Cystine Depletion Research Articles

Abstract 3073: Targeting chronic lymphocytic leukemia by interfering glutathione synthesis using a novel therapeutic enzyme cyst(e)inase (AEB3103)

Abstract Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the Western countries. Despite recent advance in new therapeutic agents that have improved treatment outcomes, CLL remains incurable due in part to the inability to completely eradicate the leukemia cells in vivo. Previous studies showed that CLL cells have high intrinsic oxidative stress and are highly dependent on cellular antioxidant glutathione (GSH) to maintain redox balance and cell viability. One logical strategy to impact CLL cells would be to abrogate the glutathione protection of CLL cells in vivo. Recently we discovered that primary leukemia cells isolated from CLL patients were unable to effectively utilize cystine for GSH synthesis due to low expression of the cystine transporter Xc-, and that bone marrow stromal cells highly express Xc- and effectively take up cystine for conversion to cysteine (Zhang et al: Nature Cell Biology, 2012). These findings provide a biochemical basis to develop novel strategies to effectively target leukemia cells in the stromal microenvironment and improve in vivo therapeutic activity. In this study, we tested the depletion of extracellular cystine and cysteine using a novel therapeutic enzyme-cyst(e)inase (AEB3103), as a potential way to block GSH synthesis in CLL cells and abolish the stromal protection of the leukemia cells. Our study showed that AEB3103 was very effective in depleting GSH in CLL cells and caused massive CLL cell death even in the presence of stromal cells. Importantly, AEB3103 could also overcome drug resistance of CLL cells with p53 deficiency both in primary leukemia cells isolated from CLL patients with 17p deletion and mouse leukemic cells isolated from mouse model we recently reported (Liu et al: Leukemia, 2014). In addition, AEB3103 showed very low toxicity to normal cells. These promising in vitro data warrant further animal studies, which are currently on ongoing using the CLL mouse model with TCL1-Tg:p53-/- genotype. Our study suggests that AEB3103 and its combination with standard anti-CLL drugs may potentially be useful for clinical treatment of CLL, even for the more aggressive CLL subtypes with unfavorable cytogenetic alterations such as those with chromosome17p deletion and p53 mutations, and may improve in vivo therapeutic activity. Citation Format: Jinyun Liu, Li Feng, Everett M. Stone, Joseph Tyler, Scott W. Rowlinson, Michael J. Keating, Peng Huang. Targeting chronic lymphocytic leukemia by interfering glutathione synthesis using a novel therapeutic enzyme cyst(e)inase (AEB3103). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3073.

Lysosomal cystine accumulation promotes mitochondrial depolarization and induction of redox-sensitive genes in human kidney proximal tubular cells.

Cystine is a disulphide amino acid that is normally generated in the lysosomes by the breakdown of cystine-containing proteins. Previously, we demonstrated that lysosomal cystine accumulation in kidney proximal tubular epithelial cells (PTECs) dramatically reduced glutathione (GSH) levels, which may result in the disruption of cellular redox balance. In the present study, we show that lysosomal cystine accumulation following CTNS gene silencing in kidney PTECs resulted in elevated intracellular reactive oxygen species production, reduced antioxidant capacity, induction of redox-sensitive proteins, altered mitochondrial integrity and augmented cell death. These alterations may represent different facets of a unique cascade leading to tubular dysfunction initiated by lysosomal cystine accumulation and may present a clear disadvantage for cystinotic PTECs in vivo. Cystine depletion by cysteamine afforded cytoprotection in CTNS knockdown cells by reducing oxidative stress, normalizing intracellular GSH and ATP content, and preserving cell viability. Cystine is a disulphide amino acid that is normally generated within the lysosomes through lysosomal-based protein degradation and via extracellular uptake of free cystine. In the autosomal recessive disorder, cystinosis, a defect in the CTNS gene results in excessive lysosomal accumulation of cystine, with early kidney failure a hallmark of the disease. Previously, we demonstrated that silencing of the CTNS gene in kidney proximal tubular epithelial cells (PTECs) resulted in an increase in intracellular cystine concentration coupled with a dramatic reduction in the total GSH content. Because of the crucial role of GSH in maintaining the redox status and viability of kidney PTECs, we assessed the effects of CTNS knockdown-induced lysosomal cystine accumulation on intracellular reactive oxygen species (ROS) production, activity of classical redox-sensitive genes, mitochondrial integrity and cell viability. Our results showed that lysosomal cystine accumulation increased ROS production and solicitation to oxidative stress (OS). This was associated with the induction of classical redox-sensitive proteins, NF-κB, NRF2, HSP32 and HSP70. Cystine-loaded PTECs also displayed depolarized mitochondria, reduced ATP content and augmented apoptosis. Treatment of CTNS knockdown PTECs with the cystine-depleting agent cysteamine resulted in the normalization of OS index, increased GSH and ATP content, and preservation of cell viability. Taken together, the alterations observed in cystinotic cells may represent different facets of a cascade leading to tubular dysfunction and, in combination with cysteamine therapy, may offer a novel link for the attenuation of renal injury and preservation of functions of other organs affected in cystinosis.

