Unbiased Quantitation of Escherichia coli Membrane Proteome Using Phase Transfer Surfactants

We developed a sample preparation protocol for rapid and unbiased analysis of the membrane proteome using an alimentary canal-mimicking system in which proteases are activated in the presence of bile salts. In this rapid and unbiased protocol, immobilized trypsin is used in the presence of deoxycholate and lauroylsarcosine to increase digestion efficiency as well as to increase the solubility of the membrane proteins. Using 22.5 μg of Escherichia coli whole cell lysate, we quantitatively demonstrated that membrane proteins were extracted and digested at the same level as soluble proteins without any solubility-related bias. The recovery of membrane proteins was independent of the number of transmembrane domains per protein. In the analysis of the membrane-enriched fraction from 22.5 μg of E. coli cell lysate, the abundance distribution of the membrane proteins was in agreement with that of the membrane protein-coding genes when this protocol, coupled with strong cation exchange prefractionation prior to nano-LC-MS/MS analysis, was used. Because this protocol allows unbiased sample preparation, protein abundance estimation based on the number of observed peptides per protein was applied to both soluble and membrane proteins simultaneously, and the copy numbers per cell for 1,453 E. coli proteins, including 545 membrane proteins, were successfully obtained. Finally, this protocol was applied to quantitative analysis of guanosine tetra- and pentaphosphate-dependent signaling in E. coli wild-type and relA knock-out strains.Despite the importance of cell surface biology, the conventional shotgun proteomics strategy generally underrepresents the membrane proteome because of inadequate solubilization and protease digestion (1, 2). The ageless gel strategy, consisting of SDS-PAGE followed by in-gel digestion, can partially solve this problem (3–5), but the recovery from in-gel digestion is generally lower than that from in-solution digestion, and this approach is far from suitable for a rapid, simple, and high throughput automated system. Numerous approaches have been reported to overcome the difficulties in membrane proteome analysis, such as the use of surfactants (2, 6–11), organic solvents (6, 7, 12–15), or chaotropic reagents (2, 6, 16). Acid-labile surfactants, such as RapiGest SF, are among the most promising additives to enhance protein solubilization without interfering with LC-MS performance (6, 10, 17–19). However, the cleavage step at acidic pH causes loss of hydrophobic peptides because of coprecipitation with the hydrophobic part of RapiGest SF (20). Recently, we developed a new protocol to dissolve and digest membrane proteins with the aid of a removable phase transfer surfactant (PTS),1 such as sodium deoxycholate (SDC) (20). The solubility of membrane proteins with SDC was comparable to that with sodium dodecyl sulfate. In addition, the activity of trypsin was enhanced ∼5-fold in the presence of 1% SDC because this rapid PTS method mimics conditions in the alimentary canal in which bile salts such as cholate and deoxycholate are secreted together with trypsin. After tryptic digestion, SDC is removed prior to LC-MS/MS analysis by adding an organic solvent followed by pH-induced transfer of the surfactant to the organic phase, whereas tryptic peptides remain in the aqueous phase. This protocol offers a significant improvement in identifying membrane proteins by increasing the recovery of hydrophobic tryptic peptides compared with the protocols using urea and RapiGest SF.The goal of this study is to establish a membrane proteomics method that is unbiased with respect to protein solubility, hydrophobicity, and protein abundance; i.e. membrane proteins can be as efficiently extracted and digested as soluble proteins. So far, to our knowledge, little information about the recovery of the membrane proteome has been reported. Instead, the number of identified membrane proteins or the content of membrane proteins identified in the membrane-enriched fraction has been used as an indicator of the efficiency of procedures for membrane proteome analysis (4, 5, 21–23). However, these parameters usually depend on the experimental conditions, including the sample preparation procedure and LC-MS instrument used. Therefore, it is difficult to compare data obtained with these protocols except in the case of direct comparison. Furthermore, there has been no report quantitatively comparing the recovery of membrane proteome with that of soluble proteins.In this study, we used a modified version of our PTS protocol with immobilized trypsin columns to reduce the digestion time and evaluated its suitability for unbiased quantitation of the membrane proteome. In addition, we applied this protocol to estimate the copy numbers per cell of 1,453 proteins, including 545 membrane proteins, using the exponentially modified protein abundance index (emPAI). Finally, this rapid and unbiased PTS protocol was applied to the quantitative analysis of Escherichia coli BW25113 wild-type and relA knock-out (KO) strains.     

