CATs and HATs: the SLC7 family of amino acid transporters

The SLC7 family is divided into two subgroups, the cationic amino acid transporters (the CAT family, SLC7A1–4) and the glycoprotein-associated amino acid transporters (the gpaAT family, SLC7A5–11), also called light chains or catalytic chains of the hetero(di)meric amino acid transporters (HAT). The associated glycoproteins (heavy chains) 4F2hc (CD98) or rBAT (D2, NBAT) form the SLC3 family. Members of the CAT family transport essentially cationic amino acids by facilitated diffusion with differential trans-stimulation by intracellular substrates. In some cells, they may regulate the rate of NO synthesis by controlling the uptake of l-arginine as the substrate for nitric oxide synthase (NOS). The heterodimeric amino acid transporters are, in contrast, quite diverse in terms of substrate selectivity and function (mostly) as obligatory exchangers. Their selectivity ranges from large neutral amino acids (system L) to small neutral amino acids (ala, ser, cys-preferring, system asc), negatively charged amino acid (system x_c^–) and cationic amino acids plus neutral amino acids (system y⁺L and b^0,+-like). Cotransport of Na⁺ is observed only for the y⁺L transporters when they carry neutral amino acids. Mutations in b^0,+-like and y⁺L transporters lead to the hereditary diseases cystinuria and lysinuric protein intolerance (LPI), respectively.     

Neurodegeneration in the Brain Tumor Microenvironment: Glutamate in the Limelight

Malignant brain tumors are characterized by destructive growth and neuronal cell death making them one of the most devastating diseases. Neurodegenerative actions of malignant gliomas resemble mechanisms also found in many neurodegenerative diseases such as Alzheimer''s and Parkinson''s diseases and amyotrophic lateral sclerosis. Recent data demonstrate that gliomas seize neuronal glutamate signaling for their own growth advantage. Excessive glutamate release via the glutamate/cystine antiporter xCT (system xc-, SLC7a11) renders cancer cells resistant to chemotherapeutics and create the tumor microenvironment toxic for neurons. In particular the glutamate/cystine antiporter xCT takes center stage in neurodegenerative processes and sets this transporter a potential prime target for cancer therapy. Noteworthy is the finding, that reactive oxygen species (ROS) activate transient receptor potential (TRP) channels and thereby TRP channels can potentiate glutamate release. Yet another important biological feature of the xCT/glutamate system is its modulatory effect on the tumor microenvironment with impact on host cells and the cancer stem cell niche. The EMA and FDA-approved drug sulfasalazine (SAS) presents a lead compound for xCT inhibition, although so far clinical trials on glioblastomas with SAS were ambiguous. Here, we critically analyze the mechanisms of action of xCT antiporter on malignant gliomas and in the tumor microenvironment. Deciphering the impact of xCT and glutamate and its relation to TRP channels in brain tumors pave the way for developing important cancer microenvironmental modulators and drugable lead targets.     

Differentiation of substrate and non-substrate inhibitors of transport system xc(-): an obligate exchanger of L-glutamate and L-cystine

In addition to the well-characterized sodium-dependent excitatory amino acid transporters (EAATs) present in the mammalian CNS, a chloride-dependent, sodium-independent transporter has also been identified that is capable of mediating the uptake of L-glutamate. Named system x(c)(-), this transporter is an obligate exchanger that normally couples the export of intracellular L-glutamate with the import of extracellular L-cystine. Two cell lines that express high levels of system x(c)(-) are used to delineate the pharmacology of the transporter and demonstrate that it is distinct from both the EAATs and EAA ionotropic receptors. Potent competitive inhibitors of system x(c)(-) include: L-homocysteate, ibotenate, L-serine-O-sulphate, (RS)-4-bromohomoibotenate, quisqualate, and (S)-4-carboxyphenylglycine. A fluorescent-based assay that allows system x(c)(-)-mediated exchange of L-glutamate and L-cystine to be followed in real time is used to assess substrate activity. Interestingly, those compounds that proved to be the most potent competitive inhibitors (e.g. L-quisqualate and 4-S-CPG) also proved to be the least active as substrates, suggesting that distinct structural features may control binding and translocation. Lastly, the finding that a number of system x(c)(-) inhibitors are also commonly used as probes of excitotoxic pathology (e.g., L-quisqualate, ibotenate and L-homocysteate) raises some interesting questions regarding the mechanisms through which these analogues produce CNS damage.     