Cystinosin is a Component of the Vacuolar H+-ATPase-Ragulator-Rag Complex Controlling Mammalian Target of Rapamycin Complex 1 Signaling.

Cystinosis is a rare autosomal recessive storage disorder characterized by defective lysosomal efflux of cystine due to mutations in the CTNS gene encoding the lysosomal cystine transporter, cystinosin. Lysosomal cystine accumulation leads to crystal formation and functional impairment of multiple organs. Moreover, cystinosis is the most common inherited cause of renal Fanconi syndrome in children. Oral cysteamine therapy delays disease progression by reducing intracellular cystine levels. However, because cysteamine does not correct all complications of cystinosis, including Fanconi syndrome, we hypothesized that cystinosin could have novel roles in addition to transporting cystine out of the lysosome. By coimmunoprecipitation experiments and mass spectrometry, we found cystinosin interacts with almost all components of vacuolar H(+)-ATPase and the Ragulator complex and with the small GTPases Ras-related GTP-binding protein A (RagA) and RagC. Furthermore, the mammalian target of rapamycin complex 1 (mTORC1) pathway was downregulated in proximal tubular cell lines derived from Ctns(-/-) mice. Decrease of lysosomal cystine levels by cysteamine did not rescue mTORC1 activation in these cells, suggesting that the downregulation of mTORC1 is due to the absence of cystinosin rather than to the accumulation of cystine. Our results show a dual role for cystinosin as a cystine transporter and as a component of the mTORC1 pathway, and provide an explanation for the appearance of Fanconi syndrome in cystinosis. Furthermore, this study highlights the need to develop new treatments not dependent on lysosomal cystine depletion alone for this devastating disease.

Cystinosis: renal glomerular and renal tubular function in relation to compliance with cystine-depleting therapy.

Nephropathic cystinosis is a lysosomal storage disorder characterized by renal tubular Fanconi syndrome in infancy and glomerular damage leading to renal failure at ∼10 years of age. Therapy with the cystine-depleting agent cysteamine postpones renal failure, but the degree of compliance with this treatment has not been correlated with preservation of kidney function. We assessed leucocyte cystine depletion by cysteamine and created the composite compliance score that incorporates the extent of leucocyte cystine depletion, as well as duration of cysteamine treatment, into a single integer. Age at renal failure was used to gauge preservation of renal function, and the Fanconi syndrome index (FSI), a measure of aminoaciduria, was used to assess renal tubular Fanconi syndrome. Age at renal failure varied directly and linearly with the composite compliance score (y = 0.3x +8.8; R(2) = 0.61). The slope indicated that for every year of excellent cystine depletion, nearly 1 year of renal function was preserved. Age at renal failure correlated roughly with mean leucocyte cystine level, but not with mean cysteamine dosage. There was no correlation between the FSI and the composite compliance score. Greater compliance with oral cysteamine therapy yields greater preservation of renal glomerular, but not tubular, function. Oral cysteamine therapy should be given at the maximum tolerated dose, within the recommended limits.

