Search

Cystinosis is an autosomal recessive disease caused by mutations in the CTNS gene encoding a protein called cystinosine, which is a lysosomal cystine transporter. Disease-causing mutations lead to accumulation of cystine crystals in the lysosomes, thereby causing dysfunction of vital organs. Determination of the increased leukocyte cystine level is one of the most used methods for diagnosis. However, this method is expensive, difficult to perform, and may yield different results in different laboratories. In this study, a disease model was created with CTNS gene-silenced HK2 cells, which can mimic cystinosis in cell culture, and multiomics methods (ie, proteomics, metabolomics, and fluxomics) were implemented at this cell culture to investigate new biomarkers for the diagnosis. CTNS-silenced cell line exhibited distinct metabolic profiles compared with the control cell line. Pathway analysis highlighted significant alterations in various metabolic pathways, including alanine, aspartate, and glutamate metabolism; glutathione metabolism; aminoacyl-tRNA biosynthesis; arginine and proline metabolism; beta-alanine metabolism; ascorbate and aldarate metabolism; and histidine metabolism upon CTNS silencing. Fluxomics analysis revealed increased cycle rates of Krebs cycle intermediates such as fumarate, malate, and citrate, accompanied by enhanced activation of inorganic phosphate and ATP production. Furthermore, proteomic analysis unveiled differential expression levels of key proteins involved in crucial cellular processes. Notably, peptidyl-prolyl cis-trans isomerase A, translation elongation factor 1-beta (EF-1beta), and 60S acidic ribosomal protein decreased in CTNS-silenced cells. Additionally, levels of P0 and tubulin α-1A chain were reduced, whereas levels of 40S ribosomal protein S8 and Midasin increased. Overall, our study, through the utilization of an in vitro cystinosis model and comprehensive multiomics approach, led to the way toward the identification of potential new biomarkers while offering valuable insights into the pathogenesis of cystinosis., (Copyright © 2023 United States & Canadian Academy of Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Cystinuria is an autosomal recessive disorder characterized by a cystine transport deficiency in the renal tubules due to mutations in two genes: SLC3A1 and SLC7A9. Cystinuria can be classified into three forms based on the genotype: type A, due to mutations in the SLC3A1 gene; type B, due to mutations in the SLC7A9 gene; and type AB, due to mutations in both genes., Methods: We report a 12-year-old boy from central China with cystine stones. He was from a non-consanguineous family that had no known history of genetic disease. A physical examination showed normal development and neurological behaviors. Whole-exome and Sanger sequencing were used to identify and verify the suspected pathogenic variants., Results: The compound heterozygous variants c.898_905del (p.Arg301AlafsTer6) is located in exon5 and c.1898_1899insAT (p.Asp634LeufsTer46) is located in exon10 of SLC3A1 (NM_000341.4) were deemed responsible for type A cystinuria family. The variant c.898_905del was reported in a Japanese patient in 2000, and the variant c.1898_1899insAT is novel., Conclusion: A novel pathogenic heterozygous variant pair of the SLC3A1 gene was identified in a Chinese boy with type A cystinuria, enriching the mutational spectrum of the SLC3A1 gene. We attempted to find a pattern for the association between the genotype of SLC3A1 variants and the manifestations of cystinuria in patients with different onset ages. Our findings have important implications for genetic counseling and the early clinical diagnosis of cystinuria., (© 2023. The Author(s).)

Background: Cystinosis, a rare lysosomal storage disease caused by mutations in the CTNS gene, is characterized by cystine crystallization and accumulation within multiple tissues, including kidney and brain. Its impact on neural function appears mild relative to its effects on other organs during early disease, but since therapeutic advances have led to substantially increased life expectancy, neurological implications are of increasing interest, necessitating deeper understanding of the impact of cystinosis on neurocognitive function. Behavioral difficulties have been reported in cystinosis in the visual domain. Very little is known, however, about how the brains of people living with cystinosis process visual information. This is especially interesting given that cystine accumulation in the cornea and posterior ocular structures is a hallmark of cystinosis., Methods: Here, high-density scalp electrophysiology was recorded to visual stimuli (during a Go/No-Go task) to investigate visual processing in individuals with cystinosis, compared to age-matched controls. Analyses focused on early stages of cortical visual processing., Results: The groups differed in their initial cortical response, with individuals with cystinosis exhibiting a significantly larger visual evoked potential (VEP) in the 130-150 ms time window. The groups also differed in the associations between neural responses and verbal abilities: While controls with higher IQ scores presented larger neural responses, that relationship was not observed in cystinosis., Conclusions: The enlarged VEP in cystinosis could be the result of cortical hyperexcitability and/or differences in attentional engagement and explain, at least partially, the visual and visual-spatial difficulties described in this population., (© 2023. The Author(s).)

