Josef Van Damme

Human 2-D PAGE databases for proteome analysis in health and disease: http ://biobase.dk/cgi-bin/celis

Human 2‐D PAGE Databases established at the Danish Centre for Human Genome Research are now available on the World Wide Web (http://biobase.dk/cgi‐bin/celis). The databanks, which offer a comprehensive approach to the analysis of the human proteome both in health and disease, contain data on known and unknown proteins recorded in various IEF and NEPHGE 2‐D PAGE reference maps (non‐cultured keratinocytes, non‐cultured transitional cell carcinomas, MRC‐5 fibroblasts and urine). One can display names and information on specific protein spots by clicking on the image of the gel representing the 2‐D gel map in which one is interested. In addition, the database can be searched by protein name, keywords or organelle or cellular component. The entry files contain links to other databases such as Medline, Swiss‐Prot, PIR, PDB, CySPID, OMIM, Methabolic pathways, etc. The on‐line information is updated regularly.

The atypical kinase Cdk5 is activated by insulin, regulates the association between GLUT4 and E-Syt1, and modulates glucose transport in 3T3-L1 adipocytes.

Here, we report that Cdk5 activation is stimulated by insulin and plays a key role in the regulation of GLUT4-mediated glucose uptake in 3T3-L1 adipocytes. Insulin activation of Cdk5 requires PI3K signaling. Insulin-activated Cdk5 phosphorylates E-Syt1, a 5 C2-domain protein-related to the synaptotagmins that is induced during adipocyte differentiation. Phosphorylated E-Syt1 associates with GLUT4, an event inhibited by the Cdks inhibitor roscovitine. Cdk5 silencing inhibits glucose uptake by 3T3-L1 adipocytes. These studies elucidate a previously unknown activity of Cdk5 and demonstrate the involvement of this kinase in the regulation of insulin-dependent glucose uptake in adipocytes.

Analysis of the catalytic site of the actin ADP-ribosylating Clostridium perfringens iota toxin

The enzyme component of the actin ADP‐ribosylating Clostridium perfringens iota toxin was affinity labelled by UV irradiation in the presence of [carbonyl‐14C]NAD. A peptide containing the radiolabel was generated by CNBr cleavage and subsequent proteolysis with trypsin. Its amino acid sequence is Gly‐Ser‐Pro‐Gly‐Ala‐Tyr‐Leu‐Ser‐Ala‐Ile‐Pro‐Gly‐Tyr‐Ala‐Gly‐X‐Tyr‐Glu‐Val‐Leu‐Leu‐Asn‐His‐Gly‐Ser‐Lys corresponding with the region Gly‐363 through Lys‐388 in the C. perfringens iota toxin. Mass spectrometric data as well as the results of the PTH‐amino acid analysis are in line with a modification of a glutamic acid side chain located at position 378. Therefore, in addition to Glu‐380, as could be concluded by analogy with other ADP‐ribosyltransferases, Glu‐378 may play a pivotal role in the active site of C. perfringens iota toxin.

Evidence for new phosphorylation sites for protein kinase C and cyclic AMP-dependent protein kinase in bovine heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

Bovine heart 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase (PFK‐2/FBPase‐2) was phosphorylated by incubation with [γ‐32P]MgATP and cyclic AMP‐dependent protein kinase (PKA) or protein kinase C (PKC). After digestion with chymotrypsin, the phosphorylation sites for the two protein kinases were identified by peptide mapping, and microsequencing. Evidence for new phosphorylation sites for PKA (Ser‐483) and PKC (Ser‐84 and Ser‐466) was obtained.

Human Cellular Protein Patterns and Their Link to Genome DNA Mapping and Sequencing Data: Towards an Integrated Approach to the Study of Gene Expression

Analysis of cellular protein patterns by computer-aided two-dimensional gel electrophoresis together with recent advances in protein sequence analysis and expression systems have made possible the establishment of comprehensive two-dimensional gel protein databases that may link protein and DNA mapping and sequence information and that offer an integrated approach to the study of gene expression. With the integrated approach offered by two-dimensional gel protein databases it is now possible to reveal phenotype-specific protein(s), to microsequence them, to search for homology with previous identified proteins, to clone the cDNAs, to assign partial protein sequences to genes for which the full DNA sequence and the chromosome location are known, and to study the regulatory properties and function of groups of proteins that are coordinately expressed in a given biological process. Comprehensive two-dimensional gel protein databases will provide an integrated picture of the expression levels and properties of the thousands of protein components of organelles, pathways, and cytoskeletal systems, both under physiological and abnormal conditions, and are expected to lead to the identification of new regulatory networks. So far, about 20% (600 out of 2,980) of the total number of proteins recorded in the human keratinocyte protein database have been identified and we are actively gathering qualitative and quantitative biological data on all resolved proteins. Given the current improvements on microsequencing as well as the availability of specific antibodies, it seems feasible to expect that most known keratinocyte proteins will be identified in the very near future. This feast will reveal a wealth of new proteins that will become amenable to experimentation both at the biochemical and molecular biology level.