Improving the prognosis of nephropathic cystinosis.

Cystinosis is an autosomal recessive inherited lysosomal storage disease. It is characterized by generalized proximal tubular dysfunction known as renal Fanconi syndrome and causes end-stage renal disease by the age of about 10 years if left untreated. Extrarenal organs are also affected, including the thyroid gland, gonads, pancreas, liver, muscle, and brain. Treatment consists of administration of cysteamine, resulting in depletion of cystine that is trapped inside the lysosomes. Since cysteamine has a short half-life, it should be administered every 6 hours. Recently, a new delayed-release formulation was marketed, that should be administered every 12 hours. The first studies comparing both cysteamine formulations show comparable results regarding white blood cell cystine depletion (which serves as a measure for cystine accumulation in the body), while a slightly lower daily dose of cysteamine can be used.

Quality of Life is Improved and Kidney Function Preserved in Patients with Nephropathic Cystinosis Treated for 2 Years with Delayed-Release Cysteamine Bitartrate

ObjectivesTo determine the long-term effects of delayed-release cysteamine bitartrate (DR-CYS) based on our previous work that established the short-term noninferiority of DR-CYS every 12 hours compared with immediate-release cysteamine bitartrate every 6 hours.Study designWe conducted a prospective, controlled, open label, single-arm study of DR-CYS for 2 years in 40 patients to assess efficacy in depletion of cystine in peripheral white blood cells, to assess the dose required to maintain white blood cell content of cystine <1 nmol ½ cystine/mg protein, to measure quality of life using the Pediatric Quality of Life Inventory, change in estimated glomerular filtration rate, and change in height Z-score.ResultsThrough 24 months of study, the mean white blood cell content of cystine was always <1 nmol ½ cystine/mg protein, and the dose of DR-CYS decreased from 43.5-40.1 mg/kg/d (P = .05), and the significant improvement in social function, school function, and in total function scores on the Pediatric Quality of Life Inventory remained. The estimated glomerular filtration rate was maintained and growth velocity was maintained at 24 months compared with the baseline height Z-score.ConclusionsThe use of a DR-CYS administered every 12 hours to patients with cystinosis is of great benefit to their quality of life and to important biomarkers of disease control, when studied in a prospective, controlled fashion. We suggest that DR-CYS should be considered for substrate depletion in patients with cystinosis.

Modulation of CD8+ T-cell activation events by monocytic and granulocytic myeloid-derived suppressor cells

Myeloid-derived suppressor cells are immature myeloid cells, consisting of a monocytic and a granulocytic fraction, that are known to suppress anti-tumor immune responses. Important targets of the immunosuppressive capacity of MDSC are CD8+ T cells, which are crucial cytotoxic effector cells in immunotherapeutic settings. CD8+ T-cell activation and differentiation comprises a well-orchestrated series of events, starting from early TCR-mediated signaling and leading to cytokine secretion, the expression of activation markers, proliferation and the differentiation into several subsets of effector and memory cells. In this review, we summarize the available data on how the production of reactive oxygen species, nitric oxide, the arginase-mediated depletion of l-arginine and Cystine depletion by MDSCs interfere with the signaling molecules necessary for normal CTL differentiation and activation.

In vitro correction of disorders of lysosomal transport by microvesicles derived from baculovirus-infected Spodoptera cells

Infection of Spodoptera frugiperda (Sf9) cells by baculovirus (BV) is well established for transgene expression of soluble proteins, but few correctly folded transmembrane proteins have been so produced. We here report the use of the BV/Sf9 (BVES) method for the expression and transfer, via microvesicles, of the exclusive lysosomal exporters for cystine and sialic acid, human cystinosin and sialin. These proteins and their mRNA are released into the culture medium as very low-density microvesicles (~1.05g/ml), which do not label for lysobisphosphatidic acid. The presence of the human transgene proteins in the vesicles was confirmed by western blotting and confirmed and quantified by mass spectrometry. Addition of vesicles to cultures of human fibroblast lines deficient in either cystinosin or sialin produced a progressive depletion of stored lysosomal cystine or sialic acid, respectively. The depletion effect was slow (T1/2 ~48h), saturable (down to ~40% of initial after 4days) and stable (>one week). Surprisingly, BV infection of Spodoptera appeared to induce expression and release into microvesicles of the insect orthologue of cystinosin, but not of sialin. We conclude that BVES is an effective method to express and transfer functional transmembrane proteins so as to study their properties in mammalian cells, and has a generic potential for transport protein replacement therapy.

