A gene encoding a novel RFX-associated transactivator is mutated in the majority of MHC class II deficiency patients.

Major histocompatibility class II (MHC-II) molecules are transmembrane proteins that have a central role in development and control of the immune system. They are encoded by a multigene family and their expression is tightly regulated. MHC-II deficiency (OMIM 209920) is an autosomal recessive immunodeficiency syndrome resulting from defects in trans-acting factors essential for transcription of MHC-II genes. There are four genetic complementation groups (A, B, C and D), reflecting the existence of four MHC-II regulators. The factors defective in groups A (CIITA), C (RFX5) and D (RFXAP) have been identified. CIITA is a non-DNA-binding co-activator that controls the cell-type specificity and inducibility of MHC-II expression. RFX5 and RFXAP are two subunits of RFX, a multi-protein complex that binds the X box motif of MHC-II promoters. Mutations in the genes encoding RFX5 (RFX5) or RFXAP (RFXAP) abolish binding of RFX (refs 7,8,12). Similar to groups C and D, group B is characterized by a defect in RFX binding, and although it accounts for the majority of patients, the factor defective in group B has remained unknown. We report here the isolation of RFX by a novel single-step DNA-affinity purification approach and the identification of RFXANK, the gene encoding a third subunit of RFX. RFXANK restores MHC-II expression in cell lines from patients in group B and is mutated in these patients. RFXANK contains a protein-protein interaction region consisting of three ankyrin repeats. Its interaction with RFX5 and RFXAP is essential for binding of the RFX complex to MHC-II promoters.

Changes of the cortex proteome and Apolipoprotein E in transgenic mouse models of Alzheimer's Disease.

Transgenic mice carrying human Amyloid Precursor Protein mutations present amyloid plaque deposition in the brain upon aging. In this study, we characterized the changes of cortex proteome and endogenous Apolipoprotein E in these mice. Differential analysis of two-dimensional electrophoresis images revealed spots altered upon aging, transgene addition and plaque deposition. Alpha-synuclein and cytochrome oxidase polypeptide Va were up-regulated in transgenic mice. Upon aging, expression of ATP synthase alpha, alpha enolase, UMP-CMP kinase, and dihydropyrimidinase like-2 protein was modified. These proteins and their modification probably play a role in the amyloid aggregate formation in these mice.

Protein identification with N and C-terminal sequence tags in proteome projects.

Genome sequences are available for increasing numbers of organisms. The proteomes (protein complement expressed by the genome) of many such organisms are being studied with two-dimensional (2D) gel electrophoresis. Here we have investigated the application of short N-terminal and C-terminal sequence tags to the identification of proteins separated on 2D gels. The theoretical N and C termini of 15, 519 proteins, representing all SWISS-PROT entries for the organisms Mycoplasma genitalium, Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae and human, were analysed. Sequence tags were found to be surprisingly specific, with N-terminal tags of four amino acid residues found to be unique for between 43% and 83% of proteins, and C-terminal tags of four amino acid residues unique for between 74% and 97% of proteins, depending on the species studied. Sequence tags of five amino acid residues were found to be even more specific. To utilise this specificity of sequence tags for protein identification, we created a world-wide web-accessible protein identification program, TagIdent (http://www.expasy.ch/www/tools.html), which matches sequence tags of up to six amino acid residues as well as estimated protein pI and mass against proteins in the SWISS-PROT database. We demonstrate the utility of this identification approach with sequence tags generated from 91 different E. coli proteins purified by 2D gel electrophoresis. Fifty-one proteins were unambiguously identified by virtue of their sequence tags and estimated pI and mass, and a further 11 proteins identified when sequence tags were combined with protein amino acid composition data. We conlcude that the TagIdent identification approach is best suited to the identification of proteins from prokaryotes whose complete genome sequences are available. The approach is less well suited to proteins from eukaryotes, as many eukaryotic proteins are not amenable to sequencing via Edman degradation, and tag protein identification cannot be unambiguous unless an organism's complete sequence is available.

High-throughput mass spectrometric discovery of protein post-translational modifications.

