Functional Proteomics

This paper presents a new solid-phase extraction and elution platform based on surface-functionalized diamond nanocrystallites. Compared with conventional methods, the platform facilitates purification and concentration of intact proteins and their enzymatic digests for ensuing sodium dodecyl sulfate-polyacrylamide gel electrophoresis or matrix-assisted laser desorption/ionization mass spectrometry analysis without prior removal of the adsorbent. One-pot work flow involving reduction of disulfide bonds, protection of free cysteine residues, washing off residual chemicals, and proteolytic digestion of adsorbed proteins can be performed directly on the particles. The utility and versatility of this protein workup platform were demonstrated with liquid chromatography-electrospray ionization tandem mass spectrometry in proteome analysis of human urine. The proteome analysis of each urine sample can be completed in 8 h.

This report describes a draft genome sequence of Lactobacillus fermentum strain 3872. The data revealed remarkable similarity to and dissimilarity with the published genome sequences of other strains of the species. The absence of and variation in structures of some adhesins and the presence of an additional adhesin may reflect adaptation of the bacterium to different host systems and may contribute to specific properties of this strain as a new probiotic.

Proteases drive the life cycle of all proteins, ensuring the transportation and activation of newly minted, would-be proteins into their functional form while recycling spent or unneeded proteins. Far from their image as engines of protein digestion, proteases play fundamental roles in basic physiology and regulation at multiple levels of systems biology. Proteases are intimately associated with disease and modulation of proteolytic activity is the presumed target for successful therapeutics. "Proteases: Pivot Points in Functional Proteomics" examines the crucial roles of proteolysis across a wide range of physiological processes and diseases. The existing and potential impacts of proteolysis-related activity on drug and biomarker development are presented in detail. All told the decisive roles of proteases in four major categories comprising 23 separate subcategories are addressed. Within this construct, 15 sets of subject-specific, tabulated data are presented that include identification of proteases, protease inhibitors, substrates and their actions. Said data are derived from and confirmed by over 300 references. Cross comparison of datasets indicates that proteases, their inhibitors/promoters and substrates intersect over a range of physiological processes and diseases, both chronic and pathogenic. Indeed, "Proteases: Pivot Points …" closes by dramatizing this very point through association of (pro)Thrombin and Fibrin(ogen) with: hemostasis, innate immunity, cardiovascular and metabolic disease, cancer, neurodegeneration and bacterial self-defense.

Urokinase plasminogen activator receptor (uPAR) and the epithelial integrin αvβ6 are thought to individually play critical roles in cancer metastasis. These observations have been highlighted by the recent discovery (by proteomics) of an interaction between these two molecules, which are also both implicated in the epithelial−mesenchymal transition (EMT) that facilitates escape of cells from tissue barriers and is a common signature of cancer metastases. In this study, orthogonal in cellulo and in vitro functional proteomic approaches were used to better characterize the uPAR•αvβ6 interaction. Proximity ligation assays (PLA) confirmed the uPAR•αvβ6 interaction on OVCA429 (ovarian cancer line) and four different colon cancer cell lines including positive controls in cells with de novo β6 subunit expression. PLA studies were then validated using peptide arrays, which also identified potential physical sites of uPAR interaction with αvβ6, as well as verifying interactions with other known uPAR ligands (e.g., uPA, vitronectin) and individual integrin subunits (i.e., αv, β1, β3, and β6 alone). Our data suggest that interaction with uPAR requires expression of the complete αβ heterodimer (e.g., αvβ6), not individual subunits (i.e., αv, β1, β3, or β6). Finally, using in silico structural analyses in concert with these functional proteomics studies, we propose and demonstrate that the most likely unique sites of interaction between αvβ6 and uPAR are located in uPAR domains II and III.

Specific elution of captured proteins greatly improves the quality of proteomic data obtained from pulldowns by avoiding signals from nonspecific proteins, thus allowing more sensitive identification of target proteins. This is important in activity-based proteomics or drug target identification. However, commonly used chemically cleavable linkers can only be cleaved at close to neutral pH, which prevents them from being used for proteins binding only at lower pH when no cross-linking is applied. On the other hand, elution of common acid-cleavable labels can also coelute proteins bound by ionic interactions. Here, we report the synthesis and application of a label readily cleavable by mild oxidation at moderately acidic pH for the noncovalent labeling and pull-down of intracellular aspartic proteases. Using specific release, target proteins cathepsin D and napsin A were identified from human kidney with much higher confidence and without any nontarget hits.

Conformational variants of the unique family of protease inhibitors annotated as SERPINs are most often underrepresented in proteomic analyses. This limits understanding the complex regulation that this family of proteins presents to the networks within the protease web of interactions. Using bead-based separation provided by a family of proteomic enrichment products-notably AlbuVoid™ and AlbuSorb™, we demonstrate their utility to satisfy investigations of serum SERPINs. We also suggest their use to develop functional profiles of the SERPIN proteoforms, and how those can establish relationships to disease phenotypes, gene mutations, and dysregulated mechanisms.