Cysteamine restores glutathione redox status in cultured cystinotic proximal tubular epithelial cells

Recent evidence implies that impaired metabolism of glutathione has a role in the pathogenesis of nephropathic cystinosis. This recessive inherited disorder is characterized by lysosomal cystine accumulation and results in renal Fanconi syndrome progressing to end stage renal disease in the majority of patients. The most common treatment involves intracellular cystine depletion by cysteamine, delaying the development of end stage renal disease by a yet elusive mechanism. However, cystine depletion does not arrest the disease nor cures Fanconi syndrome in patients, indicating involvement of other yet unknown pathologic pathways. Using a newly developed proximal tubular epithelial cell model from cystinotic patients, we investigate the effect of cystine accumulation and cysteamine on both glutathione and ATP metabolism. In addition to the expected increase in cystine and defective sodium-dependent phosphate reabsorption, we observed less negative glutathione redox status and decreased intracellular ATP levels. No differences between control and cystinosis cell lines were observed with respect to protein turnover, albumin uptake, cytosolic and mitochondrial ATP production, total glutathione levels, protein oxidation and lipid peroxidation. Cysteamine treatment increased total glutathione in both control and cystinotic cells and normalized cystine levels and glutathione redox status in cystinotic cells. However, cysteamine did not improve decreased sodium-dependent phosphate uptake. Our data implicate that cysteamine increases total glutathione and restores glutathione redox status in cystinosis, which is a positive side-effect of this agent next to cystine depletion. This beneficial effect points to a potential role of cysteamine as anti-oxidant for other renal disorders associated with enhanced oxidative stress.

PEGylated derivatives of cystamine as enhanced treatments for nephropathic cystinosis

The genetic disease, nephropathic cystinosis is characterized by lysosomal accumulation of the amino acid cystine. Crystallization of cystine in affected organs, if untreated, results in mortality of the affected individuals by their middle to late teens. The only approved treatment for cystinosis is administration of cysteamine. However, cysteamine is associated with an offending odor and taste and this, coupled to a rapid first pass metabolism and a 6h dosing regimen, suggest a clear need to improve the therapy. A number of PEGylated derivatives of cystamine, the disulfide counterpart of cysteamine, have been synthesised and evaluated in cultured cystinotic fibroblasts for toxicity and efficacy. All of the tested compounds were non-cytotoxic and displayed a remarkable depletion of intralysosomal cystine.

Downregulation of Cystine Transporter xc– by Irinotecan in Human Head and Neck Cancer FaDu Xenografts

Background: The purpose of this study was: (1) to document the critical requirement of cystine for growth of human tumor cells in vitro, and (2) to determine the effect of the anticancer agent irinotecan on the cystine transporter x_c^– in head and neck FaDu xenografts. Methods: Cell growth was measured by sulforhodamine B assay. xCT protein, glutathione (GSH) and DNA damage were determined using Western blot, spectrophotometry, and immunohistochemistry, respectively. Results: Depletion of cystine from the medium inhibited tumor cell growth. Treatment of FaDu tumor with a therapeutic dose of irinotecan resulted in depression of xCT protein levels, leading to tumor growth retardation and downregulation of GSH with increased reactive oxygen species (ROS). The accumulation of ROS correlated with increased DNA damage as evidenced by increased H2AX. Conclusion: Depression of xCT protein by irinotecan resulted in downregulation of GSH and increase in ROS, which could be the other possible mechanisms of DNA damage by irinotecan.

