Wolbachia pipientis, a widespread vertically transmitted intracellular bacterium, provides a tool for insect control through manipulation of host-microbe interactions. We report proteomic characterization of wStr, a Wolbachia strain associated with a strong cytoplasmic incompatibility phenotype in its native host, Laodelphax striatellus. In the Aedes albopictus?C/wStr1 mosquito cell line, wStr maintains a robust, persistent infection. MS/MS analyses of gel bands revealed a protein 'footprint' dominated by Wolbachia-encoded chaperones, stress response and cell membrane proteins, including the surface antigen WspA, a peptidoglycan-associated lipoprotein and a 73?kDa outer membrane protein. Functional classifications and estimated abundance levels of 790 identified proteins suggested that expression, stabilization and secretion of proteins predominate over bacterial genome replication and cell division. High relative abundances of cysteine desulphurase, serine/glycine hydroxymethyl transferase, and components of the ?-ketoglutarate dehydrogenase complex in conjunction with above average abundances of glutamate dehydrogenase and proline utilization protein A support Wolbachia genome-based predictions for amino acid metabolism as a primary energy source. wStr expresses 15 Vir proteins of a Type IV secretion system and its transcriptional regulator. Proteomic characterization of a robust insect-associated Wolbachia strain provides baseline information that will inform further development of in vitro protocols for Wolbachia manipulation.
Protein prenylation is a post-translational modification required for proper cellular localization and activity of many important eukaryotic proteins. Farnesyltransferase inhibitors (FTIs) have been explored extensively for their antitumor activity. To assist in identifying potentially new and more useful markers for therapeutic applications, we developed a strategy that uses a combination of metabolic labeling and 2D DIGE (differential gel electrophoresis) to discover new prenylated proteins whose cellular levels are influenced by FTIs. In this approach, metabolic labeling of prenylated proteins was first carried out with an alkyne-modified isoprenoid analog, C15Alk, in the presence or absence of the FTI L-744,832. The resulting alkyne-tagged proteins were then labeled with Cy3-N3 and Cy5-N3 and subjected to 2D-DIGE. Multiple spots having altered levels of labeling in presence of the FTI were observed. Mass spectrometric analysis of some of the differentially labeled spots identified several known prenylated proteins, along with HisRS, PACN-3, GNAI-1 and GNAI-2, which are not known to be prenylated. In vitro farnesylation of a C-terminal peptide sequence derived from GNAI-1 and GNAI-2 produced a farnesylated product, suggesting GNAI-1 and GNAI-2 are potential novel farnesylated proteins. These results suggest that this new strategy could be useful for the identification of prenylated proteins whose level of post-translational modification has been modulated by the presence of an FTI. Additionally, this approach, which decreases sample complexity and thereby facilitates analysis, should be applicable to studies of other post-translational modifications as well.
There is accumulating evidence that mesenchymal stem cells (MSCs) have their origin as perivascular cells (PVCs) in vivo, but precisely identifying them has been a challenge, as they have no single definitive marker and are rare. We have developed a fluorescent transgenic vertebrate model in which PVC can be visualized in vivo based upon sdf1 expression in the zebrafish. Prospective isolation and culture of sdf1(DsRed) PVC demonstrated properties consistent with MSC including prototypical cell surface marker expression; mesodermal differentiation into adipogenic, osteogenic, and chondrogenic lineages; and the ability to support hematopoietic cells. Global proteomic studies performed by two-dimensional liquid chromatography and tandem mass spectrometry revealed a high degree of similarity to human MSC (hMSC) and discovery of novel markers (CD99, CD151, and MYOF) that were previously unknown to be expressed by hMSC. Dynamic in vivo imaging during fin regeneration showed that PVC may arise from undifferentiated mesenchyme providing evidence of a PVC-MSC relationship. This is the first model, established in zebrafish, in which MSC can be visualized in vivo and will allow us to better understand their function in a native environment.
Development of a vaccine against congenital infection with human cytomegalovirus (HCMV) is a major public health priority. A potential vaccine target receiving considerable recent attention is the pentameric complex (PC) of HCMV proteins consisting of gL, gH, UL128, UL130, and UL131, since some antibodies against these target proteins are capable of potently neutralizing virus at epithelial and endothelial cell surfaces. Recently, homologous proteins have been described for guinea pig cytomegalovirus (GPCMV), consisting of gH, gL, and the GPCMV proteins GP129, GP131, and GP133. To investigate these proteins as potential vaccine targets, expression of GP129-GP133 transcripts was confirmed by reverse-transcriptase PCR. Mass spectrometry combined with western blot assays demonstrated the presence of GP129, GP131, and GP133 proteins in virus particles. Recombinant proteins corresponding to these PC proteins were generated in baculovirus, and as GST fusion proteins. Recombinant proteins were noted to be immunoreactive with convalescent sera from infected animals, suggesting that these proteins are recognized in the humoral immune response to GPCMV infection. These analyses support the study of PC-based recombinant vaccines in the GPCMV congenital infection model.
