Cystine Transport Research Articles

Structural basis for proton coupled cystine transport by cystinosin

Amino acid transporters play a key role controlling the flow of nutrients across the lysosomal membrane and regulating metabolism in the cell. Mutations in the gene encoding the transporter cystinosin result in cystinosis, an autosomal recessive metabolic disorder characterised by the accumulation of cystine crystals in the lysosome. Cystinosin is a member of the PQ-loop family of solute carrier (SLC) transporters and uses the proton gradient to drive cystine export into the cytoplasm. However, the molecular basis for cystinosin function remains elusive, hampering efforts to develop novel treatments for cystinosis and understand the mechanisms of ion driven transport in the PQ-loop family. To address these questions, we present the crystal structures of cystinosin from Arabidopsis thaliana in both apo and cystine bound states. Using a combination of in vitro and in vivo based assays, we establish a mechanism for cystine recognition and proton coupled transport. Mutational mapping and functional characterisation of human cystinosin further provide a framework for understanding the molecular impact of disease-causing mutations.

A Fe(III)-porphyrin-oxaliplatin(IV) nanoplatform for enhanced ferroptosis and combined therapy.

Since there are several limitations in cancer treatment for traditional chemotherapy, such as side effects, poor prognosis and drug resistance, developing new combined therapy is urgently needed. In this work, a biocompatible, simple and tumor microenvironment-responsive nanotheranostics (PCN-Oxpt/PEG) was built to favor the chemotherapy/ferroptosis/immunomodulation synergism in cancer. This nanotheranostics is constructed by modifying oxaliplatin prodrug and PEG on Fe(III)-porphyrin metal-organic frameworks (PCN(Fe) MOFs). After intravenous injection, the cloak of PEG leads to long circulation, and the Fe(III)-porphyrin MOFs enables dual-model guidance with fluorescence (FL) and magnetic resonance imaging (MRI). Inside the tumor, the intracellular H2O2 would be transferred into hydroxyl radicals (•OH) by iron ions released from MOFs, which could trigger the lethal ferroptosis to cancer cells. Meanwhile, oxaliplatin(II) transformed from the loaded oxaliplatin prodrug would result in the chemotherapy, as well as immunogenic cell death (ICD), and the prodrug strategy could also avoid the occurring of liver damage by the direct administration of oxaliplatin(II). It was noticed that the ferroptosis effect was enhanced by triple-assistance during the combined therapy, as followed: (1) glutathione (GSH) would be consumed in the process of oxaliplatin(II) generation from oxaliplatin prodrug; (2) the increased CD8+ T cells induced by ICD were able to produce interferon-γ (IFN-γ), which could inhibit the transport of cystine by tumor cells, and impair the activation of glutathione peroxidase 4 (GPX4); (3) the amount of H2O2 could be increased by the internalized oxaliplatin and thus further promote the Fenton reaction and ferroptosis. Both in vivo and in vitro results revealed that tumor growing was significantly inhibited by PCN-Oxpt/PEG, taken together, the concomitant of oxaliplatin-mediated chemotherapy and ICD with triple-enhanced ferroptosis offer great prospect in the clinical treatment of cancer.

Relationship between age at initiation of cysteamine treatment, adherence with therapy, and glomerular kidney function in infantile nephropathic cystinosis.

Infantile nephropathic cystinosis, due to impaired transport of cystine out of lysosomes, occurs with an incidence of 1 in 100-200,000 live births. It is characterized by renal Fanconi syndrome in the first year of life and glomerular dysfunction progression to end-stage kidney disease by approximately 10 years of age. Treatment with oral cysteamine therapy helps preserve glomerular function, but affected individuals eventually require kidney replacement therapy. This is because glomerular damage had already occurred by the time a child is diagnosed with cystinosis, typically in the second year of life. We performed a retrospective multicenter study to investigate the impact of initiating cysteamine treatment within the first 2 months of life in some infants and comparing two different levels of adherence in patients diagnosed at the typical age. We collected 3983 data points from 55 patients born between 1997 and 2020; 52 patients with 1592 data points could be further evaluated. These data were first analyzed by dividing the patient cohort into three groups: (i) standard treatment start with good adherence, (ii) standard treatment start with less good adherence, and (iii) early treatment start. At every age, mean estimated glomerular filtration rate (eGFR) was higher in early-treated patients than in later-treated patients. Second, a generalized additive mixed model (GAMM) was applied showing that patients with initiation of treatment before 2 months of age are expected to have a 34 ml/min/1.73 m2 higher eGFR than patients with later treatment start while controlling for adherence and patients' age. These data strongly suggest that oral cysteamine treatment initiated within 2 months of birth preserves kidney function in infantile nephropathic cystinosis and provide evidence of the utility of newborn screening for this disease.