Stomatal Ultrastructure, Molecular Phylogeny, and Description of Parasitodiplogaster laevigata n. sp. (Nematoda: Diplogastridae), a Parasite of Fig Wasps

Parasitodiplogaster comprises a potentially large radiation of nematode species that appear to be parasitically bound to their Agaonid fig wasp hosts, which are mutualistically associated in the syconia (figs) of the diverse plant genus Ficus. Parasitodiplogaster laevigata n. sp. is described and illustrated as an associate of the fig wasp, Pegoscapus sp. from Ficus laevigata from southern Florida. It is the first species of Parasitodiplogaster reported from North America and is closest to P. trigonema from F. trigonata from Panama. Parasitodiplogaster laevigata n. sp. can be differentiated from all described species of Parasitodiplogaster based on stomatal morphology (presence of a large dorsal and a right subventral tooth) in the adults of both sexes, molecular comparisons of two expansion segments (D2,D3) of the large subunit (LSU) rRNAgene, and fig-fig wasp host affinities. The ultrastructure of P. laevigata n. sp. was elucidated using TEM and SEM for comparisons with other species of Parasitodiplogaster. The stoma of P. laevigata n. sp. possesses a nonsegmented cheilostomal ring that connects to the longitudinal body musculature per- and interradially, a claw-like dorsal tooth, a right subventral tooth, and telostegostomatal apodemes arising from the dorsal side of each subventral sector. The unification of the pro-, meso-, and metastegostom with the gymnostom in P. laevigata n. sp. and further simplification in other described species may be due to derived adaptations associated with the internal parasitism of fig wasps.     

E3 ubiquitin ligase synoviolin is involved in liver fibrogenesis

Background and Aim

Chronic hepatic damage leads to liver fibrosis, which is characterized by the accumulation of collagen-rich extracellular matrix. However, the mechanism by which E3 ubiquitin ligase is involved in collagen synthesis in liver fibrosis is incompletely understood. This study aimed to explore the involvement of the E3 ubiquitin ligase synoviolin (Syno) in liver fibrosis.

Methods

The expression and localization of synoviolin in the liver were analyzed in CCl₄-induced hepatic injury models and human cirrhosis tissues. The degree of liver fibrosis and the number of activated hepatic stellate cells (HSCs) was compared between wild type (wt) and Syno^+/− mice in the chronic hepatic injury model. We compared the ratio of apoptosis in activated HSCs between wt and Syno^+/− mice. We also analyzed the effect of synoviolin on collagen synthesis in the cell line from HSCs (LX-2) using siRNA-synoviolin and a mutant synoviolin in which E3 ligase activity was abolished. Furthermore, we compared collagen synthesis between wt and Syno^−/− mice embryonic fibroblasts (MEF) using quantitative RT-PCR, western blotting, and collagen assay; then, we immunohistochemically analyzed the localization of collagen in Syno^−/− MEF cells.

Results

In the hepatic injury model as well as in cirrhosis, synoviolin was upregulated in the activated HSCs, while Syno^+/− mice developed significantly less liver fibrosis than in wt mice. The number of activated HSCs was decreased in Syno^+/− mice, and some of these cells showed apoptosis. Furthermore, collagen expression in LX-2 cells was upregulated by synoviolin overexpression, while synoviolin knockdown led to reduced collagen expression. Moreover, in Syno^−/− MEF cells, the amounts of intracellular and secreted mature collagen were significantly decreased, and procollagen was abnormally accumulated in the endoplasmic reticulum.

Conclusion

Our findings demonstrate the importance of the E3 ubiquitin ligase synoviolin in liver fibrosis.     