Early Onset of Age-Related Cataracts in Cystine/Glutamate Antiporter Knockout Mice

PurposeThe purpose of this study was to determine the importance of the xCT is a subunit. The cystine/glutamate antiporter is actually system xc-xCT subunit of the cystine/glutamate antiporter in maintaining redox balance by investigating the effects of the loss of xCT on lens transparency and cystine/cysteine balance in the aqueous humour.MethodsC57Bl/6 wild-type and xCT knockout mice at five age groups (6 weeks to 12 months) were used. Lens transparency was examined using a slit-lamp and morphological changes visualized by immunolabelling and confocal microscopy. Quantification of glutathione in lenses and cysteine and cystine levels in the aqueous was conducted by liquid chromatography tandem mass spectrometry (LC-MS/MS).ResultsSlit-lamp examinations revealed that 3-month-old wild-type mice and xCT knockout mice lenses exhibited an anterior localized cataract. The frequency of this cataract significantly increased in the knockout mice compared to the wild-type mice. Morphological studies revealed a localized swelling of the lens fiber cells at the anterior pole. Glutathione levels in whole lenses were similar between wild-type and knockout mice. However, glutathione levels were significantly decreased at 3 months in the knockout mice in the lens epithelium compared to the wild-type mice. Aqueous cysteine levels remained similar between wild-type and knockout mice at all age groups, whereas cystine levels were significantly increased in 3-, 9-, and 12-month-old knockout mice compared to wild-type mice.ConclusionsLoss of xCT resulted in the depletion of glutathione in the epithelium and an oxidative shift in the cysteine/cystine ratio of the aqueous. Together, these oxidative changes may contribute to the accelerated development of an anterior cataract in knockout mice, which appears to be a normal feature of aging in wild-type mice.     

Mutation of ataxia–telangiectasia mutated is associated with dysfunctional glutathione homeostasis in cerebellar astroglia

Astroglial dysfunction plays an important role in neurodegenerative diseases otherwise attributed to neuronal loss of function. Here we focus on the role of astroglia in ataxia–telangiectasia (A–T), a disease caused by mutations in the ataxia–telangiectasia mutated (ATM) gene. A hallmark of A–T pathology is progressive loss of cerebellar neurons, but the mechanisms that impact neuronal survival are unclear. We now provide a possible mechanism by which A–T astroglia affect the survival of cerebellar neurons. As astroglial functions are difficult to study in an in vivo setting, particularly in the cerebellum where these cells are intertwined with the far more numerous neurons, we conducted in vitro coculture experiments that allow for the generation and pharmacological manipulation of purified cell populations. Our analyses revealed that cerebellar astroglia isolated from Atm mutant mice show decreased expression of the cystine/glutamate exchanger subunit xCT, glutathione (GSH) reductase, and glutathione‐S‐transferase. We also found decreased levels of intercellular and secreted GSH in A–T astroglia. Metabolic labeling of l ‐cystine, the major precursor for GSH, revealed that a key component of the defect in A–T astroglia is an impaired ability to import this rate‐limiting precursor for the production of GSH. This impairment resulted in suboptimal extracellular GSH supply, which in turn impaired survival of cerebellar neurons. We show that by circumventing the xCT‐dependent import of l ‐cystine through addition of N‐acetyl‐l ‐cysteine (NAC) as an alternative cysteine source, we were able to restore GSH levels in A–T mutant astroglia providing a possible future avenue for targeted therapeutic intervention. GLIA 2016;64:227–239     

xCT缺陷的Sut黑色素细胞蛋白质组学研究

目的 为研究xCT缺陷引起细胞生长抑制的机理提供理论依据.方法 提取xCT缺陷的Sut黑色素细胞和野生型Mela黑色素细胞2组细胞总蛋白,分别进行二维凝胶电泳,分离2组细胞总蛋白.以Mela细胞做对照,以PDQuest软件分析Sut细胞蛋白质表达谱的变化.选择部分表达量明显变化的蛋白质进行质谱分析,将质谱分析结果输入M...     

