Garry L. Corthals

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 multi-gene 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 7,12). 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.

Expression of a tyrosine phosphorylated, DNA binding Stat3β dimer in bacteria

The signal transducer and activator of transcription (STAT) proteins deliver signals from the cell membrane to the nucleus. An N‐terminally truncated fragment of murine Stat3β, Stat3βtc (127–722), was produced in bacteria. STAT proteins must be specifically phosphorylated at a single tyrosine residue for dimerization and DNA binding. Therefore, Stat3βtc was coexpressed with the catalytic domain of the Elk receptor tyrosine kinase. Stat3βtc was quantitatively phosphorylated by this kinase domain. Gel filtration chromatography revealed a Stat3βtc dimer. Y705 was identified as the major phosphorylated residue of Stat3βtc. This corresponds to the tyrosine residue which is phosphorylated by the Janus kinase in vivo. The phosphorylated Stat3βtc specifically bound to DNA binding sites. The described protocol allows the production of large amounts of activated protein for biochemical and pharmaceutical studies.

Towards an Integrated Analytical Technology for the Generation of Multidimensional Protein Expression Maps

A proteome has been defined as the protein complement expressed by the genome of an organism, or, in multicellular organisms, as the protein complement expressed by a tissue or differentiated cell [1]. In the most commonly used approach to proteome analysis the proteins extracted from the cell or tissue to be analyzed are separated by high resolution two dimensional gel electrophoresis (2DE), detected in the gel by staining or, if radiolabeled, by autoradiography, and identified by their amino acid sequence. The ease, sensitivity and speed with which gel separated proteins can be identified by the use of recently developed mass spectrometric techniques (MS) has led to a dramatic increase in interest in proteome studies (reviewed in [2]). One of the most attractive features of such analyses is that complex biological systems can potentially be studied as complete systems, rather than as a conglomerate of individual components.

Strategies and Methods for Proteome Analysis

A proteome has been defined as the protein complement expressed by the genome of an organism (Wilkins, et al. 1996). In multicellular organisms the proteome is the protein complement expressed by a tissue or differentiated cell. The most common approach to proteome analysis involves separation of proteins by one- or two-dimensional gel electrophoresis (IEF/SDS-PAGE), enzymatic cleavage of selected proteins, tandem mass spectrometry (MS/MS) of peptides, and finally data interpretation by computer routines which also search databases (Fig. 1.1). In most cases the availability of protein and DNA sequences in public databases eliminates the need for complete protein sequence analysis. Protein sequences are more rapidly identified by partial sequence analysis using tandem mass spectrometry which allows rapid, complete gene identification. As reliable as protein identification by mass spectrometry has become there are still many obstacles that prevent proteome analysis from becoming as automated and routine as genome analysis.