The Development of a Dual-Tag Affinity Purification System and its Application to Elucidate the Interacting Protein Network Surrounding the Human Telomere Binding Proteins TRF1, TRF2, and POT1

Protein-protein interactions (PPI) play a vital role in almost every cellular process. Although many methodologies exist to probe PPIs, one of the most successful and widely employed is tandem affinity purification coupled with liquid chromatography and tandem mass spectrometry (LC-MS/MS). Although best demonstrated in yeast, TAP has encountered significant hurdles in its application to mammalian systems, especially the observed low yield of bait protein and its interacting partners after two consecutive purifications. To address these issues, a novel dual-tag affinity purification (DAP) system was developed that not only enhances bait protein recovery, but also allows for rapid evaluation of dual-tag compatibility with a protein of interest based on its known subcellular localization. In addition, several tags of varying composition were constructed to allow for maximal bait protein compatibility. With this system, mammalian bait protein yield was improved by more than 200% relative to previously published results. Capitalizing on this success, DAP was applied to human telomere binding proteins TRF1, TRF2, and POT1 to garner a greater understanding of the protein networks that involve the telomere. Expectedly, all the members of the telosome complex were identified at frequencies that lend evidence towards the currently accepted architecture. Also identified were several other novel proteins and subcomplexes that may enhance our understanding of telomere maintenance / length regulation. For instance, members of the classical nuclear import system co-purified with both TRF1 and TRF2. Although previously documented for TRF1, TRF2’s association with importin alpha (KPNA2) and beta (KPNB1) has not been demonstrated till now. Interestingly, further study revealed that KPNA2 acts as a negative regulator of TRF2 nuclear localization. This observation could have far-reaching implications as TRF2 is thought to be also heavily involved in the DNA damage response. Along these lines, a more indepth MS analysis revealed several putative phosphorylation sites along TRF2’s sequence. One site, pS380, seems to be phosphorylated by the DNA-damage kinase ATM and plays a role in a cell’s proliferative capacity, possibly affecting telomere length regulation. The studies contained here within demonstrate the efficacy of DAP-LC-MS/MS to provide useful leads with regards to the study of PPIs

Role of the CipA Scaffoldin Protein in Cellulose Solubilization, as Determined by Targeted Gene Deletion and Complementation in Clostridium thermocellum

The CipA scaffoldin protein plays a key role in the Clostridium thermocellum cellulosome. Previous studies have revealed that mutants deficient in binding or solubilizing cellulose also exhibit reduced expression of CipA. To confirm that CipA is, in fact, necessary for rapid solubilization of crystalline cellulose, the gene was deleted from the chromosome using targeted gene deletion technologies. The CipA deletion mutant exhibited a 100-fold reduction in cellulose solubilization rate, although it was eventually able to solubilize 80% of the 5 g/liter cellulose initially present. The deletion mutant was complemented by a copy of cipA expressed from a replicating plasmid. In this strain, Avicelase activity was restored, although the rate was 2-fold lower than that in the wild type and the duration of the lag phase was increased. The cipA coding sequence is located at the beginning of a gene cluster containing several other genes thought to be responsible for the structural organization of the cellulosome, including olpB, orf2p, and olpA. Tandem mass spectrometry revealed a 10-fold reduction in the expression of olpB, which may explain the lower growth rate. This deletion experiment adds further evidence that CipA plays a key role in cellulose solubilization by C. thermocellum, and it raises interesting questions about the differential roles of the anchor scaffoldin proteins OlpB, Orf2p, and SdbA

Identification, cloning and characterization of SpEX exotoxin produced by Staphylococcus pseudintermedius