Enhanced production and usage of silver nanoparticles (AgNPs) raise concerns about their potential impacts in aquatic ecosystems. Effects of AgNPs and Ag+ were assessed based on the variations in the overall proteome and the activities of selected antioxidant enzymes in two fungal strains of Articulospora tetracladia, one isolated from a non-polluted stream (At72) and the other from a metal-polluted stream (At61). For that, fungi were exposed to concentrations of AgNPs and Ag+ affecting 20% of growth (EC20). A total of 432 proteins were identified, of which 172 belonged to At72 and 260 to At61; 71 were expressed in both strains. At72 had 58% and 52% of the proteins induced by AgNPs and Ag+, respectively. For At61, the percentages were higher (˜77%). The higher percentages of proteins suppressed by either Ag forms suggest higher stress in At72 than in At61, which is consistent with the background of this fungal strain. Major groups of proteins were related to carbohydrate metabolism, amino-acid and protein biosynthesis. Both Ag forms also induced stress-responsive proteins, including catalase and superoxide dismutase which remained consistent with the profile of their enzymatic activities. These results supported the ability of these fungi in initiating an efficient antioxidant response to cope with Ag-induced toxicity. Overall, the functional proteomic approach can be useful to get a mechanistic insight on the stress induced by AgNPs or Ag+ in aquatic fungi that play a key role in plant litter decomposition in streams.

The toxic heavy metal cadmium (Cd) appears as one of the major global threats to human and animal health. Human being and aquatic life are exposed to Cd by breathing, eating, or drinking when industrial effluents released into environment. The study was aimed to identify cadmium-binding Lactobacillus strain to reduce its bioaccessibility in in vitro digestion model. In this context, forty-eight lactobacilli strains isolated and characterized from fermented dairy products and human origin were screened for their Cd biosorption potential using Flame Atomic Absorption Spectroscopy (FAAS). The present study revealed that Cd biosorption potential of 48 lactobacilli strains ranged from 1.0832 ± 0.012 to 3.562 ± 0.03 mg Cd g(-1) of cells from initial 10 mg L(-1) cadmium chloride (CdCl2) aqueous solution. Lactobacillus plantarum strain HD 48 demonstrated highest biosorption of 3.562 ± 0.03 mg Cd g(-1) of cells. Lactobacilli-Cd complex stability indicated its strong stability as even after t...

This report describes a draft genome sequence of Lactobacillus fermentum strain 3872. The data revealed remarkable similarity to and dissimilarity with the published genome sequences of other strains of the species. The absence of and variation in structures of some adhesins and the presence of an additional adhesin may reflect adaptation of the bacterium to different host systems and may contribute to specific properties of this strain as a new probiotic.

To enable high-throughput screening of molecular phenotypes, multi-parameter uorescence microscopy is applied. Object of our study is lymphocytes which invade human tissue. One important basis for our collaborative project is the development of methods for automatic and accurate evaluation of uorescence micrographs. As a part of this, we focus on the question of how to measure the accuracy of microscope image interpretation, by human experts or a computer system. Following standard practice we use methods motivated by receiver operator characteristics to discuss the accuracies of human experts and of neural network-based algorithms. For images of good quality the algorithms achieve the accuracy of the medium-skilled experts. In images with increased noise, the classiÿers are outperformed by some of the experts. Furthermore, the neural network-based cell detection is much faster than the human experts.

Amelogenins are a set of low molecular-weight enamel proteins belonging to a group of extracellular matrix (ECM) proteins with a key role in tooth enamel development and in other regeneration processes, such as wound healing and angiogenesis. Since only few data are actually available to unravel amelogenin mechanism of action in chronic skin healing restoration, we moved to the full characterization of the human amelogenin isoform 2 interactome in the secretome and lysate of Human Umbilical Vein Endothelial cells (HUVEC), using a functional proteomic approach. Trombospondin-1 has been identified as a novel and interesting partner of human amelogenin isoform 2 and their direct binding has been validated thought biophysical orthogonal approaches.

GTPase ras-related C3 botulinum toxin substrate 1 (Rac1) plays a role in various cellular processes pertinent to cancer development. In the present study, we investigated the molecular mechanisms underlying apoptosis regulation by Rac1 through functional proteomic analysis of three human melanoma M14 cell lines stably transfected with constitutively active Rac1V12, dominant negative Rac1N17, and empty vector (pIRES), respectively. We found that paclitaxel evoked apoptosis in the melanoma cell lines through the intrinsic (mitochondria) pathway in a caspsae-3-dependent manner. Compared to the Rac1pIRES and Rac1V12 cells, Rac1N17 cells were more resistant to paclitaxel-triggered caspase-3 activation and apoptosis. Protein composition comparisons amongst the three cell lines identified two peptide spots of interest. One was Hsp27, which was upregulated in Rac1N17 cells as assessed in our gel image interpretation, PMFand Western blot analysis. The other was identified as SR-25 protein (also known as the ADP-ribosylation factor-like factor 6-interacting protein 4; ARL6IP4) using PMF, which was separated only from the Rac1N17 cells under the experimental conditions. Moreover, knockdown of the protein level of Hsp27 using small interfering RNA in Rac1N17 cells significantly increased the paclitaxel-elicited caspase-3 activation and apoptosis. In conclusion, our results implicate that Hsp27 and SR-25 are mediators in Rac1 signaling pathway(s). It appears that the dominant negative Rac1N17 reduces the apoptosis sensitivity toward paclitaxel in the melanoma cells through upregulation of Hsp27, which inhibits its down stream drug-elicited caspase-3 activation.