Soy protein isolate (SPI) and ?-conglycinin- and glycinin-rich fractions were hydrolyzed using papain and pepsin. Protein denaturation, profiling, and peptide identification were carried out following DSC, SDS-PAGE, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The in vitro antihypertensive activity of the hydrolysates was compared by determining the angiotensin converting enzyme (ACE) inhibitory activity. SDS-PAGE and LC-MS/MS analysis confirmed pepsin selectivity to glycinin and papain partial selectivity to ?-conglycinin when the protein is least denatured. Both the papain-hydrolyzed SPI and the papain-hydrolyzed ?-conglycinin-rich fraction had more than double the ACE inhibitory activity of that of pepsin-hydrolyzed SPI and pepsin-hydrolyzed glycinin-rich fraction. This observation indicated that ?-conglycinin is a better precursor for antihypertensive peptides than glycinin. Additionally, the inhibitory activity of the papain-hydrolyzed SPI was thermally stable. This work demonstrated, for the first time, that selective hydrolysis to release peptides with ACE inhibitory activity can be accomplished without inducing extensive hydrolysis and performing unnecessary fractionation.
Wolbachia are obligate intracellular bacteria that cause cytoplasmic incompatibility in mosquitoes. In an incompatible cross, eggs of uninfected females fail to hatch when fertilized by sperm from infected males. We used polyacrylamide gel electrophoresis and tandem mass spectrometry to identify Wolbachia proteins in infected mosquito gonads. These included surface proteins with masses of 25 and 18 kDa and the DNA binding protein, HU beta. Using reverse transcriptase polymerase chain reaction, we showed that the HU gene is transcribed in Wolbachia-infected Culex pipiens and Aedes albopictus mosquitoes. We sequenced HU genes from four Wolbachia strains and compared deduced protein sequences with additional homologs from the databases. Among the Rickettsiales, Wolbachia HU has distinct N- and C-terminal basic/acidic amino acid motifs as well as a pair of conserved, cysteine residues.
To sensitively analyze complex protein mixtures by mass spectrometry-based shotgun proteomics, researchers have employed platforms that couple orthogonal peptide fractionation methods using nanoscale HPLC. Commonly used platforms have coupled either strong cation exchange (SCX) HPLC or preparative isoelectric focusing (IEF) with nanoscale reversed-phase (nanoRP) HPLC fractionation of peptides. Coupling two dimensions of peptide fractionation, prior to mass spectrometric analysis, increases the sensitivity for identifying low abundance proteins. However, the large dynamic range of protein abundance and high level of complexity of protein mixtures derived from many biological sources, such as bodily fluids, require additional steps of peptide fractionation. To address this shortcoming, we have developed a platform combining three dimensions of peptide fractionation as follows: (1) preparative IEF; (2) SCX HPLC; and (3) nanoRP HPLC. This platform significantly increases the sensitivity of shotgun proteomic analysis in complex protein mixtures. Here, we describe the implementation of this three-dimensional peptide fractionation platform for proteomic studies of complex mixtures.
Trinucleotide expansions cause disease by both protein- and RNA-mediated mechanisms. Unexpectedly, we discovered that CAG expansion constructs express homopolymeric polyglutamine, polyalanine, and polyserine proteins in the absence of an ATG start codon. This repeat-associated non-ATG translation (RAN translation) occurs across long, hairpin-forming repeats in transfected cells or when expansion constructs are integrated into the genome in lentiviral-transduced cells and brains. Additionally, we show that RAN translation across human spinocerebellar ataxia type 8 (SCA8) and myotonic dystrophy type 1 (DM1) CAG expansion transcripts results in the accumulation of SCA8 polyalanine and DM1 polyglutamine expansion proteins in previously established SCA8 and DM1 mouse models and human tissue. These results have implications for understanding fundamental mechanisms of gene expression. Moreover, these toxic, unexpected, homopolymeric proteins now should be considered in pathogenic models of microsatellite disorders.