A major advance in treating cystinosis

Although many pediatricians have not heard of it, cysteamine bitartrate (Cystagon) can be considered one of pediatrics' “miracle drugs.” By promoting the movement of cystine out of the cell, the drug can arrest the ongoing organ damage in cystinosis. Prior to the development of this drug, renal failure in cystinosis was inevitable; today, with early introduction of cysteamine treatment, it can be postponed indefinitely. Unfortunately, the drug has a number of side effects that make adherence with therapy quite difficult. The recent development of an enteric coated product had been hoped to increase compliance by virtue of less frequent dosing (twice daily, instead of every 6 hours) and less troublesome gastrointestinal distress. The current issue of The Journal reports the long term (10 to 27 months) experience with enteric coated cysteamine in 5 children with cystinosis. Depletion of intracellular cystine was equivalent with this regimen to that achieved with every 6 hour dosing of the current product, and there was a trend toward less gastrointestinal side effects. Cystinosis may not be a common disease, but the development of a simple therapy that can promote growth and prevent kidney transplantation is a tribute to the efforts of basic scientists. This paper also provides the generalist with a nice review of the basic clinical chemistry involved. Long-Term Treatment of Cystinosis in Children with Twice-Daily CysteamineThe Journal of PediatricsVol. 156Issue 5PreviewCystinosis causes renal and other organ failure. Treatment with 6-hourly cysteamine bitartrate (Cystagon, Mylan, Morgantown, West Virginia) reduces intracellular cystine and the rate of organ deterioration. A recent study showed that an enteric-release cysteamine required less frequent daily dosing. This report describes the long-term use of enteric-coated (EC) cysteamine bitartrate (Cystagon) in children with cystinosis. Full-Text PDF

O.359 The effects of distractive osteogenesis on blood circulation in great vessels of maxillo-facial area

To characterize the pulmonarydysfunction in patients with nephropathic cystinosis after renaltransplantation.Cross-sectional analysis ofconsecutive adult patients.Twelve adult, nephropathic cystinosis patients and 3 adult, ocular, nonnephropathiccystinosis patients admitted to the National Institutes of Health Clinical Center.The 12 nephropathiccystinosis patients (age range, 21 to 40 years) showed anextraparenchymal pattern of restrictive lung disease, with inspiratoryand expiratory dysfunction. Specifically, the mean FVC was 58% ofpredicted, the mean FEV1 was 57% of predicted, and themean total lung capacity was 66% of predicted, while the mean residualvolume was normal. Furthermore, the mean maximal inspiratory pressurefor the eight patients tested was 40% of predicted, and the meanmaximal expiratory pressure was 26% of predicted. Two patients died ofrespiratory insufficiency. All the patients had lived at least 17years, while lacking compliant cystine-depleting therapy with oralcysteamine. Seven patients had a conical chest, restricting excursion, and 10 of the 12 patients had evidence of the myopathy that typifieslate cystinosis. In fact, the severity of pulmonary disease correlateddirectly with the severity of myopathy in our group of 12 patients. Incontrast, the lung parenchyma was essentially normal, as gauged bychest radiographs and CT scans of the lung. The three patients withnonnephropathic cystinosis displayed entirely normal pulmonaryfunction.The distal myopathycharacteristic of nephropathic cystinosis results in anextraparenchymal pattern of restrictive lung disease in adults who havenot received long-term cystine depletion. Whether or not oralcysteamine therapy can prevent this complication remains to bedetermined.

Understanding intestinal cysteamine bitartrate absorption

To test the hypothesis that a controlled-release preparation of cysteamine, with fewer daily administrations, would improve the quality of life for patients with cystinosis. A specifically designed nasoenteric tube was used to administer cysteamine directly into the stomach, small intestine (SI) and colon and serial plasma cysteamine, serum gastrin and leukocyte cystine levels were measured. Eight control subjects (mean age 23.2 years) and 6 subjects with cystinosis (mean age 15.2 years) were studied. Cysteamine absorption (maximum concentration and area under the curve of the concentration-time gradient) was greater from the SI than stomach or cecum (P < .01). Leukocyte cystine depletion was greater after delivery of cysteamine into the SI than stomach or cecum; this effect was associated with the plasma cysteamine maximum concentration and area under the curve (P < .001 and < .02, respectively). Gastrin levels were not affected by site of drug delivery and were elevated only in patients with cystinosis with gastrointestinal symptoms. The absorption of cysteamine and the effect of this agent on leukocyte cystine depletion are more profound after SI administration. Enteric-coated cysteamine, targeted for SI release, may require fewer daily dosages. Not all patients with cystinosis require acid-suppression therapy.

