Formation of heterodimers between wild type and mutant trp aporepressor polypeptides of Escherichia coli

Availability of the three-dimensional structure of the trp repressor of Escherichia coli and a large group of repressor mutants has permitted the identification and analysis of mutants with substitutions of the amino acid residues that from the tryptophan binding pocket. Mutant aporepressors selected for study were overproduced using a multicopy expression plasmid. Equilibrium dialysis with 14 C-tryptophan and purified mutant and wild type aporepressors was employed to determine tryptophan binding constants. The results obtained indicate that replacement of theronine 44 by methionine (TM44) or arginine 84 by histidine (RH84) lowers the affinity for tryptophan approximately two-and four-fold, respectively. Replacement of ariginine 54 by histidine (RH84) or glycine 85 by ariginine (GR85) results in complete loss of tryptophan binding activity. Purified mutant and wild type aporepressors were used in vitro heterodimer studies. The trp repressor of E. coli functions as a stable dimer. A large number of trp repressor mutants prduces defective repressors that are transdominant to the wild type repressor in vivo. The transdominance presumably results from the formation of inactive or slightly active heterodimers between the mutant and wild type polypeptide subunits. An in vitro assay was developed to detect and measure heterodimer formation. Heterodimer formation was thermally induced, and heterodimers were separated on nondenaturing polyacrylamide gels. Aporepressors readily formed heterodimer formation upon treatment at 65°C for 3 minutes. Heterodimer formation was significantly retarded by the presence of the corepressor, L-tryptophan. Indole-3-propionic acid, 5-methyl tryptophan, and other analogs of tryptophan, as well as indole, also inhibited heterodimer formation. These results indicate that the presence of the indole moiety in the corepressor binding pocket increases the stability of the dimer.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/38518/1/340040304_ftp.pd

Bioinformatic identification of proteins with tissue-specific expression for biomarker discovery

<p>Abstract</p> <p>Background</p> <p>There is an important need for the identification of novel serological biomarkers for the early detection of cancer. Current biomarkers suffer from a lack of tissue specificity, rendering them vulnerable to non-disease-specific increases. The present study details a strategy to rapidly identify tissue-specific proteins using bioinformatics.</p> <p>Methods</p> <p>Previous studies have focused on either gene or protein expression databases for the identification of candidates. We developed a strategy that mines six publicly available gene and protein databases for tissue-specific proteins, selects proteins likely to enter the circulation, and integrates proteomic datasets enriched for the cancer secretome to prioritize candidates for further verification and validation studies.</p> <p>Results</p> <p>Using colon, lung, pancreatic and prostate cancer as case examples, we identified 48 candidate tissue-specific biomarkers, of which 14 have been previously studied as biomarkers of cancer or benign disease. Twenty-six candidate biomarkers for these four cancer types are proposed.</p> <p>Conclusions</p> <p>We present a novel strategy using bioinformatics to identify tissue-specific proteins that are potential cancer serum biomarkers. Investigation of the 26 candidates in disease states of the organs is warranted.</p

DNA vaccination for prostate cancer: key concepts and considerations

While locally confined prostate cancer is associated with a low five year mortality rate, advanced or metastatic disease remains a major challenge for healthcare professionals to treat and is usually terminal. As such, there is a need for the development of new, efficacious therapies for prostate cancer. Immunotherapy represents a promising approach where the host’s immune system is harnessed to mount an anti-tumour effect, and the licensing of the first prostate cancer specific immunotherapy in 2010 has opened the door for other immunotherapies to gain regulatory approval. Among these strategies DNA vaccines are an attractive option in terms of their ability to elicit a highly specific, potent and wide-sweeping immune response. Several DNA vaccines have been tested for prostate cancer and while they have demonstrated a good safety profile they have faced problems with low efficacy and immunogenicity compared to other immunotherapeutic approaches. This review focuses on the positive aspects of DNA vaccines for prostate cancer that have been assessed in preclinical and clinical trials thus far and examines the key considerations that must be employed to improve the efficacy and immunogenicity of these vaccines

Non-conventional sources of peptides presented by MHC class I

Effectiveness of immune surveillance of intracellular viruses and bacteria depends upon a functioning antigen presentation pathway that allows infected cells to reveal the presence of an intracellular pathogen. The antigen presentation pathway uses virtually all endogenous polypeptides as a source to produce antigenic peptides that are eventually chaperoned to the cell surface by MHC class I molecules. Intriguingly, MHC I molecules present peptides encoded not only in the primary open reading frames but also those encoded in alternate reading frames. Here, we review recent studies on the generation of cryptic pMHC I. We focus on the immunological significance of cryptic pMHC I, and the novel translational mechanisms that allow production of these antigenic peptides from unconventional sources

Identification and characterization of the membrane components of the LIV-I transport system of Escherichia coli.

The work described in this thesis concerns the identification, characterization, and expression of the membrane components (LivH, LivM, LivG, and LivF) of the high affinity, periplasmic binding protein-dependent, branched-chain amino acid LIV-I transport system of Escherichia coli. The livH gene has previously been established to be essential for LIV-I transport function. Recombinant DNA methods have permitted the generation of antibody specific to LivH protein, which has assisted in identifying the polypeptide product of the livH gene. Amplified expression of plasmid encoded LivH protein seems to require additional membrane components, LivM and LivG, suggesting they form a complex in the membrane. The identity of the LivH protein is firmly established by amino-terminal and internal amino acid sequence analysis. An amino-terminal block of LivH, believed to be a formyl moiety, must be removed prior to Edman degradation in order to obtain amino-terminal sequence information. A plasmid system was developed that permitted expression and identification of the livM, livG, and livF gene products. The livM gene was demonstrated to be essential to the function of the LIV-I transport system. The LivM protein has been tentatively identified to be a polypeptide band with an apparent Mr of 32 kD when assayed by SDS-PAGE. The identity of the livG gene product was confirmed by amino acid composition and amino-terminal amino acid sequence analysis. A new gene product essential to the function of LIV-I transport system has been designated LivF and was discovered by protein expression studies and subsequently confirmed by nucleotide sequence analysis of the livF gene. The identity of the LivF protein is confirmed by amino acid composition and amino-terminal amino acid sequence analysis. Subcellular fractionation studies establish LivH, LivG and LivF to be integral inner membrane proteins. Regulated expression of the LIV-I proteins was analyzed in vivo by a translational gene fusion between livH and lacZ genes and by expressing plasmid encoded LIV-I genes in minicells. The plasmid encoded expression of the LivH\sp\prime-\sp\primeLacZ fusion protein and the gene products of the livKHMGF operon is regulated by sub-millimolar concentration of leucine present in the medium.Ph.D.Biological ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/102949/1/9023555.pdfDescription of 9023555.pdf : Restricted to UM users only

Selection against metabolic diseases

Abstract: Metabolic diseases, such as ketosis and milk fever, are among the most common diseases affecting dairy cattle. Genetic improvement of ability to resist metabolic diseases can be achieved by direct selection with genetic evaluation based on clinically observed traits, or by indirect selection based on indicators or predictors of metabolic diseases. The most prevalent metabolic diseases in dairy cattle, for which genetic parameters have been published, are ketosis, displaced abomasum, milk fever, and tetany. In this review we present genetic parameters for these metabolic diseases, give a status of genetic and genomic evaluations, and discuss possible indicator traits.acceptedVersio