The availability of genome sequences, affordable mass spectrometers and high-resolution two-dimensional gels has made possible the identification of hundreds of proteins from many organisms by peptide mass fingerprinting. However, little attention has been paid to how information generated by these means can be utilised for detailed protein characterisation. Here we present an approach for the systematic characterisation of proteins using mass spectrometry and a software tool FindMod. This tool, available on the internet at http://www.expasy.ch/sprot/findmod.html , examines peptide mass fingerprinting data for mass differences between empirical and theoretical peptides. Where mass differences correspond to a post-translational modification, intelligent rules are applied to predict the amino acids in the peptide, if any, that might carry the modification. FindMod rules were constructed by examining 5153 incidences of post-translational modifications documented in the SWISS-PROT database, and for the 22 post-translational modifications currently considered (acetylation, amidation, biotinylation, C-mannosylation, deamidation, flavinylation, farnesylation, formylation, geranyl-geranylation, gamma-carboxyglutamic acids, hydroxylation, lipoylation, methylation, myristoylation, N -acyl diglyceride (tripalmitate), O-GlcNAc, palmitoylation, phosphorylation, pyridoxal phosphate, phospho-pantetheine, pyrrolidone carboxylic acid, sulphation) a total of 29 different rules were made. These consider which amino acids can carry a modification, whether the modification occurs on N-terminal, C-terminal or internal amino acids, and the type of organisms on which the modification can be found. We illustrate the utility of the approach with proteins from 2-D gels of Escherichia coli and sheep wool, where post-translational modifications predicted by FindMod were confirmed by MALDI post-source decay peptide fragmentation. As the approach is amenable to automation, it presents a potentially large-scale means of protein characterisation in proteome projects.

Elevation of apolipoprotein E in the CSF of cattle affected by BSE.

The cerebrospinal fluid (CSF) of patients suffering from Creutzfeldt-Jakob disease (CJD) display two unique polypeptide chains by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). In the absence of a well-defined ante-mortem diagnostic test for bovine spongiform encephalopathy (BSE), spinal fluid samples of eight normal cows and eight cows known to carry BSE by post-mortem histological analysis were investigated to verify if equivalent polypeptides were present. Proteins with similar migration to human CJD polypeptides were not detected. But surprisingly, a cluster of polypeptide spots that was faint or not detected in normal bovine CSF samples was found to be elevated or massively increased in BSE CSF samples (more than 10-fold increase). These elevated polypeptide chains were identified as apolipoprotein E.

CSF detection of the 14-3-3 protein in unselected patients with dementia.

OBJECTIVE: To determine the usefulness of the 14-3-3 test in patients with dementia of various causes. BACKGROUND: Recent reports have suggested that the detection of the 14-3-3 protein in the CSF of patients with Creutzfeldt--Jakob disease is a highly sensitive and specific marker of the disease that might be used as a diagnostic criterion. We examined the validity of this test when applied to a cohort of unselected patients prospectively examined for an ongoing dementing process. METHODS: One hundred patients underwent an extensive neurologic examination for dementia, including a CSF 14-3-3 protein immunoblotting assay. Final clinical diagnoses were compared with the qualitative results of the test, and statistical measures of test validity were carried out. RESULTS: We found a positive test in 14 of 100 patients, only two of whom had definite Creutzfeldt--Jakob disease. Positive results were found in patients with various degenerative dementias, including AD (4), frontotemporal dementia (2), and dementia with Lewy body (1), and in patients with vascular dementia (1), carcinomatous meningitis (1), and anoxic encephalopathy (1). In two other positive patients, the dementia could not be confidently classified. Sensitivity, specificity, and negative predictive value were fairly good, but positive predictive value was poor. Similar results were found independently of the disease duration. There was no correlation between intensity nor pattern of the 14-3-3 protein expression and diagnosis. CONCLUSIONS: The 14-3-3 test is not valid for discriminating between Creutzfeldt--Jakob disease and non-Creutzfeldt--Jakob disease in unselected patients with dementia. Positive results are found in various degenerative and secondary, prion-unrelated dementias.

Two-dimensional polyacrylamide gel electrophoresis analysis of cryoglobulins and identification of an IgM-associated peptide.

The clonality of immunoglobulins (Igs) in cryoprecipitates (n = 41) was studied by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Our series included 24 cryoglobulins characterized by immunofixation electrophoresis (IF), 12 'trace amount' cryoglobulins, defined by a protein content in the precipitate of less than 0.05 mg/ml of serum, and five cryoglobulins of undetermined protein composition by IF. 2-D PAGE analysis showed polyclonal IgG associated either with monoclonal Igs (type II cryoglobulins; n = 14) or with polyclonal IgM (type III cryoglobulins; n = 14). In ten cryoprecipitates (two 'trace amount' cryoglobulins as well as seven of 19 type II and as one of five type III cryoglobulins by IF) polyclonal IgG were associated with a mixture of polyclonal and monoclonal IgM. These cryoglobulins were tentatively named type II-III cryoglobulins. A monoclonal IgM was observed in one cryoprecipitate (type I cryoglobulins). Two cryoglobulins presented unexpected 2-D patterns, characterized by the presence of oligoclonal IgM, with trace amounts of Igs of different isotypes (tentatively named type II-III(variant) cryoglobulins). A peptide of 44 kDa with a pI of 5.45 was observed in all cryoglobulins containing IgM (n = 40). This peptide was also present in purified monoclonal or polyclonal IgM fractions. N-terminal microsequencing (12 amino acid residues) revealed that this IgM-associated peptide was an unknown protein. Our results highlight the role of 2-D PAGE as an aid in the analysis of cryoglobulins.