Beneficial effects of starting oral cysteamine treatment in the first 2 months of life on glomerular and tubular kidney function in infantile nephropathic cystinosis.

Nephropathic cystinosis is a rare lysosomal storage disease whose basic defect, impaired transport of cystine out of lysosomes, results in intracellular cystine storage. Affected individuals exhibit renal Fanconi Syndrome in infancy, end-stage kidney disease at approximately 10 years of age, and many other systemic complications. Oral cysteamine therapy mitigates the detrimental effects on glomerular function and prevents most of the late complications of the disease but has not shown benefit with respect to the early tubular damage of cystinosis. This is because cystinosis is generally diagnosed in the second year of life, after the damage to kidney tubular function has already occurred. We longitudinally evaluated 6 infants diagnosed and treated with cysteamine from before 2 months of age. The 4 infants with good compliance with cysteamine and consistently low leucocyte cystine levels maintained normal eGFR values, exhibited only minor degrees of renal Fanconi Syndrome, and maintained normal serum levels of potassium, bicarbonate, phosphate, and calcium without electrolyte or mineral supplementation through 2, 4, 10 and 16 years of age. Thus, renal Fanconi syndrome can be attenuated by early administration of cysteamine and renew the call for molecular-based newborn screening for cystinosis.

Evaluation of NACA and diNACA in human cystinosis fibroblast cell cultures as potential treatments for cystinosis

BackgroundCystinosis is a rare autosomal recessive lysosomal storage disease, associated with high morbidity and mortality. Mutations in the CTNS gene disable a membrane protein responsible for the transport of cystine out of the lysosome. Loss of transporter function leads to intralysosomal cystine accumulation and long-term damage to various tissues and organs, including the kidneys, eyes, liver, muscles, pancreas, and brain. The only cystine-depletion therapy for treatment of cystinosis is cysteamine which requires frequent administration of high doses and often causes gastrointestinal pain as well as pungent sulfurous odor in patients. The current in vitro study evaluated antioxidants, N-acetylcysteine amide (NACA; NPI-001) and (2R,2R′)-3,3′-disulfanediyl bis(2-acetamidopropanamide) (diNACA; NPI-002), as potential treatments for cystinosis.MethodsCytotoxicity of cysteamine, NACA and diNACA was evaluated in cultured human cystinotic fibroblasts (HCFs). HCFs were cultured in 96 well plates incubated for 0–72 h in the presence of 25, 50 or 75 μM each of either cysteamine, NACA or diNACA along with an untreated control. Media was removed and cell viability assessed. Next, cystine-depleting activities of cysteamine, NACA and diNACA were screened in HCFs cell culture utilizing an inexpensive, proven colorimetric assay. HCFs were seeded and allowed to reach approximately 80% confluence before the addition of the test articles: 50 μM of either cysteamine, NACA or diNACA in media along with an untreated control. HCFs were incubated, harvested, and cystine was reduced to cysteine, the concentration of which was then determined per quantity of protein compared to a cysteine standard. Statistically significant cystine depletion was determined by paired t-test versus untreated control (p < 0.05).ResultsNeither cysteamine, NACA nor diNACA at 25, 50 or 75 μM caused cytotoxicity in HCFs. Treatment with all tested concentrations (25, 50 or 75 µM) of either NACA or diNACA at 48 or 72 h resulted in statistically significant increases in cell viability, relative to untreated control, whereas the higher concentrations (50 or 75 µM) of cysteamine achieved statistical significance at both timepoints but not the lowest concentration (25 µM). All test articles depleted cystine from HCFs compared to control. NACA depletion of cystine was statistically superior to cysteamine at 6, 24 and 48 h and numerically greater at 72 h. DiNACA depletion of cystine was statistically superior to cysteamine at 6 and 48 h, slightly numerically greater at 24 h and slightly less at 72 h.ConclusionsNACA and diNACA were non cytotoxic to HCFs and significantly increased cell viability. Cystine reduction was determined as percent of control after incubation with 50 µM of NACA, diNACA or cysteamine in HCFs cell culture for 6, 24, 48 and 72 h. Of the three test articles, NACA exhibited most rapid and greatest potency in cystine reduction. Rank order potency for cystine reduction over time was observed, NACA > diNACA ≥ cysteamine. Therefore, further study of NACA and diNACA as potential treatments for cystinosis is warranted.

