Expression clustering reveals detailed co-expression patterns of functionally related proteins during B cell differentiation: a proteomic study using a combination of one-dimensional gel electrophoresis, LC-MS/MS, and stable isotope labeling by amino acids in cell culture (SILAC)

B cells play an essential role in the immune response. Upon activation they may differentiate into plasma cells that secrete specific antibodies against potentially pathogenic non-self antigens. To identify the cellular proteins that are important for efficient production of these antibodies we set out to study the B cell differentiation process at the proteome level. We performed an in-depth proteomic study to quantify dynamic relative protein expression patterns of several hundreds of proteins at five consecutive time points after lipopolysaccharide-induced activation of B lymphocytes. The proteome analysis was performed using a combination of stable isotope labeling using [13C6]leucine added to the murine B cell cultures, one-dimensional gel electrophoresis, and LC-MS/MS. In this study we identified 1,001 B cell proteins. We were able to quantify the expression levels of a quarter of all identified proteins (i.e. 234) at each of the five different time points. Nearly all proteins revealed changes in expression patterns. The quantitative dataset was further analyzed using an unbiased clustering method. Based on their expression profiles, we grouped the entire set of 234 quantified proteins into a limited number of 12 distinct clusters. Functionally related proteins showed a strong correlation in their temporal expression profiles. The quality of the quantitative data allowed us to even identify subclusters within functionally related classes of proteins such as in the endoplasmic reticulum proteins that are involved in antibody production.     

Leucine aminopeptidase is not essential for trimming peptides in the cytosol or generating epitopes for MHC class I antigen presentation

To detect viral infections and tumors, CD8+ T lymphocytes monitor cells for the presence of antigenic peptides bound to MHC class I molecules. The majority of MHC class I-presented peptides are generated from the cleavage of cellular and viral proteins by the ubiquitin-proteasome pathway. Many of the oligopeptides produced by this process are too long to stably bind to MHC class I molecules and require further trimming for presentation. Leucine aminopeptidase (LAP) is an IFN-inducible cytosolic aminopeptidase that can trim precursor peptides to mature epitopes and has been thought to play an important role in Ag presentation. To examine the role of LAP in generating MHC class I peptides in vivo, we generated LAP-deficient mice and LAP-deficient cell lines. These mutant mice and cells are viable and grow normally. The trimming of peptides in LAP-deficient cells is not reduced under basal conditions or after stimulation with IFN. Similarly, there is no reduction in presentation of peptides from precursor or full-length Ag constructs or in the overall supply of peptides from cellular proteins to MHC class I molecules even after stimulation with IFN. After viral infection, LAP-deficient mice generate normal CTL responses to seven epitopes from three different viruses. These data demonstrate that LAP is not an essential enzyme for generating most MHC class I-presented peptides and reveal redundancy in the function of cellular aminopeptidases.     

Mass tag-assisted identification of naturally processed HLA class II-presented meningococcal peptides recognized by CD4+ T lymphocytes

The meningococcal class I outer membrane protein porin A plays an important role in the development of T cell-dependent protective immunity against meningococcal serogroup B infection and is therefore a major component of candidate meningococcal vaccines. T cell epitopes from porin A are poorly characterized because of weak in vitro memory T cell responses against purified Ag and strain variation. We applied a novel strategy to identify relevant naturally processed and MHC class II-presented porin A epitopes, based on stable isotope labeling of Ag. Human immature HLA-DR1-positive dendritic cells were used for optimal uptake and MHC class II processing of (14)N- and (15)N-labeled isoforms of the neisserial porin A serosubtype P1.5-2,10 in bacterial outer membrane vesicles. HLA-DR1 bound peptides, obtained after 48 h of Ag processing, contained typical spectral doublets in mass spectrometry that could easily be assigned to four porin A regions, expressed at diverging densities ( approximately 30-4000 copies/per cell). Epitopes from two of these regions are recognized by HLA-DR1-restricted CD4(+) T cell lines and are conserved among different serosubtypes of meningococcal porin A. This mass tag-assisted approach provides a useful methodology for rapid identification of MHC class II presented bacterial CD4(+) T cell epitopes relevant for vaccine development.     

Absence of thrombin-activatable fibrinolysis inhibitor protects against sepsis-induced liver injury in mice

Thrombin-activatable fibrinolysis inhibitor (TAFI), also known as carboxypeptidase R, has been implicated as an important negative regulator of the fibrinolytic system. In addition, TAFI is able to inactivate inflammatory peptides such as complement factors C3a and C5a. To determine the role of TAFI in the hemostatic and innate immune response to abdominal sepsis, TAFI gene-deficient (TAFI-/-) and normal wild-type mice received an i.p. injection with Escherichia coli. Liver TAFI mRNA and TAFI protein concentrations increased during sepsis. In contrast to the presumptive role of TAFI as a natural inhibitor of fibrinolysis, TAFI-/- mice did not show any difference in E. coli-induced activation of coagulation or fibrinolysis, as measured by plasma levels of thrombin-anti-thrombin complexes and D-dimer and the extent of fibrin depositions in lung and liver tissues. However, TAFI-/- mice were protected from liver necrosis as indicated by histopathology and clinical chemistry. Furthermore, TAFI-/- mice displayed an altered immune response to sepsis, as indicated by an increased neutrophil recruitment to the peritoneal cavity and a transiently increased bacterial outgrowth together with higher plasma TNF-alpha and IL-6 levels. These data argue against an important part for TAFI in the regulation of the procoagulant-fibrinolytic balance in sepsis and reveals a thus far unknown role of TAFI in the occurrence of hepatic necrosis.     

