Category D. Protein Structure & Function
D1. Characterization of the Salicylate Adenylase in Mycobacterium tuberculosis
Blake Balcomb and Audrey L. Lamb1
1Department of Molecular Biosciences, University of Kansas, Lawrence, KS
Arguably the greatest risk of contemporary medicine is the growing concern of antibiotic resistance and its public health implications. The majority of current antibiotics target cell wall/membrane synthesis, folate synthesis and or bacterial protein/DNA/RNA synthesis. The development of antibiotic resistance pathogens warrants the need to assess other bacterial pathways that could be used as alternative targets. Pathways associated with bacterial nutrient uptake and virulence have been identified as attractive targets of inhibition. The utilization of iron in basic metabolic pathways is essential to both the human host and pathogenic microorganism. Mycobacterium tuberculosis is known to utilize the host’s iron sources under iron limiting conditions for survival and virulence. One of the mechanisms M. tuberculosis employs in acquiring this iron is through the production of iron-chelating molecules known as siderophores. M. tuberculosis produces the siderophore mycobactin. This siderophore is synthesized by multimodular nonribosomal peptide synthetases and several accessory proteins. One of the initial steps involved in the biosynthetic pathway of mycobactin is carried out by MbtA. This protein activates the initial substrate salicylate through its adenylation domain to form salicyl-AMP. This activated salicylate allows for transfer of the salicyl moiety to the peptidyl carrier domain in MbtE and subsequently through multiple enzymes to the final formation of mycobactin. We aim to characterize MbtA through x-ray crystallography and enzyme kinetics. This information will assist us in our long term goal of designing highly specific inhibitors of the enzymes involved in siderophore biosynthesis as a form of antibiotic therapy. In addition, we will provide the first structure of a salicylate adenylase.
D2. Identification of Protein Targets for Isoniazid Reactive Metabolites
Yakov Koen1, Nadezhda Galeva1, Robert Hanzlik1, Imir Metushi2, Jack Uetrecht2, Jingtao Lu3, Paul Watkins3
1The University of Kansas, Lawrence (KS);2The University of Toronto (CA);3The Hamner Institute (Research Triangle Park, NC)
Isoniazid (isonicotinic acid hydrazide, INH) has been used for long-term treatment of tuberculosis since 1952. Approximately 1% of INH patients experience idiosyncratic drug-induced liver injury (IDILI), but when INH is co-administered with rifampicin (a P450-inducer) the incidence of DILI rises to almost 100%. The mechanisms underlying IDILI are largely unknown, and there is no suitable animal model for INH hepatotoxicity. However, for many other small hepatotoxic molecules, protein covalent binding (CVB) by chemically reactive metabolites (CRMs) is thought to initiate hepatotoxicity. CVB of INH to human liver microsomes was recently attributed to the acylation of lysine side chains by a diazohydroxide metabolite of INH (Metushi et al., 2012). An antibody to isonicotinoyl-lysine residues (raised by treating rabbits with INA-KLH) detected numerous INA-adducted proteins in livers of rats and mice treated with INH for five weeks.
We obtained samples of the S9 fraction of the livers of INH-treated rats and mice from Metushi et al. and subjected them to "shotgun" proteomic analysis by LC-MS/MS. More than 630 individual proteins were identified, ONE of which, D-dopachrome decarboxylase, showed unequivocal evidence for an isonicotinoyl-lysine residue. Similar analysis of extracts of INH-treated human hepatocyte-like cells revealed INA-adducted peptides derived from Prohibitin 2 and Macrophage Migration Inhibitory Factor (MIF). To improve analytical sensitivity and detect more INA-adducted peptides, we are attempting to apply immunoaffinity enrichment to protein digests prior to LC-MS/MS. Immunospecific IgG was isolated from antiserum using an INH hapten column prepared by coupling isoniazid to NHS-activated Sepharose 4 Fast Flow. From 7.1 mL antiserum (337 mg protein) we isolated 8.8 mg of IgG that showed very high potency in a dot-blot assay. The use of this antibody for pre-concentrating INA-adducted peptides is currently under evaluation. (Supported in part by NIH-GM-21784).
D3. Protein Production Group of the COBRE in Protein Structure and Function
Anne Cooper, Philip Gao and Robert Hanzlik
Protein Structure Group, Center of Biomedical Research Excellence in Protein Structure and Function, 2034 Becker Drive, The University of Kansas, Lawrence, KS, 66047
The Protein Production Group is one of the three core laboratories of the Kansas COBRE in Protein Structure and Function. The lab focuses on the cloning, expression and purification of prokaryotic and eukaryotic proteins for COBRE and other investigators in Kansas and the region who need to obtain proteins for their research. It also works closely with the Bio-NMR lab, the PSL, and the KU High Throughput Screening Lab. The PPG provides a one-stop menu of services to investigators, including protocol development, cloning, mutagenesis, expression system development or improvement, large or small scale protein expression and/or purification, protein binding studies, and technical training for researchers in any of these areas. Both prokaryotic and eukaryotic proteins can be produced in E. coli, yeast, insect cell, mammalian and cell-free expression systems. Most of the preparations and purifications are conducted in conjunction with automated equipment to ensure precision and efficiency. The facility also has the capacity for expanding on existing purifications schemes for large scale preparations. In addition, the facility also provides advice on the generation and analysis of proteins depending on their physicochemical properties and the needs of the investigator.
D4. The Retinaldehyde Reductase DHRS3 Is Essential for Preventing the Formation of Excess Retinoic Acid during Embryonic Development
Suya Wang,a Sara E. Billings,a Keely Pierzchalski,bNaomi E. Butler Tjaden,cd Xiaoyan Pang,a Paul A. Trainor,cd Maureen A. Kane,b Alexander R. Moisea,a
aDepartment of Pharmacology and Toxicology, School of Pharmacy, University of Kansas, Lawrence, Kansas, 66045, USA; bDepartment of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Maryland, 21201, USA; cStowers Institute for Medical Research, Kansas City, Missouri, 64110, USA;
dDepartment of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, Kansas, 66160, USA.
All-trans-retinoic acid (ATRA), a metabolite of vitamin A, retinol, plays an important role in embryonic development, whose homeostasis is sustained by a precise balance between its synthesis and degradation. Previous studies of the regulation of ATRA metabolism have focused on the enzymes involved in its synthesis, namely the oxidation of retinol and retinaldehyde and the degradation by cytochrome P450 enzymes. In addition, in vitro studies have identified several enzymes that catalyze the reduction of retinaldehyde to retinol. However, it is still unknown if such conversion affects ATRA levels in vivo. DHRS3 is a ubiquitously expressed short chain dehydrogenase/ reductase (SDR) enzyme which reduces retinaldehyde to retinol and whose expression is controlled by ATRA. Here, we investigate the physiological role of Dhrs3 by employing a Dhrs3-deficient mouse model. Ours studies reveal that Dhrs3-deficient embryos exhibit a statistically significant reduction in the levels of retinol and retinyl esters and an increase in ATRA, compared to wild-type embryos. Such an accumulation of ATRA in Dhrs3-/- mice leads to metabolic compensation through both upregulation of the expression of the ATRA catabolic enzyme Cyp26a1 and reduction in the expression of genes involved in ATRA synthesis. Despite such compensation, Dhrs3-/- mice show altered ATRA signaling and developmental programming during embryogenesis. As the result of these alterations, Dhrs3-/- mice present with cardiac malformations, cleft palate and defects in skeletal development and are not viable. These results provide evidence of the critical function of DHRS3 in reducing retinaldehyde to retinol to safeguard against excess formation of ATRA during embryonic development