Staphylococci have evolved numerous strategies to evade their hosts’ immune systems. Some staphylococcal toxins target essential components of host innate immunity, one of the two main branches of the immune system. Analysis of the Staphylococcus pseudintermedius secretome using liquid chromatography mass spectrometry guided by genomic data, was used to identify an S. pseudintermedius exotoxin provisionally named SpEX. This exoprotein has low overall amino acid identity with the Staphylococcus aureus group of proteins named staphylococcal superantigen like proteins (SSLs) and staphylococcal enterotoxin- like toxin X (SEIX), but predictive modeling showed that it shares similar folds and domain architecture to these important virulence factors. In this study, we found SpEX binds to complement component C5, prevents complement mediated lysis of sensitized bovine red blood cells, kills polymorphonuclear leukocytes and monocytes and inhibits neutrophil migration at sub-lethal concentrations. A mutant version of SpEX, produced through amino acid substitution at selected positions, had diminished cytotoxicity. Anti-SpEX produced in dogs reduced the inhibitory effect of native SpEX on canine neutrophil migration and protected immune cells from the toxic effects of the native recombinant protein. These results suggest that SpEX likely plays an important role in S. pseudintermedius virulence and that attenuated SpEX may be an important candidate for inclusion in a vaccine against S. pseudintermedius infections

Characterization of a leukocidin identified in Staphylococcus pseudintermedius

Bacterial infections from Staphylococcus pseudintermedius are the most common cause of skin infections (pyoderma) affecting dogs. Two component pore-forming leukocidins are a family of potent toxins secreted by staphylococci and consist of S (slow) and F (fast) components. They impair the innate immune system, the first line of defense against these pathogens. Seven different leukocidins have been characterized in Staphylococcus aureus, some of which are host and cell specific. Through genome sequencing and analysis of the S. pseudintermediussecretome using liquid chromatography mass spectrometry we identified two proteins, named “LukS-I” and “LukF-I”, encoded on a degenerate prophage contained in the genome of S. pseudintermedius isolates. Phylogenetic analysis of LukS-I components in comparison to the rest of the leukocidin family showed that LukS-I was most closely related to S. intermediusLukS-I, S. aureus LukE and LukP, whereas LukF-I was most similar to S. intermedius LukF-I S. aureus gamma hemolysin subunit B. The killing effect of recombinant S. pseudintermediusLukS-I and LukF-I on canine polymorphonuclear leukocytes was determined using a flow cytometry cell permeability assay. The cytotoxic effect occurred only when the two recombinant proteins were combined. Engineered mutant versions of the two-component pore-forming leukocidins, produced through amino acids substitutions at selected points, were not cytotoxic. Anti-Luk-I produced in dogs against attenuated proteins reduced the cytotoxic effect of native canine leukotoxin which highlights the importance of Luk-I as a promising component in a vaccine against canine S. pseudintermedius infections

Coculture with hemicellulose-fermenting microbes reverses inhibition of corn fiber solubilization by Clostridium thermocellum at elevated solids loadings

Background: The cellulolytic thermophile Clostridium thermocellum is an important biocatalyst due to its ability to solubilize lignocellulosic feedstocks without the need for pretreatment or exogenous enzyme addition. At low concentrations of substrate, C. thermocellum can solubilize corn fiber \u3e 95% in 5 days, but solubilization declines markedly at substrate concentrations higher than 20 g/L. This differs for model cellulose like Avicel, on which the maximum solubilization rate increases in proportion to substrate concentration. The goal of this study was to examine fermentation at increasing corn fiber concentrations and investigate possible reasons for declining performance. Results: The rate of growth of C. thermocellum on corn fiber, inferred from CipA scaffoldin levels measured by LC–MS/MS, showed very little increase with increasing solids loading. To test for inhibition, we evaluated the effects of spent broth on growth and cellulase activity. The liquids remaining after corn fiber fermentation were found to be strongly inhibitory to growth on cellobiose, a substrate that does not require cellulose hydrolysis. Additionally, the hydrolytic activity of C. thermocellum cellulase was also reduced to less-than half by adding spent broth. Noting that \u3e 15 g/L hemicellulose oligosaccharides accumulated in the spent broth of a 40 g/L corn fiber fermentation, we tested the effect of various model carbohydrates on growth on cellobiose and Avicel. Some compounds like xylooligosaccharides caused a decline in cellulolytic activity and a reduction in the maximum solubilization rate on Avicel. However, there were no relevant model compounds that could replicate the strong inhibition by spent broth on C. thermocellum growth on cellobiose. Cocultures of C. thermocellum with hemicellulose-consuming partners—Herbinix spp. strain LL1355 and Thermoanaerobacterium thermosaccharolyticum—exhibited lower levels of unfermented hemicellulose hydrolysis products, a doubling of the maximum solubilization rate, and final solubilization increased from 67 to 93%. Conclusions: This study documents inhibition of C. thermocellum with increasing corn fiber concentration and demonstrates inhibition of cellulase activity by xylooligosaccharides, but further work is needed to understand why growth on cellobiose was inhibited by corn fiber fermentation broth. Our results support the importance of hemicellulose-utilizing coculture partners to augment C. thermocellum in the fermentation of lignocellulosic feedstocks at high solids loading

