Cystine

Cystine transporter SLC7A11 (xCT)

The cystine/glutamate antiporter SLC7A11 (also commonly known as xCT) functions to import cystine for glutathione biosynthesis and antioxidant defense and is overexpressed in multiple human cancers. …

However, most cancer cells largely depend on the cystine transporter system xc− to import cystine, which is then converted to cysteine in the cytosol through an NADPH-consuming reduction reaction (R3)

system x-c consists of two subunits: SLC7A11 and SLC3A2

It also depends in chloride:

xCT is the functional light chain subunit of system x−c , which is a sodium-independent, chloride-dependent, anionic L-cystine/L-glutamate antiporter on the cell surface.

The SLC7A11 protein requires either of the two heavy chain subunits of SLC3A2 to import extracellular cystine in exchange for intracellular glutamate, at a molar ratio of 1:1 (R4)

Regulation of xCT

Transcriptional repression: Under basal conditions, p53 and ATF3 repress SLC7A11 transcription. BAP1 deubiquitinates H2Aub on the SLC7A11 promoter and subsequently represses SLC7A11, whereas H2A ubiquitination by PRC1 on the SLC7A11 promoter also represses SLC7A11.

p53-mediated nuclear translocation of USP7 results in decreased H2Bub occupancy on the SLC7A11 promoter via deubiquitination, resulting in transcriptional repression.

Post-translational regulation:

OTUB1 and CD44 form a trimeric complex with SLC7A11 to deubiquitinate SLC7A11 and inhibit SLC7A11 degradation in proteasome, thereby stabilizing SLC7A11 protein, whereas mTORC1 promotes SLC7A11 protein stability by inhibiting its lysosomal degradation.

High cell density inhibits mTORC1 and promotes SLC7A11 degradation in lysosomes.

Both mTORC2 and AKT inhibit SLC7A11 transporter activity by phosphorylating SLC7A11 at serine 26. (R3)

Intracellular buffering in lysosomes

Cystine is extracted from proteins within lysosomes, but also cystine taken up by xCT antiporter is processed inside the lysosomes:

Cystine is a disulphide amino acid that is normally generated inside the lysosomes by a cathepsin‐catalysed breakdown of cystine‐containing proteins. … experimental data on cystinotic leukocytes and fibroblasts have shown that part of the lysosomal cystine pool originates from the uptake of extracellular non‐protein cystine. (R1)

Once released from the lysosome, cystine can be reduced by a reaction that requires NADPH:

Extracellular cystine is imported into the cell through SLC7A11 and is converted to cysteine through a NADPH-consuming reduction reaction in the highly reducing atmosphere of the cytosol. (R4)

Open question: what enzyme does it?

Most of intracellular cystine is found in lysosomes

the cystine compartmentalized in the lysosome represents > 90% of the intracellular cystine pool (R1)

Consequences of defective export of cystine

• Lysosomal cystine accumulation increases intracellular O2 − and NO production but does not alter basal OS index
• Lysosomal cystine accumulation promotes nitrosative damage under oxidizing conditions
• Lysosomal cystine accumulation promotes induction of ROS‐generating enzymes
• Lysosomal cystine accumulation increases intracellular Ca2+ flux
• Lysosomal cystine accumulation promotes induction redox‐sensitive transcription factors and cytoprotective proteins
• Lysosomal cystine accumulation results in the depolarization of ΔΨm and in the reduction of intracellular ATP content
• Lysosomal cystine accumulation does not alter autophagic activity
• Lysosomal cystine accumulation augments apoptosis and reduces cell viability
• Cysteamine treatment reverses some effects associated with lysosomal cystine accumulation

Reduction of cystine to cysteine requires NADPH and GSH

Using metabolomic profiling, we found that cells undergoing glucose deprivation-induced cell death exhibited dramatic accumulation of intracellular L-cysteine and its oxidized dimer, L-cystine, and depletion of the antioxidant glutathione.

Building on this observation, we show that glucose deprivation-induced cell death is driven not by lack of glucose but rather by L-cystine import.

Following glucose deprivation, the import of L-cystine and subsequent reduction to L-cysteine depleted both NADPH and glutathione, thereby allowing toxic accumulation of reactive oxygen species. (R5)

But only NADPH was the limiting factor

We found that levels of NADPH decreased dramatically within 10 min of glucose deprivation and were undetectable after 60 min.

Notably, this short time scale is similar to the time required for L-cystine and L-cysteine accumulation following glucose deprivation. In contrast, intracellular pools of GSH and NADH were not depleted until later time points (90-120 min). These results suggest that NADPH, rather than NADH or GSH, is the limiting reducing agent when cancer cells are deprived of glucose. (R5)

Cystinosin (CTNS)

Cystine/H+ symporter that mediates export of cystine, the oxidized dimer of cysteine, from lysosomes.

Plays an important role in melanin synthesis by catalyzing cystine export from melanosomes, possibly by inhibiting pheomelanin synthesis (R2).

In addition to cystine export, also acts as a positive regulator of mTORC1 signaling in kidney proximal tubular cells, via interactions with components of the v-ATPase and Ragulator complexes.

Also involved in small GTPase-regulated vesicle trafficking and lysosomal localization of LAMP2A, independently of cystine transporter activity. (Uniprot)

Cystinosin is also located in melanosomes (see below).

Defective melanogenesis is observed in cystinosis

Patients with cystinosis frequently exhibit blond hair and fair complexion, suggesting an alteration in melanogenesis.

Analysis of the hair melanin content in these patients by HPLC demonstrated a 50% decrease in eumelanin (4360 vs. 9360 ng/mg), and a 2-fold increase in pheomelanin (53 vs. 20 ng/mg), the yellow/red pigments. Cystinosin-deficient mice also showed a 4-fold increase in hair pheomelanin content. In vitro studies showed that cystinosin was located at melanosomes.

CTNS silencing led to a 75% reduction of melanin synthesis that was caused by a degradation of tyrosinase by lysosomal proteases.

Our results objectify the pigmentation defect in patients with cystinosis. We also identify the role of CTNS in melanogenesis and add a new gene to the list of the genes involved in the control of skin and hair pigmentation. (R2)