Lipidomics characterization of the lipid metabolism profiles in a cystinuria rat model: Precalculus damage in the kidney of cystinuria

Cystinuria is a genetic disorder of cystine transport, including defective protein b0,+AT (encoded by SLC7A9), and/or rBAT (encoded by SLC3A1). Patients present hyperexcretion of cystine in the urine, recurrent cystine lithiasis, and progressive decline in kidney function. Moreover, heterodimer transport is defective. To date, little omics data are accessible regarding this metabolic disease caused by membrane proteins. Since membrane function is closely related to changes in the lipidome, we decided to explore the changes in kidney tissue of a self-established cystinuria rat model by performing lipidomic analysis by LC-MS/MS. Our results demonstrated that Slc7a9 deficiency changed the lipid profile of the renal cortex and induced vital modifications in the lipidome, including major alterations in ChE, LPA, and PA. Among those alterations, this lipidomic study highlights the lipid changes that participate in inflammatory responses during cystinuria. As a result, lipid research, perhaps has great potential, for it may lead to the identification of novel therapeutic targets for the prevention and treatment of cystinuria.

Melatonin modulates Nrf2 activity to protect porcine pre-pubertal Sertoli cells from the abnormal H2 O2 generation and reductive stress effects of cadmium.

Melatonin (MLT) is a cytoprotective agent holding potential to prevent cadmium (Cd) toxicity and its impact in testicular function and fertility. In this study, we explored such potential in porcine pre‐pubertal Sertoli cells (SCs). Cd toxicity resulted in impaired SC viability and function, abnormal cellular H2O2 generation and efflux, and induction of reductive stress by the upregulation of Nrf2 expression and activity, cystine uptake and glutathione biosynthesis, glutathione‐S‐transferase P (GSTP) expression, and protein glutathionylation inhibition. Cd toxicity also stimulated the activity of cellular kinases (MAPK‐ERK1/2 and Akt) and NFkB transcription factor, and cJun expression was increased. MLT produced a potent cytoprotective effect when co‐administered with Cd to SCs; its efficacy and the molecular mechanism behind its cytoprotective function varied according to Cd concentrations. However, a significant restoration of cell viability and function, and of H2O2 levels, was observed both at 5 and 10 μM Cd. Mechanistically, these effects of MLT were associated with a significant reduction of the Cd‐induced activation of Nrf2 and GSTP expression at all Cd concentrations. CAT and MAPK‐ERK1/2 activity upregulation was associated with these effects at 5 μM Cd, whereas glutathione biosynthesis and efflux were involved at 10 μM Cd together with an increased expression of the cystine transporter xCT, of cJun and Akt and NFkB activity. MLT protects SCs from Cd toxicity reducing its H2O2 generation and reductive stress effects. A reduced activity of Nrf2 and the modulation of other molecular players of MLT signaling, provide a mechanistic rational for the cytoprotective effect of this molecule in SCs.

Molecular characterization of CTNSmutations in Tunisian patients with ocular cystinosis.