Microbiota functional activity biosensors for characterizing nutrient metabolism in vivo

Methods for measuring gut microbiota biochemical activities in vivo are needed to characterize its functional states in health and disease. To illustrate one approach, an arabinan-containing polysaccharide was isolated from pea fiber, its structure defined, and forward genetic and proteomic analyses used to compare its effects, versus unfractionated pea fiber and sugar beet arabinan, on a human gut bacterial strain consortium in gnotobiotic mice. We produced \u27Microbiota Functional Activity Biosensors\u27 (MFABs) consisting of glycans covalently linked to the surface of fluorescent paramagnetic microscopic glass beads. Three MFABs, each containing a unique glycan/fluorophore combination, were simultaneously orally gavaged into gnotobiotic mice, recovered from their intestines, and analyzed to directly quantify bacterial metabolism of structurally distinct arabinans in different human diet contexts. Colocalizing pea-fiber arabinan and another polysaccharide (glucomannan) on the bead surface enhanced in vivo degradation of glucomannan. MFABs represent a potentially versatile platform for developing new prebiotics and more nutritious foods

A critical review of the Exit-Voice-Loyalty-Neglect literature: limitations, key challenges and directions for future research

The study of human behaviour holds a prominent role in organizational behavior literature. For almost 45 years, the exit, voice, loyalty and neglect typology has attracted scholars' interest and has been linked to employee responses towards dissatisfaction and problematic events in the workplace. This paper reviews the literature and identifies and addresses key theoretical and methodological deficiencies that the exit, voice, loyalty and neglect typology faces that have been either ignored or undeveloped. Moreover, by unpicking this typology as currently portrayed in the existing literature, it proposes key challenges that need to be addressed and provides directions for future research

Impact of Fatty-Acid Labeling of Bacillus subtilis Membranes on the Cellular Lipidome and Proteome

Developing cultivation methods that yield chemically and isotopically defined fatty acid (FA) compositions within bacterial cytoplasmic membranes establishes an in vivo experimental platform to study membrane biophysics and cell membrane regulation using novel approaches. Yet before fully realizing the potential of this method, it is prudent to understand the systemic changes in cells induced by the labeling procedure itself. In this work, analysis of cellular membrane compositions was paired with proteomics to assess how the proteome changes in response to the directed incorporation of exogenous FAs into the membrane of Bacillus subtilis. Key findings from this analysis include an alteration in lipid headgroup distribution, with an increase in phosphatidylglycerol lipids and decrease in phosphatidylethanolamine lipids, possibly providing a fluidizing effect on the cell membrane in response to the induced change in membrane composition. Changes in the abundance of enzymes involved in FA biosynthesis and degradation are observed; along with changes in abundance of cell wall enzymes and isoprenoid lipid production. The observed changes may influence membrane organization, and indeed the well-known lipid raft-associated protein flotillin was found to be substantially down-regulated in the labeled cells – as was the actin-like protein MreB. Taken as a whole, this study provides a greater depth of understanding for this important cell membrane experimental platform and presents a number of new connections to be explored in regard to modulating cell membrane FA composition and its effects on lipid headgroup and raft/cytoskeletal associated proteins

追加1. 胃切除術後の鼻腔ゾンデ早期抜去について(シンポジウム 中山式切除術をめぐって,第473回千葉医学会例会,第4回佐藤外科例会)

Additional file 4. Proteomic data of strains wild-type, AG553, AG553, AG601 and LL1210 of Clostridium thermocellum