Allan Christian Shaw

Hybrid Receptors Formed by Insulin Receptor (IR) and Insulin-like Growth Factor I Receptor (IGF-IR) Have Low Insulin and High IGF-1 Affinity Irrespective of the IR Splice Variant

Insulin receptor (IR) and insulin-like growth factor I receptor (IGF-IR) are both from the same subgroup of receptor tyrosine kinases that exist as covalently bound receptor dimers at the cell surface. For both IR and IGF-IR, the most described forms are homodimer receptors. However, hybrid receptors consisting of one-half IR and one-half IGF-IR are also present at the cell surface. Two splice variants of IR are expressed that enable formation of two isoforms of the IGF-IR/IR hybrid receptor. In this study, these two splice variants of hybrid receptors were studied with respect to binding affinities of insulin, insulin-like growth factor I (IGF-I), and insulin-like growth factor II (IGF-II). Unlike previously published data, in which semipurified receptors have been studied, we found that the two hybrid receptor splice variants had similar binding characteristics with respect to insulin, IGF-I, and IGF-II binding. We studied both semipurified and purified hybrid receptors. In all cases we found that IGF-I had at least 50-fold higher affinity than insulin, irrespective of the splice variant. The binding characteristics of insulin and IGF-I to both splice variants of the hybrid receptors were similar to classical homodimer IGF-IR.

A novel high-affinity peptide antagonist to the insulin receptor

In this publication we describe a peptide insulin receptor antagonist, S661, which is a single chain peptide of 43 amino acids. The affinity of S661 for the insulin receptor is comparable to that of insulin and the selectivity for the insulin receptor versus the IGF-1 receptor is higher than that of insulin itself. S661 is also an antagonist of the insulin receptor of other species such as pig and rat, and it also has considerable affinity for hybrid insulin/IGF-1 receptors. S661 completely inhibits insulin action, both in cellular assays and in vivo in rats. A biosynthetic version called S961 which is identical to S661 except for being a C-terminal acid seems to have properties indistinguishable from those of S661. These antagonists provide a useful research tool for unraveling biochemical mechanisms involving the insulin receptor and could form the basis for treatment of hypoglycemic conditions.

Characterization of a secreted Chlamydia protease

Chlamydiae are obligate intracellular bacteria that are important human pathogens. The Chlamydia genomes contain orthologues to secretion apparatus proteins from other intracellular bacteria, but only a few secreted proteins have been identified. Most likely, effector proteins are secreted in order to promote infection. Effector proteins cannot be identified by motif or similarity searches. As a new strategy for identification of secreted proteins we have compared 2D‐PAGE profiles of [35S]‐labelled Chlamydia proteins from whole lysates of infected cells to 2D‐PAGE profiles of proteins from purified Chlamydia. Several secretion candidates from Chlamydia trachomatis D and Chlamydia pneumoniae were detected by this method. Two protein spots were identified among the candidates. These represent fragments of the ‘chlamydial protease‐ or proteasome‐like activity factor’ (CPAF) and were clearly present in 2D‐PAGE profiles of whole lysates of infected cells but absent from purified Chlamydia. CPAF was recently identified by Zhong and colleagues as a secreted protease which cleaves host cell transcription factors essential for MHC class I and II antigen presentation. The identification of CPAF in this paper verifies the applicability of the described method for the identification of secreted proteins. We extend the findings by Zhong et al. by proteome studies of expression and turnover of C. trachomatis CPAF showing that the degradation of C. trachomatis D CPAF in the host cell is very limited. Furthermore, we show that two fragments of CPAF exist in C. pneumoniae as well as in C. trachomatis.