BackgroundOcular cystinosis is a rare autosomal recessive disorder characterized by intralysosomal cystine accumulation in renal, ophthalmic (cornea, conjunctiva), and other organ abnormalities. Patients with ocular cystinosis are mostly asymptomatic and typically experience mild photophobia due to cystine crystals in the cornea observed accidently during a routine ocular examination. The ocular cystinosis is associated with different mutations in CTNS gene. Cysteamine therapy mostly corrects the organ abnormalities.MethodsThis study was performed in collaboration with the department of ophthalmology of Farhat Hached Hospital. The Optical Coherence Tomography (OCT) of the cornea and retinal photography were used to search cystine crystals within the corneas and conjunctiva in eight Tunisian patients. Screening for the common 57-kb deletion was performed by standard multiplex PCR, followed by direct sequencing of the entire CTNS gene.ResultsThe studied patients were found to have cystine crystal limited anterior corneal stroma and the conjunctiva associated with retinal crystals accumulation. CTNS gene sequencing disclosed 7 mutations: three missense mutations (G308R, p.Q88K, and p.S139Y); one duplication (C.829dup), one framshift mutation (p.G258f), one splice site mutation (c.681 + 7delC) and a large deletion (20,327-bp deletion). Crystallographic structure analysis suggests that the novel mutation p.S139Y is buried in a first transmembrane helix closed to the lipid bilayer polar region, introducing a difference in hydrophobicity which could affect the hydrophobic interactions with the membrane lipids. The second novel mutation p.Q88K which is located in the lysosomal lumen close to the lipid membrane polar head region, introduced a basic amino acid in a region which tolerate only uncharged residue. The third missense mutation introduces a positive change in nonpolar tail region of the phospholipid bilayer membrane affecting the folding and stability of the protein in the lipid bilayer.ConclusionsOur data demonstrate that impaired transport of cystine out of lysosomes is the most common, which is obviously associated with the mutations of transmembrane domains of cystinosine resulting from a total loss of its activity.

MO038: Mineral bone density assessment in patients affected by cystinuria

Abstract BACKGROUND AND AIMS Cystinuria is a rare genetic disease characterized by impaired tubular transport of cystine and other dibasic amino acids that leads to cystine renal calculi. Although being a rare condition, cystinuria is the most frequent monogenic cause of nephrolithiasis accounting for 1% of kidney stones in adults and 8% in children with an average prevalence of 1 in 7000 births. Up to date, clinical features of cystinuria are believed to be attributable only to nephrolithiasis although cystinuric patients may present other comorbidities (chronic kidney disease, progression toward end-stage kidney disease and hypertension). All these features may be partially explained by recurrent nephrolithiasis. There are currently no data on bone features of patients with cystinuria. Data on a murine model demonstrated the presence of reduced bone mineral density irrespective of the presence or absence of uremia, suggesting bone metabolic abnormalities in cystinuria even in the absence of reduced glomerular filtration rate (GFR). Our aim is to characterize bone mineral density (BMD) in patients affected by cystinuria. METHOD The present study included 13 patients affected by cystinuria followed at the Rare Kidney Diseases outpatient clinic of our Institution. All eligible patients were more than 18 years of age with an estimated GFR ≥15 mL/min/1.73 m2 and provided written informed consent. Patients who did not sign informed consent, pregnant women, patients undergoing chronic hemodialysis or with a previous kidney transplant were excluded. History and laboratory data were collected during scheduled outpatient visits from September 2021 to December 2021, and all patients underwent a dual-energy X-ray absorptiometry (DEXA). One sample t-tests were used to assess whether BMD Z-scores were significantly different from 0. RESULTS Patients’ characteristics are summarized in Table 1. Of the enrolled patients, six (46%) were female and four of them (67%) were in menopause, four (31%) had active nephrolithiasis described as at least two episodes in the last 3 months, and two (15%) had a self-reported diagnosis of osteoporosis. Moreover, six patients (46%) had a positive history for bone fractures that were all self-reported as traumatic. Femoral Z-scores were significantly lower than 0 overall (P = 0.015) and across femoral sites, whereas lumbar Z-scores, although low on average, did not meet statistical significance (Figure 1). Interestingly, reduced BMD was very prevalent, with 10 patients (77%) showing osteopenia and 3 (23%) showing osteoporosis. CONCLUSION Our data show a high prevalence of osteopenia and osteoporosis in cystinuric patients, especially when considering that our population was rather young and with a normal or moderately impaired kidney function. Of further interest appears also the differential distribution of decreased BMD that seems to be affecting the femur rather than the lumbar spine. To our knowledge, these are the first data assessing the prevalence and the characteristics of BMD disorder in cystinuric patients.

Clinical and genetic characteristics of Tunisian children with infantile nephropathic cystinosis.