Unintentional introductions of microscopic organisms associated with forest insects

International trade and travel are devastating native flora and fauna in many countries through the intentional and/or unintentional introduction of exotic organisms. Pathway control appears to be particularly effective for microscopic organisms such as mites, nematodes, and fungi that are difficult to see with the naked eye. However, taxonomic and ecological information on such organisms is scarce, sometimes causing time lags or failure in eradication programs. Several groups of mites, nematodes, and fungi commonly share a habitat with insects or use them as dispersal agents (phoresy). Some exotic mites and nematodes are introduced simultaneously with exotic insects, sometimes in wood materials. In Japan, mites, nematodes, and fungi have been collected from lucanid beetles introduced as pets from Southeast Asia. While no lethal nematode species have been collected from lucanid beetles, one hemolymph-sucking mite species, inhabiting the sub-elytral space of its native host, is able to easily switch to the Japanese beetle, Dorcus rectus, killing the insect. Yeasts have also been reported on exotic beetles and laboulbeniomycetes have been found on mites associated with the beetles, although their interactions are unknown. Despite the lack of information available about other mites, nematodes, and fungi associated with intentionally and unintentionally introduced forest insects, our analysis of insect species listed by the International Union for Conservation of Nature suggests that unintentional introductions of the microscopic organisms are quite common as a consequence of the existence of symbiotic relationships such as phoresy and parasitism.     

CREB represses p53-dependent transactivation of MDM2 through the complex formation with p53 and contributes to p53-mediated apoptosis in response to glucose deprivation

Recently, we have described that CREB (cAMP-responsive element-binding protein) has the ability to transactivate tumor suppressor p53 gene in response to glucose deprivation. In this study, we have found that CREB forms a complex with p53 and represses p53-mediated transactivation of MDM2 but not of p21^WAF1. Immunoprecipitation analysis revealed that CREB interacts with p53 in response to glucose deprivation. Forced expression of CREB significantly attenuated the up-regulation of the endogenous MDM2 in response to p53. By contrast, the mutant form of CREB lacking DNA-binding domain (CREBΔ) had an undetectable effect on the expression level of the endogenous MDM2. During the glucose deprivation-mediated apoptosis, there existed an inverse relationship between the expression levels of MDM2 and p53/CREB. Additionally, p53/CREB complex was dissociated from MDM2 promoter in response to glucose deprivation. Collectively, our present results suggest that CREB preferentially down-regulates MDM2 and thereby contributing to p53-mediated apoptosis in response to glucose deprivation.     

C5a controls TLR-induced IL-10 and IL-12 production independent of phosphoinositide 3-kinase

The complement system is a classic central player in innate immunity. Most pathogens activate both complement and the toll-like receptor (TLR) pathway. Therefore, to provide a more comprehensive understanding of innate immunity, it is important to understand the crosstalk between these two systems. Mouse macrophages produce IL-12 and IL-10 in response to TLR ligands such as LPS, CpG, Poly I:C and Malp2. The TLR-induced IL-12 production was decreased, while that of IL-10 was increased by concurrent stimulation with a complement fragment C5a. Pharmacological studies have suggested that C5a regulates TLR4-induced IL-12 production in a phosphoinositide 3-kinase (PI3K)-dependent mechanism. In the present study, however, we found that the C5a-mediated changes can be observed in macrophages from mice lacking PI3K p85α or PI3K p110γ. The result indicates that the C5a action is PI3K-independent; neither class IA nor class IB PI3K subtype is involved in this regulation. The actions of C5a were sensitive to pertussis toxin and PD98059, suggesting a role of G protein-mediated activation of the Erk1/2 pathway.     

Mitochondrial DNA copy number is maintained during spermatogenesis and in the development of male larvae to sustain the doubly uniparental inheritance of mitochondrial DNA system in the blue mussel Mytilus galloprovincialis

Doubly uniparental inheritance (DUI) of mitochondrial (mt) DNA has been reported in the blue mussel Mytilus galloprovincialis. In DUI, males inherit both paternal (M type) and maternal (F type) mtDNA. Here we investigated changes in M type mtDNA copy numbers and mitochondrial mass in testicular cells by real‐time polymerase chain reaction and flow cytometry. The ratios of M type mtDNA copy numbers to nuclear DNA content were not different between haploid (1n), diploid (2n) and tetraploid (4n) spermatogenic cells. The mitochondrial mass decreased gradually during spermatogenesis. These results suggest that mtDNA and mitochondrial mass are maintained during spermatogenesis. We then traced M type mtDNA in larvae after fertilization. M type mtDNA was maintained up to 24 h after fertilization in the male‐biased crosses, but decreased significantly in female‐biased crosses (predicted by Mito Tracker staining pattern). These results are strikingly different from those reported for mammals and fish, where it is well known that the mitochondria and mtDNA are reduced during spermatogenesis and that sperm mitochondria and mtDNA are eliminated soon after fertilization. Thus, the M type mtDNA copy number is maintained during spermatogenesis and in the development of male larvae to sustain the DUI system in the blue mussel.     