Comparative proteome analysis of Chlamydia trachomatis serovar A, D and L2

Chlamydia trachomatis represents a group of human pathogenic obligate intracellular and gram‐negative bacteria. The genome of C. trachomatis D comprises 894 open reading frames (ORFs). In this study the global expression of genes in C. trachomatis A, D and L2, which are responsible for different chlamydial diseases, was investigated using a proteomics approach. Based on silver stained two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE), gels with purified elementary bodies (EB) and auto‐radiography of gels with 35S‐labeled C. trachomatis proteins up to 700 protein spots were detectable within the range of the immobilized pH gradient (IPG) system used. Using mass spectrometry and N‐terminal sequencing followed by database searching we identified 250 C. trachomatis proteins from purified EB of which 144 were derived from different genes representing 16% of the ORFs predicted from the C. trachomatis D genome and the 7.5 kb C. trachomatis plasmid. Important findings include identification of proteins from the type III secretion apparatus, enzymes from the central metabolism and confirmation of expression of 25 hypothetical ORFs and five polymorphic membrane proteins. Comparison of serovars generated novel data on genetic variability as indicated by electrophoretic variation and potentially important examples of serovar specific differences in protein abundance. The availability of the complete genome made it feasible to map and to identify proteins of C. trachomatis on a large scale and the integration of our data in a 2‐D PAGE database will create a basis for post genomic research, important for the understanding of chlamydial development and pathogenesis.

Mapping and identification of interferon gamma-regulated HeLa cell proteins separated by immobilized pH gradient two-dimensional gel electrophoresis.

Interferon gamma (IFN‐γ) is a potent immunomodulatory lymphokine, secreted by activated T‐lymphocytes and NK‐cells during the cellular immune response. Actions of IFN‐γ are mediated through binding to the IFN‐γ‐receptor, present on most cells, and the subsequent activation of a great magnitude of IFN‐γ responsive genes has been reported previously. Our goal is to identify and map IFN‐γ‐regulated HeLa cell proteins to the two‐dimensional polyacrylamide gel electrophoresis with the immobilized pH gradient (IPG) two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) system. A semiconfluent layer of HeLa cells was grown on tissue culture plates, and changes in protein expression due to 100 U/mL IFN‐γ were investigated at different periods after treatment, using pulse labeling with [35S]methionine/cysteine in combination with 2‐D PAGE (IPG). The identity of eight protein spots was elucidated by matrix‐assisted laser desorption/ionization‐mass spectrometry (MALDI‐MS), and several variants of the IFN‐γ‐inducible tryptophanyl‐tRNA synthetase (hWRS) were detected by immunoblotting.

Genetic differences in the Chlamydia trachomatis tryptophan synthase alpha-subunit can explain variations in serovar pathogenesis

The human pathogen Chlamydia trachomatis is an obligate intracellular bacterium, characterized by a developmental cycle that alternates between the infectious, extracellular elementary bodies and intracellular, metabolically active reticulate bodies. The cellular immune effector interferon gamma (IFN-gamma) inhibits chlamydial multiplication in human epithelial cells by induction of the tryptophan degrading enzyme indoleamine 2,3 dioxygenase. IFN-gamma causes persistent C. trachomatis serovar A infections with atypical reticulate bodies that are unable to redifferentiate into elementary bodies and show diminished expression of important immunogens, but not of GroEL. However, the sensitivity to IFN-gamma varies among serovars of C. trachomatis. In our previous study significant IFN-gamma-specific, but tryptophan reversible, induction of proteins in C. trachomatis A and L2 with molecular masses of approximately 30 and 40 kDa was observed on 2D-gels. The 30-kDa protein from C. trachomatis L2 migrated with a significantly lower molecular weight in C. trachomatis A. In this paper we include C. trachomatis B, C and D in our investigations and identify the proteins as alpha- and beta-subunits of the chlamydial tryptophan synthase using matrix-assisted laser desorption/ionization mass spectrometry. DNA sequencing of the trpA genes from C. trachomatis A and C shows that the TrpA in these serovars is a 7.7-kDa truncated version of C. trachomatis D and L2 TrpA. The truncation probably impairs the TrpA activity, thus elucidating a possible molecular mechanism behind variations in the pathogenesis of C. trachomatis serovars.