Molecular signature of recent thymic selection events on effector and regulatory CD4+ T lymphocytes

Natural CD4+CD25+ regulatory T lymphocytes (Treg) are key protagonists in the induction and maintenance of peripheral T cell tolerance. Their thymic origin and biased repertoire continue to raise important questions about the signals that mediate their development. We validated analysis of MHC class II capture by developing thymocytes from thymic stroma as a tool to study quantitative and qualitative aspects of the cellular interactions involved in thymic T cell development and used it to analyze Treg differentiation in wild-type mice. Our data indicate that APCs of bone marrow origin, but, surprisingly and importantly, not thymic epithelial cells, induce significant negative selection among the very autoreactive Treg precursors. This fundamental difference between thymic development of regulatory and effector T lymphocytes leads to the development of a Treg repertoire enriched in cells specific for a selected subpopulation of self-Ags, i.e., those specifically expressed by thymic epithelial cells.     

Quantitative kinetic analysis of nucleolar breakdown and reassembly during mitosis in live human cells

One of the great mysteries of the nucleolus surrounds its disappearance during mitosis and subsequent reassembly at late mitosis. Here, the relative dynamics of nucleolar disassembly and reformation were dissected using quantitative 4D microscopy with fluorescent protein-tagged proteins in human stable cell lines. The data provide a novel insight into the fates of the three distinct nucleolar subcompartments and their associated protein machineries in a single dividing cell. Before the onset of nuclear envelope (NE) breakdown, nucleolar disassembly started with the loss of RNA polymerase I subunits from the fibrillar centers. Dissociation of proteins from the other subcompartments occurred with faster kinetics but commenced later, coincident with the process of NE breakdown. The reformation pathway also follows a reproducible and defined temporal sequence but the order of reassembly is shown not to be dictated by the order in which individual nucleolar components reaccumulate within the nucleus after mitosis.     

Tissue kallikrein stimulates Ca(2+) reabsorption via PKC-dependent plasma membrane accumulation of TRPV5

The transient receptor potential vanilloid 5 (TRPV5) channel determines urinary Ca(2+) excretion, and is therefore critical for Ca(2+) homeostasis. Interestingly, mice lacking the serine protease tissue kallikrein (TK) exhibit robust hypercalciuria comparable to the Ca(2+) leak in TRPV5 knockout mice. Here, we delineated the molecular mechanism through which TK stimulates Ca(2+) reabsorption. Using TRPV5-expressing primary cultures of renal Ca(2+)-transporting epithelial cells, we showed that TK activates Ca(2+) reabsorption. The stimulatory effect of TK was mimicked by bradykinin (BK) and could be reversed by application of JE049, a BK receptor type 2 antagonist. A cell permeable analog of DAG increased TRPV5 activity within 30 min via protein kinase C activation of the channel since mutation of TRPV5 at the putative PKC phosphorylation sites S299 and S654 prevented the stimulatory effect of TK. Cell surface labeling revealed that TK enhances the amount of wild-type TRPV5 channels, but not of the TRPV5 S299A and S654A mutants, at the plasma membrane by delaying its retrieval. In conclusion, TK stimulates Ca(2+) reabsorption via the BK-activated PLC/DAG/PKC pathway and the subsequent stabilization of the TRPV5 channel at the plasma membrane.     

Characterization of single nucleotide polymorphisms in sheep and their variation as evidence of selection

The discovery of SNPs was performed using animals from eight European sheep breeds. Eleven SNPs were further characterized using about 1,700 sheep belonging to 57 breeds. A method for the identification of loci that were likely subject to selection was applied; three of the 11 SNPs lying outside the 95% confidence region of the conditional joint distribution of F(ST) and mean heterozygosity were identified as outliers.     

Alcoholic fermentation of carbon sources in biomass hydrolysates by Saccharomyces cerevisiae: current status

Fuel ethanol production from plant biomass hydrolysates by Saccharomyces cerevisiae is of great economic and environmental significance. This paper reviews the current status with respect to alcoholic fermentation of the main plant biomass-derived monosaccharides by this yeast. Wild-type S. cerevisiae strains readily ferment glucose, mannose and fructose via the Embden–Meyerhof pathway of glycolysis, while galactose is fermented via the Leloir pathway. Construction of yeast strains that efficiently convert other potentially fermentable substrates in plant biomass hydrolysates into ethanol is a major challenge in metabolic engineering. The most abundant of these compounds is xylose. Recent metabolic and evolutionary engineering studies on S. cerevisiae strains that express a fungal xylose isomerase have enabled the rapid and efficient␣anaerobic fermentation of this pentose. l-Arabinose fermentation, based on the expression of a prokaryotic pathway in S. cerevisiae, has also been established, but needs further optimization before it can be considered for industrial implementation. In addition to these already investigated strategies, possible approaches for metabolic engineering of galacturonic acid and rhamnose fermentation by S. cerevisiae are discussed. An emerging and major challenge is to achieve the rapid transition from proof-of-principle experiments under ‘academic’ conditions (synthetic media, single substrates or simple substrate mixtures, absence of toxic inhibitors) towards efficient conversion of complex industrial substrate mixtures that contain synergistically acting inhibitors.