PaxDb, a database of protein abundance averages across all three domains of life.

Although protein expression is regulated both temporally and spatially, most proteins have an intrinsic, "typical" range of functionally effective abundance levels. These extend from a few molecules per cell for signaling proteins, to millions of molecules for structural proteins. When addressing fundamental questions related to protein evolution, translation and folding, but also in routine laboratory work, a simple rough estimate of the average wild type abundance of each detectable protein in an organism is often desirable. Here, we introduce a meta-resource dedicated to integrating information on absolute protein abundance levels; we place particular emphasis on deep coverage, consistent post-processing and comparability across different organisms. Publicly available experimental data are mapped onto a common namespace and, in the case of tandem mass spectrometry data, re-processed using a standardized spectral counting pipeline. By aggregating and averaging over the various samples, conditions and cell-types, the resulting integrated data set achieves increased coverage and a high dynamic range. We score and rank each contributing, individual data set by assessing its consistency against externally provided protein-network information, and demonstrate that our weighted integration exhibits more consistency than the data sets individually. The current PaxDb-release 2.1 (at http://pax-db.org/) presents whole-organism data as well as tissue-resolved data, and covers 85,000 proteins in 12 model organisms. All values can be seamlessly compared across organisms via pre-computed orthology relationships.

A two-dimensional protein map of Caenorhabditis elegans.

A protein map of Caenorhabditis elegans was constructed by using two-dimensional gel electrophoresis followed by peptide mass fingerprinting. A whole worm extract of a mixed population was separated on immobilized pH gradient strips 4-7 L, 3-10 NL, 6-11 L and 12% sodium dodecyl sulfate-polyacrylamide gel eletrophoresis (SDS-PAGE) gels. Gels were stained with colloidal Coomassie blue and 286 spots representing 152 proteins were subsequently identified by matrix-assisted laser desorption/ionization-mass spectrometry after in-gel digestion with trypsin. Most of the identified proteins with known cellular function were enzymes related to carbohydrate and lipid metabolism, or structural proteins with subcellular locations in the cytoplasm, mitochondria or cytoskeleton.

Calsyntenin-1, a proteolytically processed postsynaptic membrane protein with a cytoplasmic calcium-binding domain.

In a screen for proteins released from synapse-forming spinal cord neurons, we found the proteolytically cleaved N-terminal fragment of a transmembrane protein localized in the postsynaptic membrane of both excitatory and inhibitory synapses. We termed this protein calsyntenin-1, because it binds synaptic Ca2+ with its cytoplasmic domain. By binding Ca2+, calsyntenin-1 may modulate Ca2+-mediated postsynaptic signals. Proteolytic cleavage of calsyntenin-1 in its extracellular moiety generates a transmembrane stump that is internalized and accumulated in the spine apparatus of spine synapses. Therefore, the synaptic Ca2+ modulation by calsyntenin-1 may be subject to regulation by extracellular proteolysis in the synaptic cleft. Thus, calsyntenin-1 may link extracellular proteolysis in the synaptic cleft and postsynaptic Ca2+ signaling.

Identification of dipeptidyl peptidase IV as the antigen of a monoclonal anti-prostasome antibody.

BACKGROUND: Our purpose was to elucidate the identity of an antigen of a monoclonal antibody against prostasomes, which are prostate-derived organelles occurring freely in human semen. METHODS: Monoclonal antibodies against prostasomes were generated by intrasplenic immunization to allow better characterization of the prostasomes. One third of the resulting antibodies bound to a 110-kDa prostasomal protein on Western blots. RESULTS: The antigen was purified from seminal prostasomes by anion exchange chromatography and gel electrophoresis. After in-gel digestion and amino-acid sequence analysis of selected peptides, it was identified as dipeptidyl peptidase IV (DPP IV), EC 3.4.14.5, also known as T-cell-activating antigen CD26. No DPP IV could be detected in the supernatant obtained after preparative ultracentrifugation of the prostasomes. CONCLUSIONS: DPP IV/CD26 is structurally bound to prostasomes rather than being free in seminal fluid. This enzyme may play a critical role in the promoting effect of prostasomes on forward motility of spermatozoa.

The axonally secreted serine proteinase inhibitor, neuroserpin, inhibits plasminogen activators and plasmin but not thrombin.

Neuroserpin is an axonally secreted serine proteinase inhibitor that is expressed in neurons during embryogenesis and in the adult nervous system. To identify target proteinases, we used a eucaryotic expression system based on the mouse myeloma cell line J558L and vectors including a promoter from an Ig-kappa-variable region, an Ig-kappa enhancer, and the exon encoding the Ig-kappa constant region (C kappa) and produced recombinant neuroserpin as a wild-type protein or as a fusion protein with C kappa. We investigated the capability of recombinant neuroserpin to form SDS-stable complexes with, and to reduce the amidolytic activity of, a variety of serine proteinases in vitro. Consistent with its primary structure at the reactive site, neuroserpin exhibited inhibitory activity against trypsin-like proteinases. Although neuroserpin bound and inactivated plasminogen activators and plasmin, no interaction was observed with thrombin. A reactive site mutant of neuroserpin neither formed complexes with nor inhibited the amidolytic activity of any of the tested proteinases. Kinetic analysis of the inhibitory activity revealed neuroserpin to be a slow binding inhibitor of plasminogen activators and plasmin. Thus, we postulate that neuroserpin could represent a regulatory element of extracellular proteolytic events in the nervous system mediated by plasminogen activators or plasmin.

Human neuroserpin (PI12): cDNA cloning and chromosomal localization to 3q26.

Neuroserpin is a novel serine protease inhibitor of the serpin family. It has been reported as a 55-kDa glycoprotein that is secreted from the axons of cultured central and peripheral nervous system neurons. In situ hybridization and Northern blot analyses at different developmental stages of the chicken revealed that neuroserpin is predominantly expressed in the nervous system and that most cells expressing neuroserpin can be qualified as bona fide neurons. We have isolated the full-length cDNA for human neuroserpin from a fetal retina cDNA library. The open reading frame of the cDNA of human neuroserpin, like that of its chicken counterpart, encodes a protein of 410 amino acids. The human and the chicken neuroserpin exhibit an amino acid sequence identity of 80%. Northern blot analysis of human organs demonstrated predominant expression of neuroserpin in the brain. By fluorescence in situ hybridization the human neuroserpin gene (HGMW-approved symbol PI12) was mapped to region q26 of chromosome 3.