Cystinosis is a lysosomal storage disease that is characterized by the accumulation of dipeptide cystine within the lumen. It is caused by mutations in the cystine exporter, cystinosin. Most of the clinically reported mutations are due to the loss of transporter function. In this study, we identified a rapidly degrading disease variant, referred to as cystinosin(7Δ). We demonstrated that this mutant is retained in the ER and degraded via the ER-associated degradation (ERAD) pathway. Using genetic and chemical inhibition methods, we elucidated the roles of HRD1, p97, EDEMs, and the proteasome complex in cystinosin(7Δ) degradation pathway. Having understood the degradation mechanisms, we tested some chemical chaperones previously used for treating CFTR F508Δ and demonstrated that they could facilitate the folding and trafficking of cystinosin(7Δ). Strikingly, chemical chaperone treatment can reduce the lumenal cystine level by approximately 70%. We believe that our study conclusively establishes the connection between ERAD and cystinosis pathogenesis and demonstrates the possibility of using chemical chaperones to treat cystinosin(7Δ).

Purpose: Cystinosis is an autosomal recessive lysosomal storage disease (LSDs) caused by mutations in the gene encoding cystinosin (CTNS) that leads to cystine crystal accumulation in the lysosome that compromises cellular functions resulting in tissue damage and organ failure, especially in kidneys and eyes. However, the underlying molecular mechanism of its pathogenesis remains elusive. Two novel mice lines created via CRISPR are used to examine the pathogenesis of cystinosis in the kidney and cornea and the treatment efficacy of corneal pathology using self-complimentary Adeno-associated viral (scAAV-CTNS) vector., Methods: The CRISPR technique generated two novel cystinotic mouse lines, Ctnsis1 (an insertional mutation) and Ctnsis2 (a nonsense mutation). Immune histochemistry, renal functions test and HRT2 in vivo confocal microscopy were used to evaluate the age-related renal pathogenesis and treatment efficacy of the scAAV-CTNS virus in corneal pathology., Results: Both mutations lead to the production of truncated Ctns proteins. Ctnsis1 and Ctnsis 2 mice exhibit the characteristic of cystinotic corneal crystal phenotype at four-week-old. Treatment with the scAAV-CTNS viral vector decreased the corneal crystals in the treated mice cornea. Ctnsis 1 show renal abnormalities manifested by increased urine volume, reduced urine osmolality, and the loss of response to Desmopressin (dDAVP) at 22-month-old but Ctnsis2 don't manifest renal pathology up to 2 years of age., Conclusions: Both Ctnsis1 and Ctnsis2 mice exhibit phenotypes resembling human intermediate nephropathic and ocular cystinosis, respectively. scAAV-CTNS viral vectors reduce the corneal cystine crystals and have a great potential as a therapeutic strategy for treating patients suffering from cystinosis., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Prostate Cancer (PCa) represents one of the most commonly diagnosed neoplasms in men and is associated with significant morbidity and mortality. Therapy resistance and significant side effects of current treatment strategies indicate the need for more effective agents to treat both androgen-dependent and androgen-independent PCa. In earlier studies, we demonstrated that depletion of L-cysteine/cystine with an engineered human enzyme, Cyst(e)inase, increased intracellular ROS levels and inhibited PCa growth in vitro and in vivo. The current study was conducted to further explore the mechanisms and potential combinatorial approaches with Cyst(e)inase for treatment of PCa., Methods: DNA single strand breaks and clustered oxidative DNA damage were evaluated by alkaline comet assay and pulsed field gel electrophoresis, respectively. Neutral comet assay and immunofluorescence staining was used to measure DNA double strand breaks. Cell survival and reactive oxygen species level were measured by crystal violet assay and DCFDA staining, respectively. Western blot was used to determine protein expression. FACS analyses were preformed for immune cell phenotyping. Allograft and xenograft tumor models were used for assessing effects on tumor growth., Results: PCa cells treated with Cyst(e)inase lead to DNA single and double strand breaks resulted from clustered oxidative DNA damage (SSBs and DSBs). Cyst(e)inase in combination with Auranofin, a thioredoxin reductase inhibitor, further increased intracellular ROS and DNA DSBs and synergistically inhibited PCa cell growth in vitro and in vivo. A combination of Cyst(e)inase with a PARP inhibitor (Olaparib) also increased DNA DSBs and synergistically inhibited PCa cell growth in vitro and in vivo without additional ROS induction. Knockdown of BRCA2 in PCa cells increased DSBs and enhanced sensitivity to Cyst(e)inase. Finally, Cyst(e)inase treatment altered tumor immune infiltrates and PD-L1 expression and sensitized PCa cells to anti-PD-L1 treatment., Conclusions: The current results demonstrate the importance of oxidative DNA damage either alone or in combination for Cyst(e)inase-induced anticancer activity. Furthermore, cysteine/cystine depletion alters the tumor immune landscape favoring enhanced immune checkpoint inhibition targeting PD-L1. Thus, combinatorial approaches with Cyst(e)inase could lead to novel therapeutic strategies for PCa., (© 2023. The Author(s).)

