Cystinosis: Status of research and treatment in India and the world.

Cystinosis is an autosomally inherited rare genetic disorder in which cystine accumulates in the lysosome. The defect arises from a mutation in the lysosomal efflux pump, cystinosin (or CTNS). Despite the disease being known for more than a century, research, diagnosis, and treatment in India have been very minimal. In recent years, however, some research on cystinosis has been carried out on understanding the pathophysiology and in the development of a humanized yeast model for interrogating the CTNS protein. There has also been a greater awareness of the disease that has been facilitated by the formation of the Cystinosis Foundation of India just over a decade ago. Awareness among primary physicians is critical for early diagnosis, which in turn is critical for proper treatment. Eight different mutations have been observed in cystinosis patients in India, and the mutation spectrum seems different to what has been seen in the US and Europe. Despite these positive developments, there are immense hurdles still to be surmounted. This includes ensuring that the diagnosis is done sooner, making cysteamine more easily available, and, also for the future, to make accessible the promise of gene therapy to cystinosis patients.

Recent advances in the in vitro and in vivo methods to assess impact of P-glycoprotein and breast cancer resistance protein transporters in central nervous system drug disposition.

One challenge in central nervous system (CNS) drug discovery has been ensuring the blood-brain barrier (BBB) penetration of compounds at an efficacious concentration that provides suitable safety margins for clinical investigation. Research providing for the accurate prediction of brain penetration of compounds during preclinical discovery is important to a CNS program. In the BBB, P-glycoprotein (P-gp) (ABCB1) and breast cancer resistance protein (BCRP) (ABCG2) transporters have been demonstrated to play a major role in the active efflux of endogenous compounds and xenobiotics out of the brain microvessel cells and back to the systemic circulation. In the past 10 years, there has been significant technological improvement in the sensitivity of quantitative proteomics methods, in vivo imaging, in vitro methods of organoid and microphysiological systems, as well as in silico quantitative physiological based pharmacokinetic and systems pharmacology models. Scientists continually leverage these advancements to interrogate the distribution of compounds in the CNS which may also show signals of substrate specificity of P-gp and/or BCRP. These methods have shown promise toward predicting and quantifying the unbound concentration(s) within the brain relevant for efficacy or safety. In this review, the authors have summarized the in vivo, in vitro, and proteomics advancements toward understanding the contribution of P-gp and/or BCRP in restricting the entry of compounds to the CNS of either healthy or special populations. Special emphasis has been provided on recent investigations on the application of a proteomics-informed approach to predict steady-state drug concentrations in the brain. Moreover, future perspectives regarding the role of these transporters in newer modalities are discussed.

Author Correction: A Genetic Screen for Investigating the Human Lysosomal CystineTransporter, Cystinosin.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A Genetic Screen for Investigating the Human Lysosomal CystineTransporter, Cystinosin.

Cystinosin, a lysosomal transporter is involved in the efflux of cystine from the lysosome to the cytosol. Mutations in the human cystinosin gene (CTNS) cause cystinosis, a recessive autosomal disorder. Studies on cystinosin have been limited by the absence of a robust genetic screen. In the present study we have developed a dual strategy for evaluating cystinosin function that is amenable to rapid genetic analysis. We show that human cystinosin expressed in this yeast confers growth on cystine when the protein is mistargeted to the plasma membrane by the deletion of the C-terminal targeting signal, GYQDL. We also screened a vacuolar protein sorting deletion library, and subsequently created multiple vps deletion mutants for kinetic studies. The double deletion, vps1Δvps17Δ, greatly enhanced uptake. This enabled validation by kinetic studies, including first studies on the WT CTNS protein (that contained the GYQDL motif). Using this screen we isolated several gain of function mutants, G131S/D, G309S/D, A137V, G197R, S270T, L274F and S312N showing enhanced growth on low concentrations of cystine. Kinetic analysis yielded insights into the role of the residues (including one of the patient mutations, G197R). The results indicate that the screen could be effectively used for interrogating and understanding the CTNS protein.

Insights into the molecular basis for substrate binding and specificity of the fungal cystine transporter CgCYN1.

