighting the need for biomarkers for early detection. To enhance the discovery of tumor-specific proteins which could represent novel serum biomarkers for ovarian cancer, we depleted serum of highly abundant proteins which can mask the detection of proteins present in serum at low concentrations. Three commercial immunoaffinity columns were used in parallel to deplete the highly abundant proteins in serum from 60 patients with serous ovarian carcinoma and 60 non-cancer controls. Medium and low abundance serum proteins from each serum pool were then evaluated by the quantitative proteomic technique of Differential-In-Gel-Electrophoresis (DIGE). The number of protein spots that were elevated in ovarian cancer sera by at least 2-fold ranged from 36 to 248, depending upon the depletion and separation methods. From the 33 spots picked for MS analysis, nine different proteins were identified, including the novel candidate ovarian cancer biomarkers leucine-rich alpha-2 glycoprotein-1 and ficolin 3. Western blotting validated the relative increases in serum protein levels for three of the proteins identified, demonstrating the utility of this approach for the identification of novel serum biomarkers for ovarian cancer.

We are interested in the biological as well as the molecular processes involved in natural killer (NK) cell development and function. Determining the proteomic complement could be a useful tool in predicting cellular function and fate. For the first time shown here, we have utilized iTRAQ, a new method that allows identification and quantification of proteins between multiple samples, to determine the expression of membrane-bound proteins in two previously characterized human NK cell populations. One population was derived from umbilical cord blood (UCB) stem cells (CD34+38-Lin-) and the other from expanded CD3-depleted adult peripheral blood. iTRAQ was employed for multiplex peptide labeling of proteins from fractionated membranes followed by two-dimensional high-performance liquid chromatography (2D-HPLC), and tandem mass spectrometry was used to identify protein signatures. We were able to identify and quantify differences in expression levels of 400-800 proteins in a typical experiment. Ontology analysis showed the majority of the proteins to be involved in cell signaling, nucleic acid binding, or mitochondrial function. Nearly all proteins were associated with the plasma membrane, membrane-bound organelle (lysosome or mitochondria), or nucleus. We found several novel proteins highly expressed in UCB stem cell derived NK cells compared to adult NK cells including CD9, alpha-2 macroglobulin, brain abundant signaling protein (BASP1), and allograft inflammatory factor-1 (AIF-1). In addition, we were able to confirm several of our iTRAQ results by RT-PCR, Western blot, and fluorescence-activated cell-sorting (FACS) analysis. This is the first demonstration and verification using iTRAQ to screen for membrane-bound protein differences in human NK cells and represents a powerful new tool in the field of proteomics.

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.