Effect of Oxygen on Induction of the Cystine Transporter by Bacterial Lipopolysaccharide in Mouse Peritoneal Macrophages

We measured sensitivity to thiol modification of the heteromeric glutamate/cystine transporter 4F2hc-xCT expressed in Xenopus oocytes. p-Chloromercuribenzoate (pCMB) and p-chloromercuribenzenesulfonate (pCMBS) rapidly blocked transport activity. Cys 327 , located in the middle of the eighth transmembrane domain of the light subunit (xCT), was found to be the main target of inactivation. Cysteine, an impermeant reducing reagent, reversed pCMB and pCMBS effects only when applied from the extracellular medium. L-Glutamate and L-cystine, but not L-arginine, protected from the inactivation with an IC 50 similar to the K m . Protection was not temperature-dependent, suggesting that it did not depend on large substrate-induced conformational changes. Mutation of Cys 327 to Ala and Ser slightly modified the K m and a C327L mutant abolished transport function without compromising transporter expression at the plasma membrane. The results indicate that Cys 327 is a functionally important residue accessible to the aqueous extracellular environment and is structurally linked to the permeation pathway and/or the substrate binding site.

supporting

confidence: 69%

Thiol Modification of Cysteine 327 in the Eighth Transmembrane Domain of the Light Subunit xCT of the Heteromeric Cystine/Glutamate Antiporter Suggests Close Proximity to the Substrate Binding Site/Permeation Pathway

Jiménez-Vidal

Gasol

Zorzano

et al. 2004

“…The sequences underlined (EpRE1to EpRE-4) indicate the EpRE-like sequences. The detailed sequence of this region was described previously (30). agents were investigated.…”

Section: Resultsmentioning

confidence: 99%

Electrophile Response Element-mediated Induction of the Cystine/Glutamate Exchange Transporter Gene Expression

Sasaki¹,

Sato²,

Kuriyama-Matsumura³

et al. 2002

Self Cite

473

332

In mammalian cultured cells, the cystine/glutamate exchange transport mediated by system x c ؊ is important to maintain intracellular GSH levels. System x c ؊ consists of two protein components, xCT and the heavy chain of 4F2 antigen. The activity of system x c ؊ is induced by various stimuli, including electrophilic agents like diethyl maleate. In the present study, we have investigated the mechanism of the transcriptional regulation of xCT mRNA by diethyl maleate. The xCT gene consisted of twelve exons and sequence analysis identified four electrophile response element (EpRE)-like sequences between ؊230 and ؊1 in the 5-flanking region, designated EpRE-1 to EpRE-4. To identify sequences mediating the constitutive and induced expression of xCT, a series of 5-deletion mutants created from the 5-flanking region were cloned into a luciferase reproter vector and transfected into BHK21 cells. The 5-deletion analysis revealed that the sequence between ؊116 and ؊82 is essential for the basal expression and the sequence between ؊226 and ؊116 containing EpRE-1 is essential in response to diethyl maleate. Mutational analysis demonstrated that EpRE-1 is critically involved in the response to diethyl maleate. Other stress agents like arsenite, cadmium, and hydroquinone seemed to induce system x c ؊ activity via the same sequence. Furthermore, the experiments using the mouse embryonic fibroblasts derived from the Nrf2-deficient mice revealed that the induction of xCT gene by electrophilic agents is mediated by Nrf2. EpRE occurs in a broad spectrum of genes for the proteins that are involved in the defense against xenobiotics and regulates their expression. The present results have demonstrated that xCT is a novel member of this protein family.

“…4). LPS is reported to stimulate cysteine transport by induction of the x c Ϫ glutamate/cystine antiporter in mouse peritoneal macrophages (34). Thus, a combination of increased synthesis and enhanced transport probably contributes to intracellular cysteine accumulation in LPS-treated cells.…”

Section: Discussionmentioning

confidence: 99%

Monocyte Differentiation, Activation, and Mycobacterial Killing Are Linked to Transsulfuration-dependent Redox Metabolism

Garg

Vitvitsky

Gendelman

et al. 2006

Modulation of the ambient redox status by mononuclear phagocytes is central to their role in health and disease. However, little is known about the mechanism of redox regulation during mononuclear phagocyte differentiation and activation, critical cellular steps in innate immunity, and microbial clearance. An important intermediate in GSH-based redox metabolism is homocysteine, which can undergo transmethylation via methionine synthase (MS) or transsulfuration via cystathionine ␤-synthase (CBS). The transsulfuration pathway generates cysteine, the limiting reagent in GSH biosynthesis. We now demonstrate that expression of CBS and MS are strongly induced during differentiation of human monocytes and are regulated at the transcriptional and posttranscriptional levels, respectively. The changes in enzyme expression are paralleled by an ϳ150% increase in S-adenosylmethionine (accompanied by a corresponding increase in phospholipid methylation) and a similar increase in GSH. Activation with lipopolysachharide or infection with Mycobacterium smegmatis diminished expression of both enzymes to a significant extent and decreased S-adenosylmethionine concentration by ϳ30% of the control value while GSH and cysteine concentrations increased ϳ100 and 300%, respectively. Blockade of the transsulfuration pathway with propargylglycine suppressed clearance of M. smegmatis by macrophages and inhibited phagolysosomal fusion, whereas N-acetylcysteine promoted phagolysosomal fusion and enhanced mycobacterial clearance 3-fold compared with untreated cells. We posit that regulation of the transsulfuration pathway during monocyte differentiation, activation, and infection can boost host defense against invading pathogens and may represent a heretofore unrecognized antimicrobial therapeutic target.