Cystine transporters are a clinically important class of transporters found in bacteria, pathogenic fungi and mammalian cells. Despite their significance, very little is known about the mechanism of substrate recognition and transport. We have carried out studies on the plasma membrane Candida glabrata cystine transporter, CgCYN1 a member of the amino acid-polyamine-organocation (APC) transporter superfamily. A homology model of CgCYN1 was generated by using crystal structures of three known bacterial APC transporters followed by further refinement using molecular dynamics simulations. This revealed a possible translocation channel lined by TMD1, TMD3, TMD6, TMD8 and TMD10 helices. In silico docking studies with cystine along with comparison with other known cystine permeases and closely related lysine permeases allowed prediction of amino acid residues specifically involved in cystine binding. To validate this model a total of 19 predicted residues were subjected to site directed mutagenesis and functionally evaluated by growth on cystine and the analogues cystathionine and seleno-dl-cystine. Biochemical evaluation by radioactive uptake assays confirmed that these mutants showed reduced cystine uptake. Detailed kinetic analysis studies for the transport defective mutants revealed the involvement of residue G255 from the conserved FAYGGTE motif of TMD 6, and T339, S340 and H347 (all from TMD 8) in cystine binding. The implications of these findings on the homologous mammalian cystine transporter, XcT are also discussed.

Thiol trapping and metabolic redistribution of sulfur metabolites enable cells to overcome cysteine overload.

Cysteine is an essential requirement in living organisms. However, due to its reactive thiol side chain, elevated levels of intracellular cysteine can be toxic and therefore need to be rapidly eliminated from the cellular milieu. In mammals and many other organisms, excess cysteine is believed to be primarily eliminated by the cysteine dioxygenase dependent oxidative degradation of cysteine, followed by the removal of the oxidative products. However, other mechanisms of tackling excess cysteine are also likely to exist, but have not thus far been explored. In this study, we use Saccharomyces cerevisiae, which naturally lacks a cysteine dioxygenase, to investigate mechanisms for tackling cysteine overload. Overexpressing the high affinity cysteine transporter, YCT1, enabled yeast cells to rapidly accumulate high levels of intracellular cysteine. Using targeted metabolite analysis, we observe that cysteine is initially rapidly interconverted to non-reactive cystine in vivo. A time course revealed that cells systematically convert excess cysteine to inert thiol forms; initially to cystine, and subsequently to cystathionine, S-Adenosyl-L-homocysteine (SAH) and S-Adenosyl L-methionine (SAM), in addition to eventually accumulating glutathione (GSH) and polyamines. Microarray based gene expression studies revealed the upregulation of arginine/ornithine biosynthesis a few hours after the cysteine overload, and suggest that the non-toxic, non-reactive thiol based metabolic products are eventually utilized for amino acid and polyamine biogenesis, thereby enabling cell growth. Thus, cells can handle potentially toxic amounts of cysteine by a combination of thiol trapping, metabolic redistribution to non-reactive thiols and subsequent consumption for anabolism.

New insights into the genetics of 5-oxoprolinase deficiency and further evidence that it is a benign biochemical condition.

UNLABELLED: Inherited 5-oxoprolinase (OPLAH) deficiency is a rare inborn condition characterised by 5-oxoprolinuria. To date, three OPLAH mutations have been described: p.H870Pfs in a homozygous state, which results in a truncated protein, was reported in two siblings, and two heterozygous missense changes, p.S323R and p.V1089I, were independently identified in two unrelated patients. We describe the clinical context of a young girl who manifested 5-oxoprolinuria together with dusky episodes and who is compound heterozygote for two novel OPLAH variations: p.G860R and p.D1241V. To gain insight into the aetiology of the 5-oxoprolinase deficiency, we investigated the pathogenicity of all the reported missense mutations in the OPLAH gene. A yeast in vivo growth assay revealed that only p.S323R, p.G860R and p.D1241V affected the activity of the enzyme. CONCLUSION: Taken together, this report further suggests that hereditary 5-oxoprolinase deficiency is a benign biochemical condition caused by mutations in the OPLAH gene, which are transmitted in an autosomal recessive manner, but 5-oxoprolinuria may be a chance association in other disorders.