Nephropathic cystinosis is an autosomal recessive disease caused by a mutation in the CTNS gene which encodes cystinosin, a lysosomal cystine transporter. The spectrum of mutations in the CTNS gene is not well defined in the North African population. Here, we investigated twelve patients with nephropathic cystinosis belonging to eight Tunisian families in order to analyze the clinical and genetic characteristics of Tunisian children with infantile nephropathic cystinosis. Clinical data were collected retrospectively. Molecular analysis of the CTNS gene was performed by Sanger sequencing. We describe a new splicing mutation c.971-1G > C in the homozygous state in 6/12 patients which seems to be a founder mutation. The reported deletion of 23nt c.771_793 Del (p.Gly258Serfs*30) was detected in a homozygous state in one patient and in a heterozygous compound state with the c.971-1G > C mutation in 3/12 patients. Two of 12 patients have a deletion of exons 4 and 5 of the CTNS gene. None of our patients had the most common 57-kb deletion. The mutational spectrum in the Tunisian population is different from previously described populations. Thus, a molecular diagnostic strategy must be implemented in Tunisia, by targeting as a priority the common mutations described in this country. Such a strategy will allow a cost-effective diagnosis confirmation as well as early administration of treatment with oral cysteamine. A higher resolution version of the Graphical abstract is available as Supplementary information.

Differences in renal cortex transcriptional profiling of wild-type and novel type B cystinuria model rats

Cystinuria is a genetic disorder of cystine transport that accounts for 1–2% of all cases of renal lithiasis. It is characterized by hyperexcretion of cystine in urine and recurrent cystine lithiasis. Defective transport of cystine into epithelial cells of renal tubules occurs because of mutations of the transport heterodimer, including protein b0,+AT (encoded by SLC7A9) and rBAT (encoded by SLC3A1) linked through a covalent disulfide bond. Study generated a novel type B cystinuria rat model by artificially deleting 7 bp of Slc7a9 gene exon 3 using the CRISPR-Cas9 system, and those Slc7a9-deficient rats were proved to be similar with cystinuria in terms of genome, transcriptome, translation, and biologic phenotypes with no off-target editing. Subsequent comparisons of renal histopathology indicated model rats gained typical secondary changes as medullary fibrosis with no stone formation. A total of 689 DEGs (383 upregulated and 306 downregulated) were differentially expressed in the renal cortex of cystinuria rats. In accordance with the functional annotation of DEGs, the potential role of glutathione metabolism processes in the kidney of cystinuria rat model was proposed, and KEGG analysis results showed that knock-out of Slc7a9 gene triggered more biological changes which has not been studied. In short, for the first time, a rat model and its transcriptional database that mimics the pathogenesis and clinical consequences of human type B cystinuria were generated.

Mitochondrial Calcium Uniporter Drives Metastasis and Confers a Targetable Cystine Dependency in Pancreatic Cancer.

Elevated mitochondrial calcium uptake in PDAC promotes metastasis but exposes cystine addiction and ferroptosis sensitivity that could be targeted to improve pancreatic cancer treatment.

Retention of higher fertility depending on ovarian follicle reserve in cystine-glutamate transporter gene-deficient mice.

The cystine-glutamate transporter (xCT) is responsible for the transport of cystine into cells. We recently found that xCT-deficient (xCTKO) aged mice maintained a higher rate of ovulation and ovarian weight compared with wild-type (WT) mice. It has been reported that a xCT deficiency in cultured cells induces autophagy through the suppression of mTOR survival pathways. We have previously reported that starvation in neonatal mice increases the number of primordial follicles with concomitant autophagy activation. Therefore, we investigated age-related changes in follicle reserve and fertility in xCTKO mice and clarified whether the PI3K/AKT/mTOR signaling pathway contributes to this. The numbers of offspring in the xCTKO mice aged 10 and 12months were significantly higher than those in the WT mice. The primordial follicle numbers in xCTKO neonatal mice tended to be higher than WT mice during all times evaluated. In contrast, the primary follicle number was significantly lower in the xCTKO mice at 60h after birth. The expression of p-AKT, which promotes follicle development, was significantly lower in xCTKO mice than that in WT mice, whereas the expression ratios of LC3-II/LC3-I were significantly higher. The xCTKO mice had significantly more primordial follicles than WT mice at 2months of age and showed a similar trend at 13-15months of age. These results suggest that the maintenance of fertility in aged xCTKO mice can be attributed to high follicle reserve after puberty by suppression of follicle activation during the neonatal period.