Purpose of Review: Servais et al. recently published clinical practice recommendations for the care of cystinuria patients. However, these guidelines were largely based on retrospective data from adults and children presenting with stones. Significant questions remain about the natural history of cystinuria in presymptomatic children., Recent Findings: We review the natural history of cystinuria in presymptomatic children followed from birth. In total, 130 pediatric patients were assigned putative genotypes based on parental urinary phenotype: type A/A (N = 23), B/B (N = 6), and B/N (N = 101). Stones were identified in 12/130 (4% of A/A, 17% of B/B, and 1% of B/N patients). Type B/B patients had lower cystine excretion than type A/A patients. Although urine cystine/creatinine fell with age, urine cystine/l rose progressively in parallel with the risk of nephrolithiasis. Each new stone was preceded by 6-12 months of urine specific gravity of more than 1.020. However, average urine specific gravity and pH were not different in stone formers vs. nonstone formers, suggesting that intrinsic stone inhibitors or other unknown factors may be the strongest determinants of individual risk., Summary: The current study reviews the clinical evolution of cystinuria in a cohort of children identified by newborn screening, who were categorized by urinary phenotype and followed from birth., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Cystinosis is a very rare autosomal recessive lysosomal storage disorder with an incidence of 1 : 150,000 - 1 : 200,000, and is caused by mutations in the CTNS gene encoding the lysosomal membrane protein cystinosin, which transports cystine out of the lysosome into the cytoplasm. As a result, accumulation of cystine occurs in almost all cells and tissues, especially in the kidneys, leading to multiple organ involvement. Introduction of drug therapy with cysteamine in the mid 1980s, along with the availability of renal replacement therapy in childhood, have dramatically improved patient outcome. Whereas patients used to die without therapy with end-stage renal failure during the first decade of life, nowadays most patients live well into adulthood without renal replacement therapy, and several reach 40 years. There is robust evidence that early initiation and sustained lifelong therapy with cysteamine are both essential for morbidity and mortality. The rarity of the disease and the multi-organ involvement present an enormous challenge for those affected and the providers of care for this patient group., Competing Interests: KH hat Drittmittel von Recordati und Chiesi erhalten. KZ hat keinen Interessenkonflikt. DH hat Forschungsförderungen und Vortragshonorare von Chiesi erhalten./KH received external funds from Recordati and Chiesi. KZ has no conflicts of interest to declare. DH received fees for lectures and support for research from Chiesi., (Thieme. All rights reserved.)

Nephropathic cystinosis is a rare autosomal recessive disease caused by mutations in the CTNS gene. This causes dysfunction of cystinosin, a protein that transports cystine out of lysosomes, causing cystine crystals to accumulate in cells in most organ systems. While renal complications predominate in the early forms of cystinosis, corneal crystal accumulation will inevitably manifest in all patients. The main symptoms are photophobia along with glare sensitivity and blepharospasm. In addition, corneal crystal accumulation can cause other complications, such as recurrent corneal erosions, punctate or filamentary keratopathy, and chronic dry eye. Eventually, peripheral corneal neovascularization and limbal stem cell deficiency may develop. Ophthalmologists play a key role in the early diagnosis of patients with cystinosis. This review aims to not only raise awareness of secondary complications of corneal crystal accumulation, but also to highlight current treatment options and challenges that ophthalmologists and pediatricians might face., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