Molecular epidemiology of Salmonella enterica serovar typhimurium isolates from cattle in hokkaido, Japan: evidence of clonal replacement and characterization of the disseminated clone

The molecular epidemiology of 545 Salmonella enterica serovar Typhimurium isolates collected between 1977 and 2009 from cattle in Hokkaido, Japan, was investigated using pulsed-field gel electrophoresis (PFGE). Nine main clusters were identified from 116 PFGE patterns. Cluster I comprised 248 isolates, 243 of which possessed a sequence specific to definitive phage type 104 (DT104) or U302. The cluster I isolates were dominant in 1993 to 2003, but their numbers declined beginning in 2004. Beginning in 2002, an increase was observed in the number of cluster VII isolates, consisting of 21 PFGE patterns comprising 165 isolates. A total of 116 isolates representative of the 116 PFGE profiles were analyzed by multilocus variable-number tandem-repeat analysis (MLVA). Other than two drug-sensitive isolates, 19 isolates within cluster VII were classified in the same cluster by MLVA. Among the cluster VII isolates, an antibiotic resistance type showing resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, tetracycline, kanamycin, cefazolin, and sulfamethoxazole-trimethoprim and a resistance type showing resistance to ampicillin, streptomycin, sulfonamides, tetracycline, and kanamycin were found in 23 and 125 isolates, respectively. In the 19 isolates representative of cluster VII, the bla(TEM-1) gene was found on a Salmonella serotype Typhimurium virulence plasmid, which was transferred to Escherichia coli by electroporation along with resistance to two to four other antimicrobials. Genomic analysis by subtractive hybridization and plasmid analysis suggested that the bla(TEM-1)-carrying virulence plasmid has a mosaic structure composed of elements of different origin. These results indicate an emerging multidrug-resistant S. Typhimurium clone carrying a virulence-resistance plasmid among cattle in Hokkaido, Japan.     

Glycoside hydrolase family 89 alpha-N-acetylglucosaminidase from Clostridium perfringens specifically acts on GlcNAc alpha1,4Gal beta1R at the non-reducing terminus of O-glycans in gastric mucin

In mammals, α-linked GlcNAc is primarily found in heparan sulfate/heparin and gastric gland mucous cell type mucin. α-N-acetylglucosaminidases (αGNases) belonging to glycoside hydrolase family 89 are widely distributed from bacteria to higher eukaryotes. Human lysosomal αGNase is well known to degrade heparin and heparan sulfate. Here, we reveal the substrate specificity of αGNase (AgnC) from Clostridium perfringens strain 13, a bacterial homolog of human αGNase, by chemically synthesizing a series of disaccharide substrates containing α-linked GlcNAc. AgnC was found to release GlcNAc from GlcNAcα1,4Galβ1pMP and GlcNAcα1pNP substrates (where pMP and pNP represent p-methoxyphenyl and p-nitrophenyl, respectively). AgnC also released GlcNAc from porcine gastric mucin and cell surface mucin. Because AgnC showed no activity against any of the GlcNAcα1,2Galβ1pMP, GlcNAcα1,3Galβ1pMP, GlcNAcα1,6Galβ1pMP, and GlcNAcα1,4GlcAβ1pMP substrates, this enzyme may represent a specific glycosidase required for degrading α-GlcNAc-capped O-glycans of the class III mucin secreted from the stomach and duodenum. Deletion of the C-terminal region containing several carbohydrate-binding module 32 (CBM32) domains significantly reduced the activity for porcine gastric mucin; however, activity against GlcNAcα1,4Galβ1pMP was markedly enhanced. Dot blot and ELISA analyses revealed that the deletion construct containing the C-terminal CBM-C2 to CBM-C6 domains binds strongly to porcine gastric mucin. Consequently, tandem CBM32 domains located near the C terminus of AgnC should function by increasing the affinity for branched or clustered α-GlcNAc-containing glycans. The agnC gene-disrupted strain showed significantly reduced growth on the class III mucin-containing medium compared with the wild type strain, suggesting that AgnC might have an important role in dominant growth in intestines.