As part of a multi-endpoint systems approach to develop comprehensive methods for assessing endocrine stressors in vertebrates, differential protein profiling was used to investigate expression patterns in the brain of the amphibian model (Xenopus laevis) following in vivo exposure to a suite of T4 synthesis inhibitors. We specifically address the application of Two Dimensional Polyacrylamide Gel Electrophoresis (2D PAGE), Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) and LC-MS/MS to assess changes in relative protein expression levels. 2D PAGE and iTRAQ proved to be effective complementary techniques for distinguishing protein changes in the developing amphibian brain in response to T4 synthesis inhibition. This information served to evaluate the use of distinctive protein profiles as a potential mechanism to screen chemicals for endocrine activity in anurans. Regulatory pathways associated with proteins expressed as a result of chemical effect are reported. To our knowledge, this is also the first account of the anuran larvae brain proteome characterization using proteomic technologies. Correlation of protein changes to other cellular and organism-level responses will aid in the development of a more rapid and cost-effective, non-mammalian screening assay for thyroid axis-disrupting chemicals.
Ovarian cancer is the fifth leading cause of cancer death for women in the US, 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 that 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. The number of protein spots that were elevated in ovarian cancer sera by at least twofold 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 alpha2 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.
Ureteral stents are susceptible to biofilm formation and crystal deposition, especially in stone formers. To identify proteins responsible for this accumulation, we compared conditioning film proteomes obtained from human ureteral stents with and without encrustation.
Many plant proteins are modified with N-linked oligosaccharides at asparagine-X-serine/threonine sites during transit through the endoplasmic reticulum and the Golgi. We have identified a number of Arabidopsis (Arabidopsis thaliana) proteins with modifications consisting of an N-linked N-acetyl-D-glucosamine monosaccharide (N-GlcNAc). Electron transfer dissociation mass spectrometry analysis of peptides bearing this modification mapped the modification to asparagine-X-serine/threonine sites on proteins that are predicted to transit through the endoplasmic reticulum and Golgi. A mass labeling method was developed and used to study N-GlcNAc modification of two thioglucoside glucohydrolases (myrosinases), TGG1 and TGG2 (for thioglucoside glucohydrolase). These myrosinases are also modified with high-mannose (Man)-type glycans. We found that N-GlcNAc and high-Man-type glycans can occur at the same site. It has been hypothesized that N-GlcNAc modifications are generated when endo-?-N-acetylglucosaminidase (ENGase) cleaves N-linked glycans. We examined the effects of mutations affecting the two known Arabidopsis ENGases on N-GlcNAc modification of myrosinase and found that modification of TGG2 was greatly reduced in one of the single mutants and absent in the double mutant. Surprisingly, N-GlcNAc modification of TGG1 was not affected in any of the mutants. These data support the hypothesis that ENGases hydrolyze high-Man glycans to produce some of the N-GlcNAc modifications but also suggest that some N-GlcNAc modifications are generated by another mechanism. Since N-GlcNAc modification was detected at only one site on each myrosinase, the production of the N-GlcNAc modification may be regulated.
Our previous studies revealed that the staphylococcal protein Gcp is essential for bacterial growth; however, the essential function of Gcp remains undefined. In this study, we demonstrated that Gcp plays an important role in the modulation of the branched-chain amino acids biosynthesis pathway. Specifically, we identified that the depletion of Gcp dramatically elevated the production of key enzymes that are encoded in the ilv-leu operon and responsible for the biosynthesis of the branched-chain amino acids isoleucine, leucine, and valine (ILV) using proteomic approaches. Using qPCR and promoter-lux reporter fusions, we established that Gcp negatively modulates the transcription of the ilv-leu operon. Gel-shift assays revealed that Gcp lacks the capacity to bind the promoter region of ilv. Moreover, we found that the depletion of Gcp did not influence the transcription level of CodY, a known repressor of the ilv-leu operon, while induced the transcription of CcpA, a known positive regulator of the ilv-leu operon. In addition, the depletion of Gcp decreased the biosynthesis of N(6)-threonylcarbamoyladenosine (t6A). To elucidate whether the essentiality of Gcp is attributable to its negative modulation of ILV biosynthesis, we determined the impact of the ilv-leu operon on the requirement of Gcp for growth, and revealed that the deletion of the ilv-leu operon did not affect the essentiality of Gcp. Taken together, our results indicate that the essentiality of Gcp isnt attributable to its negative regulation of ILV biosynthesis in S. aureus. These findings provide new insights into the biological function of the staphylococcal Gcp.
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