Clonal imbalances of serum immunoglobulins after allogeneic bone marrow transplantation: an analysis by high-resolution two-dimensional gel electrophoresis.

The clonality pattern of immunoglobulins (Igs) produced after allogeneic bone marrow transplantation (BMT) was studied by high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of serum samples and purified Igs. With this technique, the light (L) chain of a monoclonal Ig usually appears as a single spot. Thus, the degree of clonal diversity of the functional B cells can be appreciated by the electrophoretic pattern of the serum L chains. Furthermore, 2D-PAGE allows a semi-quantitative determination of prominent Ig clones according to the size of L chain spots. We found that serum electrophoretograms of 8/19 patients after BMT (5-9 months) revealed L chain patterns which were similar to those of normal polyclonal Igs, that is, less than five distinguishable small spots among a cloud-like indiscrete L chain spots region ('polyclonal' pattern). A spectrum of clonal abnormalities was observed on the electrophoretograms of 11/19 patients: in five patients, multiple small L chain spots (corresponding to Ig concentrations between 0.2 and 2 g/l) were detected ('oligoclonal' pattern), whereas in six others, 'typical' monoclonal Igs (Ig concentrations > 2 g/l) were observed with (3/19 patients) or without (3/19 patients) multiple small clonal components. Sequential analysis of serum obtained from patients at different times after BMT revealed that imbalanced clonal reconstitution was transient and evolved towards apparently normal polyclonal Ig production. Our observations show that the development of clonal 'gammopathies' after BMT is a frequent, but not obligatory phenomenon. It may reflect a transient restriction of the B cell repertoire either through a limited outgrowth of precursor cells or through selective antigenic pressures.

Human gingival crevicular fluid contains MRP8 (S100A8) and MRP14 (S100A9), two calcium-binding proteins of the S100 family.

Human gingival crevicular fluid contains unidentified proteins which might play a role as markers in periodontal diseases. Therefore, low-molecular-weight proteins found in human gingival crevicular fluid (GCF), but absent from serum, were identified in the present study by means of two-dimensional electrophoresis (2-D PAGE) analysis. GCF, serum, and whole saliva were collected from periodontitis and healthy subjects, as well as from edentulous and newborn subjects. Protein samples were separated by two-dimensional polyacrylamide gel electrophoresis, stained with silver, and compared with reference protein maps in the SWISS-2D PAGE database. In GCF and saliva from periodontitis patients and healthy subjects, four dominant low-molecular-mass (from 8 to 14 kDa) acidic spots were observed. They were not found in serum and were less visible in saliva from edentulous and newborn subjects. From N-terminal amino acid sequencing, the two 2-D protein spots of 8 kDa and isoelectric points between 6.5 and 7.0 were both identified as protein MRP8 (SI00A8), a member of the S100 family of calcium-binding proteins. Using peptide mass fingerprinting and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), we identified the other two protein spots, with mass of 14 kDa and isoelectric points between 5.5 and 6.0, as protein MRP14 (S100A9), also belonging to the S100 family. The presence of MRP8 and MRP14 in GCF was confirmed by Western blot, with monoclonal antibodies. The two polypeptides, MRP8 and MRP14, identified in GCF represent the major difference between the 2-D PAGE patterns of serum and GCF, and we hypothesize that they may play an important role in the gingival sulcus and could represent possible markers for periodontal diseases.

Large-scale amino-acid analysis for proteome studies.

Amino-acid analysis is a relatively new method for identification of proteins separated by two-dimensional gel electrophoresis and blotted onto polyvinylidene difluoride (PVDF) membranes. This article describes modified amino-acid analysis methods for this purpose. Streamlined sample handling is a key feature of the process. To minimise sample manipulation, a single vial is used for hydrolysis and the protein hydrolysate on PVDF membrane is extracted by a one-step procedure. The hydrolysate should not be stored for long periods before analysis. Applications of the technique are presented to demonstrate the identification procedure. This approach is the most cost-effective and time-effective first step in mass protein screening for a large-scale proteome project.