POS-090 CYSTINOSIS: A RARE BUT TREATABLE CAUSE OF PROXIMAL RENAL TUBULAR ACIDOSIS

Cystinosis is a rare lysosomal storage disorder which is characterized by defective cystine transporter and accumulation of cystine in lysosomes of cells of various body organs. Most common and severe presentation is in infancy as Fanconi syndrome (infantile nephropathic cystinosis). Incidence of cystinosis is 1 in 1 to 2 lakh newborns; with high incidence in Europe and UK. In India total 19 cases are registered with Cystinosis Society of India. The most common reported Indian mutation is p. Ser141Phe in contrast to 57Kb deletion affecting first 10 exons of CTNS which is commonly reported in Europe. We report a 23 months old male child of Indian origin, born out of non-consanguineous marriage, who presented with acute gastroenteritis and seizure. On examination, child had severe dehydration, pallor, signs of rickets, failure to thrive and polyuria even in the state of dehydration. Initial evaluation revealed a S. creatinine 2.62mg/dl. After fluid resuscitation renal functions improved (nadir S. creatinine 0.82mg/dl). As per mother the child had increased frequency of urination since age of 7 months with history of hospitalization twice for dehydration and dyselectrolytemia at the age of 8 months and 10 months. During follow up period, child had persistent hyperchloremic metabolic acidosis and low serum phosphate.Urine analysis revealed- proteinuria (2+, 100mg/dl), glycosuria (sugar 1+ , 100mg/dl) and phosphaturia. Urinary anion gap was positive; Urine chromatography was suggestive of generalized aminoaciduria. Ultrasound abdomen was normal. On the basis of above findings, the diagnosis of proximal renal tubular acidosis was made. Eye evaluation was suggestive of fine crystals deposition in cornea. In view of possibility of cystinosis, Genetic analysis by next generation sequencing was done which confirmed novel mutation in cystinosine gene (NM_001031681.2: c.771_793delGGGACCACGTGGCTGCAGT;p.Gly258SerfsTer30). Child was started treatment with cysteamine capsules and eyedrops along with bicarbonate, vitamin D, calcium and phosphorus supplements. On treatment child started gaining height and weight, urine output decreased and S. creatinine continued to be at nadir value of 0.8 mg/dl. Renal tubular acidosis may be just a clinical presentation of an underlying systemic disorder, therefore relevant clinical examination with need based molecular testing should be done. As early diagnosis forms the key to specific therapy for long term favorable outcome.

The profibrotic and senescence phenotype of old lung fibroblasts is reversed or ameliorated by genetic and pharmacological manipulation of Slc7a11 expression.

Increased senescence and expression of profibrotic genes in old lung fibroblasts contribute to disrepair responses. We reported that primary lung fibroblasts from old mice have lower expression and activity of the cystine transporter Slc7a11/xCT than cells from young mice, resulting in changes in both the intracellular and extracellular redox environments. This study examines the hypothesis that low Slc7a11 expression in old lung fibroblasts promotes senescence and profibrotic gene expression. The levels of mRNA and protein of Slc7a11, senescence markers, and profibrotic genes were measured in primary fibroblasts from the lungs of old (24 mo) and young (3 mo) mice. In addition, the effects of genetic and pharmacological manipulation of Slc7a11 were investigated. We found that decreased expression of Slc7a11 in old cells was associated with elevated markers of senescence (p21, p16, p53, and β-galactosidase) and increased expression of profibrotic genes (Tgfb1, Smad3, Acta2, Fn1, Col1a1, and Col5a1). Silencing of Slc7a11 in young cells replicated the aging phenotype, whereas overexpression of Slc7a11 in old cells decreased expression of senescence and profibrotic genes. Young cells were induced to express the senescence and profibrotic phenotype by sulfasalazine, a Slc7a11 inhibitor, whereas treatment of old cells with sulforaphane, a Slc7a11 inducer, decreased senescence without affecting profibrotic genes. Like aging cells, idiopathic pulmonary fibrosis fibroblasts show decreased Slc7a11 expression and increased profibrotic markers. In short, old lung fibroblasts manifest a profibrotic and senescence phenotype that is modulated by genetic or pharmacological manipulation of Slc7a11.

