Methanogens are members ofthe human microbiome and the only known source of biogenic methane. Residing in the third domain of life, Archaea, they are strict anaerobes that have evolved systems to combat periodic oxygen exposure. There is a significant gap in our knowledge of how methanogens mount a transcriptional response that ultimatley leads to cell survival during periods of oxygen stress. The overall goal of this project is to understand the role of MsvR in regulating the oxidative stress response in methanogenic Archaea through structure/function analysis. MsvR is a newly described transcripfional regulator present in select methanogens including those residing within the human intestine and oral cavity. Early characterization of MsvR has led to the development ofthe following hypotheses: HI) multiple amino acid residues in MsvR play a role in its function as a transcriptional regulator, (H2) oxidized and reduced MsvR play distinct yet integral roles in the regulation of expression of at least two promoters, and (H3) MsvR undergoes various conformational changes when shifting between the oxidized and reduced states that impact its function. To address these hypotheses the following specific aims are proposed:
(Aim 1) Determine the 3D structure of MsvR by X-ray crystallography, (Aim 2) Identify additional amino acid residues or combinations thereof in MsvR necessary for function, (Aim 3) Identification of disulfide bonds formed upon oxidative stress and the role of oxidized vs. reduced MsvR in transcription regulation, (Aim 4) Identification of conformational changes in MsvR.
As potentially secondary contributors to pathogenesis, it is critical to gain a better understanding of how methanogens withstand intermittent encounters with oxygen. Additionally these organisms likely possess very sensitive mechanisms to sense changes in cellular redox that may provide insight into subtle changes in redox balance that play a role in aging and disease.
|Hanigan, Marie H; Gillies, Elizabeth M; Wickham, Stephanie et al. (2015) Immunolabeling of gamma-glutamyl transferase 5 in normal human tissues reveals that expression and localization differ from gamma-glutamyl transferase 1. Histochem Cell Biol 143:505-15|
|Hanigan, Marie H (2014) Gamma-glutamyl transpeptidase: redox regulation and drug resistance. Adv Cancer Res 122:103-41|
|West, Matthew B; Chen, Yunyu; Wickham, Stephanie et al. (2014) Novel insights into eukaryotic ýý-glutamyltranspeptidase 1 from the crystal structure of the glutamate-bound human enzyme. J Biol Chem 289:11569|
|Ding, Jingzhen; Mooers, Blaine H M; Zhang, Zhi et al. (2014) After embedding in membranes antiapoptotic Bcl-XL protein binds both Bcl-2 homology region 3 and helix 1 of proapoptotic Bax protein to inhibit apoptotic mitochondrial permeabilization. J Biol Chem 289:11873-96|
|Sheikh, M Osman; Schafer, Christopher M; Powell, John T et al. (2014) Glycosylation of Skp1 affects its conformation and promotes binding to a model f-box protein. Biochemistry 53:1657-69|
|West, Matthew B; Chen, Yunyu; Wickham, Stephanie et al. (2013) Novel insights into eukaryotic ýý-glutamyltranspeptidase 1 from the crystal structure of the glutamate-bound human enzyme. J Biol Chem 288:31902-13|
|Wickham, Stephanie; Regan, Nicholas; West, Matthew B et al. (2013) Inhibition of human ýý-glutamyl transpeptidase: development of more potent, physiologically relevant, uncompetitive inhibitors. Biochem J 450:547-57|
|Isom, Catherine E; Turner, Jessica L; Lessner, Daniel J et al. (2013) Redox-sensitive DNA binding by homodimeric Methanosarcina acetivorans MsvR is modulated by cysteine residues. BMC Microbiol 13:163|
|Thomas, Leonard M; Harper, Angelica R; Miner, Whitney A et al. (2013) Structure of Escherichia coli AdhP (ethanol-inducible dehydrogenase) with bound NAD. Acta Crystallogr Sect F Struct Biol Cryst Commun 69:730-2|
|Hill, Heather E; Pioszak, Augen A (2013) Bacterial expression and purification of a heterodimeric adrenomedullin receptor extracellular domain complex using DsbC-assisted disulfide shuffling. Protein Expr Purif 88:107-13|