Sulfur amino acid deprivation in cadmium chloride toxicity in hepatoma cells

The aim of this study was to investigate the effect of individual sulfur amino acid deprivation in cadmium chloride toxicity. HTC cells were deprived of cystine and/or methionine for 12 h and then exposed to CdCl 2 for 12 h. HepG2 cells were deprived of cystine for 3 and 5 h and exposed to CdCl 2 for 3 h. In addition HepG2 cells were deprived of methionine for 12 h and then exposed to CdCl 2 for 5 and 12 h. Our results indicate that only cystine depletion increased cadmium toxicity in HTC cells but not in HepG2 cells as indicated by the neutral red assay. This effect was due to glutathione depletion as indicated by measurement of intracellular glutathione in HTC cells following deprivation of cystine. Methionine depletion had only a slight effect on the viability of HepG2 cells.

Nephropathic Cystinosis: Posterior Segment Manifestations and Effects of Cysteamine Therapy

Cystinosis is a rare autosomal recessive lysosomal storage disorder characterized by the intracellular accumulation of cystine. Treatment involves intracellular cystine depletion with oral cysteamine. A wide spectrum of ocular pathologic features has been associated with nephropathic cystinosis. We used the largest documented cohort of patients in the world to study the posterior segment manifestations associated with infantile nephropathic cystinosis and to determine retrospectively the effect of chronic oral cysteamine therapy on the frequency of these abnormalities. Cross-sectional study of a series of patients. Two hundred eight patients with infantile nephropathic cystinosis were studied at the National Institutes of Health between 1976 and 2004. All patients underwent an ophthalmic evaluation. Patients older than 11 years also underwent Humphrey static perimetry, and electrophysiological testing was performed when possible. Visual acuity, retina findings, visual fields, and electroretinographic (ERG) findings. Pigmentary changes with retinal pigment epithelial mottling, seen as early as infancy, were the most common posterior segment manifestations. Moderate to severe constriction of the visual fields, as well as moderate to severe reduction of rod- and cone-mediated ERG responses, was seen in older patients. The frequency of retinopathy correlated directly with time not receiving oral cysteamine therapy and inversely with time receiving oral cysteamine therapy. Infantile nephropathic cystinosis has posterior segment complications that can contribute to significant visual handicap. Early initiation of oral cysteamine therapy can reduce the frequency of posterior segment complications in cystinosis patients.

Coronary Artery and Other Vascular Calcifications in Patients with Cystinosis after Kidney Transplantation

Cystinosis, an autosomal recessive disorder of lysosomal cystine accumulation, results from mutations in the CTNS gene that encodes the lysosomal cystine transporter, cystinosin. Renal tubular Fanconi syndrome occurs in infancy, followed by rickets, growth retardation, photophobia, and renal failure, which requires renal transplantation at approximately 10 yr of age. Treatment with cysteamine decreases cellular cystine levels, retards renal deterioration, and allows for normal growth. Patients with a history of inadequate cystine depletion therapy may survive, after renal transplantation, into the third to fifth decades but will experience other, extrarenal complications of the disease. Routine chest and head computed tomography scans of 41 posttransplantation patients with cystinosis were reviewed for vascular calcification. The radiologic procedures had been performed to examine lung and brain parenchyma, so there was little ascertainment bias. Thirteen of the 41 patients had vascular calcification, including 11 with coronary artery calcification. One 25-yr-old man required three-vessel coronary artery bypass graft surgery. There were no significant differences between the 13 patients with calcification and the 28 without calcification in the following parameters: Time on dialysis, frequency of transplantation, hypertension, hypercholesterolemia, homozygosity for the 57-kb deletion in CTNS, serum creatinine, and calcium-phosphate product. However, the finding of vascular calcification correlated directly with duration of life without cysteamine therapy and inversely with duration of life under good cystine-depleting therapy. The accumulation of intracellular cystine itself maybe a risk factor for vascular calcifications, and older patients with cystinosis should be screened for this complication.