A Novel High-Throughput Screening Method for Microbial Transglutaminases with High Specificity toward Gln141 of Human Growth Hormone

PEGylation modification has been used to improve the pharmacokinetic properties of protein-based drugs. For example, PEGylated human growth hormone (hGH) has been shown to exhibit better pharmacokinetic profiles than the unmodified hGH. Unlike chemical PEGylation of hGH that is difficult to be controlled to result in homogeneity, microbial transglutaminase (mTGase) only conjugates poly(ethelene glycol) (PEG) on glutamine-40 (Q40) and glutamine-141 (Q141) of hGH, the only glutamine residues exposed. Yet, an mTGase that can selectively conjugate PEG to only 1 glutamine residue is more desirable to control the homogeneity of the product. In this study, the authors have developed a novel high-throughput assay, with which they have identified 5 mTGase mutants that are highly specific for conjugating PEG to Q141 of hGH. In this scintillation proximity assay (SPA)–based method, the authors have (1) achieved a high expression level of active mTGase, which is toxic to the living cell, directly from Escherichia coli (0.2 U/mL/OD600) by in vivo activation; (2) developed a high-throughput affinity purification method to eliminate the strong interference of cellular protein to mTGase reaction; and (3) used therapeutic protein as the substrate. This method is highly sensitive, is easily automated, and could be generally applied to screening mTGases with desired specificity targeting on different therapeutic proteins.

Comparative proteome analysis of breast cancer and normal breast

Breast cancer is a leading cause of death for women. The underlying molecular mechanism is still not well understood. In this study, two-dimensional gel electrophoresis combined with mass spectrometry was used to analyze changes in the proteome of infiltrating ductal carcinoma compared to normal breast tissue. Ten sets of two-dimensional gels per experimental condition were analyzed and more than 500 spots each were detected. This revealed 39 spots for which expression in breast cancer cells were reproducibly altered more than twofold compared to normal controls (p<0.01). These spots represented 25 different proteins after identification using the database search after mass spectrometry, comprising cell defense proteins, enzymes involved in glycolytic energy metabolism and homeostasis, protein folding and structural proteins, proteins involved in cytoskeleton and cell motility, and proteins involved in other functions. In addition, 28 nondifferentially expressed proteins with different functions were also mapped and identified, which might help to establish a two-dimensional gel electrophoresis reference map of human breast cancer. Our study shows that proteomics offers a powerful methodology to detect the proteins that show different expression patterns in breast cancer tissue and may provide an accurate molecular classification. The differentially expressed proteins may be used as potential candidate markers for diagnostic purposes or for determination of tumor sensitivity to therapy. The functional implications of the identified proteins are discussed.

Mapping and identification of HeLa cell proteins separated by immobilized pH‐gradient two‐dimensional gel electrophoresis and construction of a two‐dimensional polyacrylamide gel electrophoresis database

The HeLa cell line, a human adenocarcinoma, is used in many research fields, since it can be infected with a wide range of viruses and intracellular bacteria. Therefore, the mapping of HeLa cell proteins is useful for the investigation of parasite host cell interactions. Because of the recent improvements of two‐dimensional gel electrophoresis with immobilized pH gradients (IPG) compared to isoelectric focusing with carrier ampholytes, a highly reproducible method for examining global changes in HeLa cell protein expression due to different stimuli is now available. Therefore, we have initiated the mapping of [35S]methionine/cysteine‐labeled HeLa cell proteins with the 2‐D PAGE (IPG)‐system, using matrix‐assisted laser desorption/ionization‐mass spectrometry (MALDI‐MS) and N‐terminal sequencing for protein identification. To date 21 proteins have been identified and mapped. In order to make these and future data accessible for interlaboratory comparison, we constructed a 2‐D PAGE database on the World Wide Web.

Upregulation of MetC Is Essential for d-Alanine-Independent Growth of an alr/dadX-Deficient Escherichia coli Strain

D-Alanine is a central component of the cell wall in most prokaryotes. D-Alanine synthesis in Escherichia coli is carried out by two different alanine racemases encoded by the alr and dadX genes. Deletion of alr and dadX from the E. coli genome results in a D-alanine auxotrophic phenotype. However, we have observed growth of prototrophic phenotypic revertants during routine culturing of a D-alanine auxotrophic strain. We present a detailed comparison of the proteome and transcriptome profiles of the D-alanine auxotroph and a prototrophic revertant strain. Most noticeably, a general upregulation of genes involved in methionine synthesis in the revertant strain was detected. The appearance of the revertant phenotype was genetically linked to point mutations in the methionine repressor gene (metJ). Our results reveal an alternative metabolic pathway which can supply essential d-alanine for peptidoglycan synthesis of alr- and dadX-deficient E. coli mutants and provide evidence for significant alanine racemase coactivity of the E. coli cystathionine beta-lyase (MetC).