Sharing of worldwide spread knowledge using hypermedia facilities & fast communication protocols (Mosaic and World Wide Web): the example of ExPASy.

The sharing of knowledge worldwide using hypermedia facilities and fast communication protocols (i.e., Mosaic and World Wide Web) provides a growth capacity with tremendous versatility and efficacy. The example of ExPASy, a molecular biology server developed at the University Hospital of Geneva, is striking. ExPASy provides hypermedia facilities to browse through several up-to-date biological and medical databases around the world and to link information from protein maps to genome information and diseases. Its extensive access is open through World Wide Web. Its concept could be extended to patient data including texts, laboratory data, relevant literature findings, sounds, images and movies. A new hypermedia culture is spreading very rapidly where the international fast transmission of documents is the central element. It is part of the emerging new "information society".

A humanist's legacy in medical informatics: visions and accomplishments of Professor Jean-Raoul Scherrer.

OBJECTIVE: To report about the work of Prof. Jean-Raoul Scherrer, and show how his humanist vision, his medical skills and his scientific background have enabled and shaped the development of medical informatics over the last 30 years. RESULTS: Starting with the mainframe-based patient-centered hospital information system DIOGENE in the 70s, Prof. Scherrer developed, implemented and evolved innovative concepts of man-machine interfaces, distributed and federated environments, leading the way with information systems that obstinately focused on the support of care providers and patients. Through a rigorous design of terminologies and ontologies, the DIOGENE data would then serve as a basis for the development of clinical research, data mining, and lead to innovative natural language processing techniques. In parallel, Prof. Scherrer supported the development of medical image management, ranging from a distributed picture archiving and communication systems (PACS) to molecular imaging of protein electrophoreses. Recognizing the need for improving the quality and trustworthiness of medical information on the Web, Prof. Scherrer created the Health-On-the-Net (HON) foundation. CONCLUSIONS: These achievements, made possible thanks to his visionary mind, deep humanism, creativity, generosity and determination, have made of Prof. Scherrer a true pioneer and leader of the human-centered, patient-oriented application of information technology for improving healthcare.

Automatic learning strategies and their application to electrophoresis analysis.

Automatic learning plays an important role in image analysis and pattern recognition. A taxonomy of automatic learning strategies is presented; this categorization is based on the amount of inferences the learning element must perform to bridge the gap between environmental and system knowledge representation level. Four main categories are identified and described: rote learning, learning by deduction, learning by induction, and learning by analogy. An application of learning by induction to medical image analysis is then exposed. It consists in the classification of two-dimensional gel electrophoretograms into meaningful distinct classes, as well in their conceptual description.

Characterization of human plasma glycoproteins separated by two-dimensional gel electrophoresis.

Purification of protein isoforms for the characterization of post-translational modifications, such as glycosylation, can be laborious and demanding. We report a means of determining monosaccharide composition and the identity of glycoproteins from a single spot on a two-dimensional (2-D) gel. The sensitivity of the method depends on the degree of glycosylation of the protein. We show that bovine fetuin can be analyzed and identified at the level of 100 pmol. 2-D reference maps enable quick identification of glycoprotein isoforms, and the nature of glycosylation differences. Human sera glycoforms were isolated by micropreparative 2-D PAGE using a narrow-range immobilized pH gradient. Single spots excised from one polyvinylidene difluoride blot of a 2-D gel were used sequentially for sialic acid analysis, neutral and amino sugar analysis, and finally amino acid analysis. The glycosylation variations in isoforms of human fetuin and alpha-1-antitrypsin were determined. The amino acid composition, in conjunction with protein pI and MW, successfully identified the glycoproteins.

From proteins to proteomes: large scale protein identification by two-dimensional electrophoresis and amino acid analysis.

Separation and identification of proteins by two-dimensional (2-D) electrophoresis can be used for protein-based gene expression analysis. In this report single protein spots, from polyvinylidene difluoride blots of micropreparative E. coli 2-D gels, were rapidly and economically identified by matching their amino acid composition, estimated pI and molecular weight against all E. coli entries in the SWISS-PROT database. Thirty proteins from an E. coli 2-D map were analyzed and identities assigned. Three of the proteins were unknown. By protein sequencing analysis, 20 of the 27 proteins were correctly identified. Importantly, correct identifications showed unambiguous "correct" score patterns. While incorrect protein identifications also showed distinctive score patterns, indicating that protein must be identified by other means. These techniques allow large-scale screening of the protein complement of simple organisms, or tissues in normal and disease states. The computer program described here is accessible via the World Wide Web at URL address (http:@expasy.hcuge.ch/).