GTPase ras-related C3 botulinum toxin substrate 1 (Rac1) plays a role in various cellular processes pertinent to cancer development. In the present study, we investigated the molecular mechanisms underlying apoptosis regulation by Rac1 through functional proteomic analysis of three human melanoma M14 cell lines stably transfected with constitutively active Rac1V12, dominant negative Rac1N17, and empty vector (pIRES), respectively. We found that paclitaxel evoked apoptosis in the melanoma cell lines through the intrinsic (mitochondria) pathway in a caspsae-3-dependent manner. Compared to the Rac1pIRES and Rac1V12 cells, Rac1N17 cells were more resistant to paclitaxel-triggered caspase-3 activation and apoptosis. Protein composition comparisons amongst the three cell lines identified two peptide spots of interest. One was Hsp27, which was upregulated in Rac1N17 cells as assessed in our gel image interpretation, PMFand Western blot analysis. The other was identified as SR-25 protein (also known as the ADP-ribosylation factor-like factor 6-interacting protein 4; ARL6IP4) using PMF, which was separated only from the Rac1N17 cells under the experimental conditions. Moreover, knockdown of the protein level of Hsp27 using small interfering RNA in Rac1N17 cells significantly increased the paclitaxel-elicited caspase-3 activation and apoptosis. In conclusion, our results implicate that Hsp27 and SR-25 are mediators in Rac1 signaling pathway(s). It appears that the dominant negative Rac1N17 reduces the apoptosis sensitivity toward paclitaxel in the melanoma cells through upregulation of Hsp27, which inhibits its down stream drug-elicited caspase-3 activation.

Sodium caseinate fermented of Lactobacilli were studied for their antimicrobial activity. Out of the total 34 standard NCDC, 12 Lactobacillus cultures were selected on the basis of maximum proteolytic activity. Proteolytic property was used to study their antimicrobial activity against pathogenic and contaminating organism. This Lactobacillus was assessed for probiotic attributes. Proteolytic and probiotic strains are selected for studying antimicrobial activity using caseinate fermented. Cell free supernatant of Lactobacillus was tested for antimicrobial activity. Sodium caseinate (2.5 %) was prepared for fermentation; concentrated 10 fold at 60°C was tested for antimicrobial activity against gram positive and gram negative bacteria, yeast and mold cultures. Lb. fermentum 141 had maximum antimicrobial activity as compared to other Lactobacillus and selected for further studies. Minimum inhibitory concentration of Lb 141 fermentate (MIC) against bacterial cultures was 100 µg / ml wh...

Prolonged heat stress is one of the harsh conditions Lactobacillus casei strains encounter as non-starter lactic acid bacteria in dairy product manufacture. To understand the physiological and molecular mechanisms through which Lb. casei GCRL163 adapts to persistent elevated temperature, label-free quantitative proteomics of cell-free extracts was used to characterize the global responses of the strain cultured anaerobically in bioreactors at 30 to 45°C, pH 6.5, together with GC-MS for fatty acid methyl ester analysis at different growth phases. At higher growth temperatures, repression of energy-consuming metabolic pathways, such as fatty acid, nucleotide and amino acid biosynthesis, was observed, while PTS- and ABC-type transporter systems associated with uptake of nitrogen and carbon sources were up-regulated. Alkaline shock protein Asp23_2 was only detected at 45°C, expressed at high abundance, and presumptive α-L-fucosidase only at 40 and 45°C, with highly increased abundance (...

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.

Lactobacillus fermentum strains NCDC 400 and RS2, previously isolated from dairy sources, exhibited excellent probiotic properties were studied for the global proteomic profile. A total of 1125 proteins were identified by a high-resolution mass spectrometer, ESI-qTOF (nano-LC-MS/MS) in the strains of L. fermentum. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis resulted in 60.9% and 59.2% of the total proteins were functionally annotated for NCDC 400 and RS2 respectively. Simultaneously, a cluster of orthologous groups (COGs) and KEGG together revealed the presence of a significant number of proteins involved in transcription, translation , chaperones, peptidoglycan biosynthesis, nucleotide, amino acid and carbohydrate metabolism. Most of the proteins that play a vital role in the formation of RNA polymerase, ribosomal subunits, and aminoacyl-tRNA biosynthesis were fully mapped. Further analysis by bioinformatics tools revealed that 13.83% of the proteins were hydrophobic while 86.17% were hydrophilic in nature. The present findings represent the first draft proteome map of L. fermentum strains and demonstrate the involvement of important proteins in normal physiology and growth of potential probiotic strain.