Cystinosis is an autosomal recessive metabolic disease characterized by lysosomal accumulation of cystine in all the cells of the body. Infantile cystinosis begins in infancy by a renal Fanconi syndrome and eventually leads to multi-organ failure, including the kidney, eye, thyroid, muscle, and pancreas, eventually causing premature death in early adulthood. The current treatment is the drug cysteamine that only delays the progression of the disease. We identified the gene involved, CTNS, and showed that the encoded protein, cystinosin, is a proton-driven cystine transporter. We generated a mouse model of cystinosis, the Ctns -/- mice, that recapitulates the main disease complications. The goal was next to develop a gene therapy approach for cystinosis. We used bone marrow stem cells as a vehicle to bring the healthy CTNS gene to tissues, and we showed that wild-type hematopoietic stem and progenitor cell (HSPC) transplantation led to abundant tissue integration of bone marrow-derived cells, significant decrease of tissue cystine accumulation and long-term kidney, eye and thyroid preservation. We then developed an autologous transplantation approach of HSPCs modified ex vivo using a lentiviral vector to introduce a functional CTNS cDNA, and showed its efficacy in Ctns -/- mice. We conducted the pharmacology/toxicology studies, developed the manufacturing process using human CD34 + cells, and design the clinical trial. We received Food and Drug Administration (FDA)-clearance to start a phase 1/2 clinical trial for cystinosis in December 2018. Six patients have been treated so far. In this review, we describe the path to go from the gene to a gene therapy approach for cystinosis., (© 2023 médecine/sciences – Inserm.)

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.

Abstract: Cystinosis is an autosomal recessive lysosomal storage disorder with intracellular cystine accumulation caused by mutations in the CTNS gene. We present a case of a 48-year-old woman with a history of cystinosis and squamous cell carcinoma treated with Mohs micrographic surgery where widespread deposition of cystine crystals were noted on frozen sections of the Mohs layers. These were rectangular to polygonal refractile crystals within the cytoplasm of dermal fibroblasts and macrophages which were highlighted by polarized light microscopy. This case illustrates the use of frozen section processing to demonstrate the presence of intracellular cystine crystals. Moreover, because patients with cystinosis may be predisposed to developing carcinomas postrenal transplantation, Mohs surgeons should be aware of this unusual phenomenon when evaluating the slides., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Gastric cancer is a highly malignant tumor. Gastric cancer stem cells (GCSCs) are the main causes of drug resistance, metastasis, recurrence, and poor prognosis. As a secondary metabolite of lichen, Atranorin has a variety of biological effects, such as antibacterial, anti-inflammatory, analgesic, and wound healing; however, its killing effect on GCSCs has not been reported. In this study, we constructed Atranorin complexes comprising superparamagnetic iron oxide nanoparticles (SPION) (Atranorin@SPION). In vitro and in vivo experiments confirmed that Atranorin@SPION could significantly inhibit the proliferation, invasion, angiogenesis, and tumorigenicity of CD44+/ CD24+ GCSCs, and induce oxidative stress injury, Fe 2 + accumulation, and ferroptosis. Quantitative real-time reverse transcription PCR and western blotting results showed that Atranorin@SPION not only reduced the expression levels of GCSC stem cell markers and cell proliferation and division markers, but also significantly inhibited the expression levels of key molecules in the cystine/glutamate transporter (Xc-)/glutathione peroxidase 4 (GPX4) and Tet methylcytosine dioxygenase (TET) family proteins. The results of high performance liquid chromatography-mass spectrometry and Dot blotting showed that Atranorin@SPION significantly inhibited the mRNA 5‑hydroxymethylcytidine modification of GCSCs. Meanwhile, the results of RNA immunoprecipitation-PCR also indicated that Atranorin@SPIONs significantly reduced the 5-hydroxymethylcytidine modification level of GPX4 and SLC7A11 mRNA 3' untranslated region in GCSCs, resulting in a decrease in their stability, shortening their half-lives and reducing translation activity. Therefore, this study revealed that Atranorin@SPIONs induced ferroptosis of GCSCs by weakening the expression of the Xc-/GPX4 axis and the 5-hydroxymethylcytidine modification of mRNAs in the pathway, thereby achieving their therapeutic effect on gastric cancer., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