Combination of cassava flour cyanide and urinary thiocyanate measurements of school children in Mozambique

The maximum daily cassava flour intake of children may be calculated from determination of the total cyanide content of cassava flour and urinary thiocyanate levels of school children in samples collected at the same time and place. Four sites, two with and two without recent konzo cases, were chosen for study. In two sites with recent konzo cases, 84% and 93% of school children consumed cassava the previous day, and the calculated maximum daily consumption of cassava was over 700 g. In two sites without recent konzo cases, about 50% of school children consumed cassava the previous day and the calculated daily consumption of cassava flour was less than 150 g. By measurements of cyanide in flour and urinary thiocyanate we are therefore able to distinguish between communities whose diet is almost totally reliant on cassava, and who are therefore susceptible to konzo, and those who have a broader diet and are free from konzo. In another calculation it is shown that 4–23% of the essential S-containing amino acids in the cassava flour consumed by children is used up to detoxify and flour cyanide to thiocyanate. This depletion of methionine and cystine may leads to protein deficiency and may contribute to onset of konzo.

Inhibition of pyruvate kinase activity by cystine in brain cortex of rats

Cystinosis is a metabolic disturb associated with excessive lysosomal cystine accumulation secondary to defective cystine efflux. Patients affected by this disease develop a variable degree of symptoms depending of the involved tissues. Accumulation of cystine in the brain may lead to severe neurological symptoms. However, the mechanisms by which cystine is neurotoxic are not fully understood. Considering that pyruvate kinase (PK) is a thiolic enzyme crucial for the glycolytic pathway, and disulfides like cystine may alter thiolic enzymes by thiol/disulfide exchange, the main objective of the present study was to investigate the effect of cystine on PK activity in the brain cortex of developing Wistar rats. We also performed kinetic studies and investigated the effects of GSH, a biologically occurring thiol groups protector, and cysteamine (CysN), the drug used for cystinosis treatment, on the enzyme activity. We observed that cystine inhibited the enzyme activity by two different mechanisms, one through the competition with ADP and phosphoenolpyruvate (PEP), and the other non-competitively, probably through oxidation of the thiol groups of PK. We also observed that GSH and cysteamine fully prevented and reversed the inhibition caused by cystine. Considering that cysteamine is used in patients with cystinosis because it causes parenkimal organ cystine depletion, the present data provides a possible new beneficial effect for the use of this drug.

Cysteamine prevents and reverses the inhibition of pyruvate kinase activity caused by cystine in rat heart.

Cystinosis is a disorder associated with excessive lysosomal cystine accumulation secondary to defective cystine efflux. Patients affected by this disease develop a variable degree of symptoms depending on the involved tissues. Accumulation of cystine in myocardium may lead to heart failure. However, the mechanisms by which cystine is toxic to the tissues are not fully understood. Considering that thiolic enzymes like pyruvate kinase (PK) may be altered by disulfides like cystine, the main objective of the present study was to investigate the effect of cystine on PK activity in the heart of developing rats. We performed kinetic studies and investigated the effects of reduced glutathione (GSH), a biologically occurring thiol groups protector, and cysteamine, the drug used for cystinosis treatment, on the enzyme activity. We observed that cystine inhibited the enzyme activity non-competitively in a dose- and time-dependent way. We also observed that GSH and cysteamine fully prevented and reversed the inhibition caused by cystine, suggesting that cystine inhibits PK activity by oxidation of the sulfhydryl groups of the enzyme. Although there is no definite proof of cystine within cytoplasm, there is indirect proof t it is able to escape lysosomes and come in contact with PK. Considering that cysteamine is used in patients with cystinosis because it causes parenchymal organ cystine depletion, the present data provide a possible new effect for this drug.