基于差异蛋白质组学技术解析xCT缺陷诱导的细胞死亡机制

目的：对Sut黑色素细胞和野生型黑色素细胞进行高分辨率差异蛋白质组学分析,探讨xCT缺陷引起细胞生长抑制的机制。方法分离Sut黑色素细胞和野生型黑色素细胞总蛋白,进行双向电泳和串联质谱分析,获得数值经Mascot软件处理后通过NCBI及Swiss-Prot蛋白质数据库检索,结合双向凝胶电泳相应点的表观等电点、相对分子质量、匹配肽段的多少和序列覆盖率等综合分析筛选差异蛋白质;逆转录-PCR分析Sut细胞差异蛋白mRNA表达水平,用蛋白免疫印迹法对xCT缺陷的Sut黑色素细胞进行自体吞噬分析。结果与结论得到20个明显上调或下调的蛋白,经MALDI-TOF质谱分析鉴定差异蛋白质,发现与自体吞噬和囊泡运输相关的调控因子（ Anxa3、Hist 1h2bk、NDRG1、CaM）以及与细胞转移相关的调控因子（S100A-4、S100A-6、波形蛋白）表达发生了改变;对自噬标志蛋白分析发现,在xCT缺陷的Sut细胞中发生了自体吞噬。结果表明,xCT缺陷可能通过与细胞外基质的黏附异常以及激活Sut黑色素细胞中自噬相关的信号通路,从而导致细胞的生长抑制和自噬性死亡。     

Biphasic induction of heme oxygenase-1 expression in macrophages stimulated with lipopolysaccharide

Time course relationship between inductions of iNOS and HO-1 was evaluated in RAW264.7 macrophages stimulated with LPS. Expression of HO-1 mRNA increased in a biphasic pattern, but that of xCT (cystine transporter) and iNOS mRNA increased in a monophasic manner. HO-1 protein level increased also in a biphasic manner, at 1-2 h and again between 8 and 24 h. However, iNOS protein began to increase at 4 h, quickly reaching a high level in a monophasic induction pattern. Production of NO* began to occur at 6 h and nitrite continued to accumulate in the culture medium. Total GSH level decreased markedly (50% of control) by 2 h, began to recover at 4 h, returned to control level by 6 h and increased above the control level during 10-24 h. Collectively, these results indicated that overproduced O2*- depletes GSH and triggers induction of xCT, HO-1, iNOS and HO-1 expression in sequence. Most notably, the second-phase induction of HO-1 was caused by overproduced NO*, resulting from LPS-derived iNOS induction. When this iNOS-derived delivery of NO* was combined with prior depletion of GSH using buthioninesulfoximine, an inhibitor of GSH biosynthesis, induction of HO-1 was potentiated. Furthermore, upon such super-induction of HO-1, NO* production was inhibited along with suppression of iNOS expression. Collectively, these results suggested that HO-1 is induced in a biphasic manner, sequentially by the overproduced O*2- and NO*, and the elevated HO-1 suppresses the production of these radicals in an auto-regulatory manner. This may allow the macrophages to survive from injuries that can be caused by concomitant oxidative and nitrosative stresses initiated by the LPS-driven oxidative burst.     

Endogenous hydrogen sulfide regulates xCT stability through persulfidation of OTUB1 at cysteine 91 in colon cancer cells

Increased xCT and transsulfuration pathway has been associated with metabolic reprogramming of colorectal cancer. However, the correlation between these 2 events and the underlying molecular mechanism remains obscure. xCT expression was determined in tissue microarrays of colorectal cancer. RNA sequencing and functional assays in vitro was adopted to delineate the involvement of transsulfuration pathway in the proper function of xCT in maintaining the chemoresistant phenotype. The synthetic lethality of blocking xCT and the transsulfuration pathway was investigated both in vitro and in vivo. The up-regulation of the transsulfuration pathway after inhibiting xCT in colon cancer cells was evident and exogenous H₂S partially reversed the loss of chemoresistance phenotype after inhibiting xCT. Mechanistically, CTH derived H₂S increased the stability of xCT through persulfidation of OTU domain-containing ubiquitin aldehyde-binding protein 1 at cysteine 91. AOAA and Erastin resulted in synthetic lethality both in vitro and in vivo, which was mediated through increased ferroptosis and apoptosis. Our findings suggest that a reciprocal regulation exists between xCT and the transsulfuration pathway, which is a targetable metabolic vulnerability. Mechanistically, CTH derived H₂S increased the stability of xCT through persulfidation of OTU domain-containing ubiquitin aldehyde-binding protein 1 at cysteine 91.