15LO1 dictates glutathione redox changes in asthmatic airway epithelium to worsen type 2 inflammation.

Altered redox biology challenges all cells, with compensatory responses often determining a cell’s fate. When 15 lipoxygenase 1 (15LO1), a lipid-peroxidizing enzyme abundant in asthmatic human airway epithelial cells (HAECs), binds phosphatidylethanolamine-binding protein 1 (PEBP1), hydroperoxy-phospholipids, which drive ferroptotic cell death, are generated. Peroxidases, including glutathione peroxidase 4 (GPX4), metabolize hydroperoxy-phospholipids to hydroxy derivatives to prevent ferroptotic death, but consume reduced glutathione (GSH). The cystine transporter SLC7A11 critically restores/maintains intracellular GSH. We hypothesized that high 15LO1, PEBP1, and GPX4 activity drives abnormal asthmatic redox biology, evidenced by lower bronchoalveolar lavage (BAL) fluid and intraepithelial cell GSH:oxidized GSH (GSSG) ratios, to enhance type 2 (T2) inflammatory responses. GSH, GSSG (enzymatic assays), 15LO1, GPX4, SLC7A11, and T2 biomarkers (Western blot and RNA-Seq) were measured in asthmatic and healthy control (HC) cells and fluids, with siRNA knockdown as appropriate. GSSG was higher and GSH:GSSG lower in asthmatic compared with HC BAL fluid, while intracellular GSH was lower in asthma. In vitro, a T2 cytokine (IL-13) induced 15LO1 generation of hydroperoxy-phospholipids, which lowered intracellular GSH and increased extracellular GSSG. Lowering GSH further by inhibiting SLC7A11 enhanced T2 inflammatory protein expression and ferroptosis. Ex vivo, redox imbalances corresponded to 15LO1 and SLC7A11 expression, T2 biomarkers, and worsened clinical outcomes. Thus, 15LO1 pathway–induced redox biology perturbations worsen T2 inflammation and asthma control, supporting 15LO1 as a therapeutic target.

Methods for accurate and reproducible studies of pharmacological effects of selenium in cancer.

Selenium compounds have pronounced effects on cell growth and proliferation. Nutritional levels induce selenoproteins. However, the antineoplastic effects of supra-nutritional selenium levels are not mediated by selenoproteins. The most studied compound, selenite, was shown in a clinical trial to possess extraordinary pharmacological properties. The uptake of selenite as for GS-Se-SG and selenocystine is dependent on the extracellular reducing environment maintained by the Xc- cystine transporter (xc- antiporter) ensuring a high level of extracellular cysteine. The expression of the xc- antiporter is vital for selenium cytotoxicity and any xenobiotic or media constituents modulating the expression of this antiporter will greatly affect the cellular response. Cytotoxicity determinations are often difficult to interpret and repeat due to differences in culture conditions. In the current chapter, factors influencing the cellular response, e.g., media composition, cell culturing conditions, assays for key enzymes of importance for selenium metabolism and effects, along with selenium mediated modulation of microRNA expression and immune responses are treated.

D-Cysteine supplementation partially protects against ferroptosis induced by xCT dysfunction via increasing the availability of glutathione.

Glutathione (GSH) is synthesized from three amino acids and the overall process is highly dependent on the availability of l-cysteine (l-Cys). GSH serves as an essential cofactor for glutathione peroxidase 4 (Gpx4), which reduces phospholipid hydroperoxides. The inactivation of Gpx4 or an insufficient supply of l-Cys results in the accumulation of lipid hydroperoxides, eventually leading to iron-dependent cell death, ferroptosis. In this study, we investigated the anti-ferroptotic properties of d-cysteine (d-Cys) under conditions of dysfunction in cystine transporter, xCT. l-Cys supplementation completely rescued ferroptosis that had been induced by the erastin-mediated inhibition of xCT in Hepa 1-6 cells. Upon d-Cys supplementation, the erastin-treated cells remained completely viable for periods of up to 24 h but eventually died after 48 h. d-Cys supplementation suppressed the production of lipid peroxides, thereby ferroptosis. The addition of d-Cys sustained intracellular Cys and GSH levels to a certain extent. When Hepa 1-6 cells were treated with a combination of buthionine sulfoximine and erastin, the anti-ferroptotic effect of d-Cys was diminished. These collective results indicate that, although d-Cys is not the direct source of GSH, d-Cys supplementation protects cells from ferroptosis in a manner that is dependent on GSH synthesis via stimulating the uptake of l-Cys.

