Overexpression of the yeast high affinity glutathione transporter, Hgt1, restores growth in cells lacking γ-glutamylcysteine synthetase

3. Kumar C, Igbaria A, D’Autreaux B, et al. Glutathione

17

revisited: a vital function in iron metabolism and ancillary role in thiol-redox control. The EMBO Journal. 30(10):2044-

Overexpression of the yeast high affinity

2056; 2011

glutathione transporter, Hgt1, restores

DOI: 10.1016/j.freeradbiomed.2017.10.030

growth in cells lacking γ-glutamylcysteine synthetase Crystal McGee1, Courtney Patterson1, and Caryn Outten1 1

18

University of South Carolina, USA

Glutathione (L-γ-glutamyl-L-cysteinylglycine) (GSH) is the most abundant low molecular weight thiol in eukaryotic cells. Its ability to serve as a reducing equivalent in several

Myristoylated

Methionine

Sulfoxide

Reductase A Interacts with the Lipid-

biological reactions offers protection against reactive oxygen

Binding Protein

species, xenobiotics, and heavy metals.

Jung Mi Lim1, Geumsoo Kim1, and Rodney L. Levine1

In addition, GSH

serves as an iron ligand for some Fe-S binding proteins involved in both iron regulation and the synthesis and maturation of Fe-S proteins. Hence, GSH is important for iron homeostasis. The biosynthesis of GSH occurs via a twostep ATP-dependent enzymatic process. In the first reaction, γ-glutamylcysteine

is

formed

by

γ-glutamylcysteine

synthetase (Gsh1). In the second step, glycine is added to the peptide via GSH synthetase (Gsh2). Deletion of the γglutamylcysteine synthase gene (GSH1) in yeast leads to growth

arrest

without

GSH

supplementation.1

In

Saccharomyces cerevisiae, extracellular GSH can be imported via a high affinity GSH transporter, Hgt1. The physiological 2

role of GSH depletion on cellular function and subcellular redox status has been well characterized. However, it has not been determined how cells depleted of GSH (∆gsh1) respond to increased GSH uptake. To study this, our lab employed a ∆gsh1 yeast strain engineered to overexpress Hgt1. Yeast strains where Hgt1 is overexpressed have been shown to overaccumulate GSH and glutathione disulfide (GSSG) when GSH is added to the media.3 Our data suggests that the overexpression of Hgt1 alone can partially rescue cells devoid of GSH in a redox-dependent manner. To future elucidate this observation, we will express subcellular targeted redox sensors developed in our lab that specifically equilibrates with GSH:GSSG redox changes. 1. Ayer A, Tan Shi-Xiong, Grant CM., Meyer AJ, Dawes IW., Perrone GC. The critical role of glutathione in maintenance of the mitochondrial genome. Free Radical Biology and Medicine. 49: 1956-1968; 2010 2. Bachhawat, A. K.; Thakur, A.; Kaur, J., Zulkifli, M. 1830: 3154-3164; 2013

28

National Heart, Lung and Blood Institute, Bethesda, MD,

USA Reactive oxygen species (ROS) are produced by a variety of cellular

processes

including

metabolic

activity,

mitochondria, cellular signaling, and oncogene activity. Methionine sulfoxide reductase A (MsrA) is an antioxidant enzyme that can reduce methionine sulfoxide (MetO) in proteins back to methionine (Met). This enzyme plays a protective role in cells against oxidative damage. Although many proteins with MetO are in vitro substrates of MsrA, no in vivo substrates have been confidently identified. In vitro, MsrA can also function as an oxidase that converts methionine to methionine sulfoxide, but it is also not known if it does so in vivo.

The cytosolic form of MsrA is

myristoylated, and overexpression of the myristoylated form protects the heart from ischemia-reperfusion injury while overexpression of the non-myristoylated form does not. Here we report a novel binding partner of MsrA, STARD3, identified by use of a human protein microarray. ProtoArray Human Protein Microarray v5.0 contains 9,483 human proteins from multiple protein classes along with 2,016 controls. STARD3 was scored as an interacting protein with high probability. STARD3 contains the star-related lipid transfer (START) domain, and proteins with that

References:

Glutathione Transporters. Biochimica

1

et Biophysica Acta

domain are implicated in lipid and sterol metabolism. STARD3 is known to bind cholesterol and transport it from the endoplasmic reticulum to the late endosome. We overexpressed both myristoylated MsrA and STARD3 in HEK293 cells and immunoprecipitated cell homogenates with

anti-STARD3

myristoylated

SfRBM 2017

MsrA

antibody. was

demonstrated

that

co-immunoprecipitated

We

with