Molecular basis for redox control by the human cystine/glutamate antiporter system xc\u2212

Cysteine plays an essential role in cellular redox homoeostasis as a key constituent of the tripeptide glutathione (GSH). A rate limiting step in cellular GSH synthesis is the availability of cysteine. However, circulating cysteine exists in the blood as the oxidised di-peptide cystine, requiring specialised transport systems for its import into the cell. System xc− is a dedicated cystine transporter, importing cystine in exchange for intracellular glutamate. To counteract elevated levels of reactive oxygen species in cancerous cells system xc− is frequently upregulated, making it an attractive target for anticancer therapies. However, the molecular basis for ligand recognition remains elusive, hampering efforts to specifically target this transport system. Here we present the cryo-EM structure of system xc− in both the apo and glutamate bound states. Structural comparisons reveal an allosteric mechanism for ligand discrimination, supported by molecular dynamics and cell-based assays, establishing a mechanism for cystine transport in human cells.

Hematopoietic Stem Cell Gene Therapy for Cystinosis: From Bench-to-Bedside.

Cystinosis is an autosomal recessive metabolic disease that belongs to the family of lysosomal storage disorders. The gene involved is the CTNS gene that encodes cystinosin, a seven-transmembrane domain lysosomal protein, which is a proton-driven cystine transporter. Cystinosis is characterized by the lysosomal accumulation of cystine, a dimer of cysteine, in all the cells of the body leading to multi-organ failure, including the failure of the kidney, eye, thyroid, muscle, and pancreas, and eventually causing premature death in early adulthood. The current treatment is the drug cysteamine, which is onerous and expensive, and only delays the progression of the disease. Employing the mouse model of cystinosis, using Ctns−/− mice, we first showed that the transplantation of syngeneic wild-type murine hematopoietic stem and progenitor cells (HSPCs) led to abundant tissue integration of bone marrow-derived cells, a significant decrease in tissue cystine accumulation, and long-term kidney, eye and thyroid preservation. To translate this result to a potential human therapeutic treatment, given the risks of mortality and morbidity associated with allogeneic HSPC transplantation, we developed an autologous transplantation approach of HSPCs modified ex vivo using a self-inactivated lentiviral vector to introduce a functional version of the CTNS cDNA, pCCL-CTNS, and showed its efficacy in Ctns−/− mice. Based on these promising results, we held a pre-IND meeting with the Food and Drug Administration (FDA) to carry out the FDA agreed-upon pharmacological and toxicological studies for our therapeutic candidate, manufacturing development, production of the GMP lentiviral vector, design Phase 1/2 of the clinical trial, and filing of an IND application. Our IND was cleared by the FDA on 19 December 2018, to proceed to the clinical trial using CD34+ HSPCs from the G-CSF/plerixafor-mobilized peripheral blood stem cells of patients with cystinosis, modified by ex vivo transduction using the pCCL-CTNS vector (investigational product name: CTNS-RD-04). The clinical trial evaluated the safety and efficacy of CTNS-RD-04 and takes place at the University of California, San Diego (UCSD) and will include up to six patients affected with cystinosis. Following leukapheresis and cell manufacturing, the subjects undergo myeloablation before HSPC infusion. Patients also undergo comprehensive assessments before and after treatment to evaluate the impact of CTNS-RD-04 on the clinical outcomes and cystine and cystine crystal levels in the blood and tissues for 2 years. If successful, this treatment could be a one-time therapy that may eliminate or reduce renal deterioration as well as the long-term complications associated with cystinosis. In this review, we will describe the long path from bench-to-bedside for autologous HSPC gene therapy used to treat cystinosis.