The lysosomal storage disease cystinosis is caused by mutations in CTNS , encoding the cystine transporter cystinosin, and in its severest form leads to proximal tubule dysfunction followed by kidney failure. Patients receive the drug-based therapy cysteamine from diagnosis. However, despite long-term treatment, cysteamine only slows the progression of end-stage renal disease. Preclinical testing in cystinotic rodents is required to evaluate new therapies; however, the current models are suboptimal. To solve this problem, we generated a new cystinotic rat model using CRISPR/Cas9-mediated gene editing to disrupt exon 3 of Ctns and measured various parameters over a 12-mo time course. Ctns -/- rats display hallmarks of cystinosis by 3-6 mo of age, as demonstrated by a failure to thrive, excessive thirst and urination, cystine accumulation in tissues, corneal cystine crystals, loss of LDL receptor-related protein 2 in proximal tubules, and immune cell infiltration. High levels of glucose, calcium, albumin, and protein were excreted at 6 mo of age, consistent with the onset of Fanconi syndrome, with a progressive diminution of urine urea and creatinine from 9 mo of age, indicative of chronic kidney disease. Kidney histology and immunohistochemistry showed proximal tubule atrophy and glomerular damage as well as classic "swan neck" lesions. Overall, Ctns -/- rats show a disease progression that more faithfully recapitulates nephropathic cystinosis than existing rodent models. The Ctns -/- rat provides an excellent new rodent model of nephropathic cystinosis that is ideally suited for conducting preclinical drug testing and is a powerful tool to advance cystinosis research. NEW & NOTEWORTHY Animal models of disease are essential to perform preclinical testing of new therapies before they can progress to clinical trials. The cystinosis field has been hampered by a lack of suitable animal models that fully recapitulate the disease. Here, we generated a rat model of cystinosis that closely models the human condition in a timeframe that makes them an excellent model for preclinical drug testing as well as being a powerful tool to advance research.

Listeria monocytogenes is a saprophyte and a human intracellular pathogen. Upon invasion into mammalian cells, it senses multiple metabolic and environmental signals that collectively trigger its transition to the pathogenic state. One of these signals is the tripeptide glutathione, which acts as an allosteric activator of L. monocytogenes's master virulence regulator, PrfA. While glutathione synthesis by L. monocytogenes was shown to be critical for PrfA activation and virulence gene expression, it remains unclear how this tripeptide is synthesized in changing environments, especially in light of the observation that L. monocytogenes is auxotrophic to one of its precursors, cysteine. Here, we show that the ABC transporter TcyKLMN is a cystine/cysteine importer that supplies cysteine for glutathione synthesis, hence mediating the induction of the virulence genes. Further, we demonstrate that this transporter is negatively regulated by three metabolic regulators, CodY, CymR, and CysK, which sense and respond to changing concentrations of branched-chain amino acids (BCAA) and cysteine. The data indicate that under low concentrations of BCAA, TcyKLMN is upregulated, driving the production of glutathione by supplying cysteine, thereby facilitating PrfA activation. These findings provide molecular insight into the coupling of L. monocytogenes metabolism and virulence, connecting BCAA sensing to cysteine uptake and glutathione biosynthesis as a mechanism that controls virulence gene expression. This study exemplifies how bacterial pathogens sense their intracellular environment and exploit essential metabolites as effectors of virulence. IMPORTANCE Bacterial pathogens sense the repertoire of metabolites in the mammalian niche and use this information to shift into the pathogenic state to accomplish a successful infection. Glutathione is a virulence-activating signal that is synthesized by L. monocytogenes during infection of mammalian cells. In this study, we show that cysteine uptake via TcyKLMN drives glutathione synthesis and virulence gene expression. The data emphasize the intimate cross-regulation between metabolism and virulence in bacterial pathogens.

Background: Ocular 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., Methods: This 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., Results: The 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., Conclusions: Our 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., (© 2022. The Author(s).)

Background: Cystinosis is an autosomal recessive disorder characterized by an accumulation of the amino acid cystine in lysosomes throughout the body. Cystinosis is an inherited disease resulting from the failure of lysosomal cystine transport. The responsible gene, Cystinosin, Lysosomal Cystine Transporter (CTNS), encodes the lysosomal cystine carrier cystinosin., Case Presentation: In this case report, we reviewed the genetic basis of cystinosis and investigated two Iranian cases affected by cystinosis, one of which revealed a rare mutation in the CTNS gene. Two patients, 9-year-old (patient A) and 11-year-old (patient B) symptomatic Iranian females with renal insufficiency, were diagnosed with cystinosis on the basis of their clinical features and laboratory tests. After genetic counseling, blood samples were obtained from the patients and their parents. Genomic Deoxyribonucleic Acid (DNA) was extracted from whole blood, and mutation analysis was performed using polymerase chain reaction and sequencing methods for all exons of the CTNS gene. At least 148 different pathogenic and deleterious mutations in the CTNS gene have been reported to date. Owing to our patient's prominent clinical features of cystinosis, we carried out a targeted search for mutations in the CTNS gene., Conclusions: This led us to confirm the existence of a homozygous DNA variation c.257_258deletionCT (p.Ser86PhefsTer38) in exon 6 of the gene in patient A and another homozygous DNA variation, c.323delA (p.Q108RfsTer10), in the same exon in patient B. As expected, the mentioned mutation existed in both her parents in a heterozygous state. Variations c.257_258delCT and c.323delA reported in three Iranian patients in the CTNS gene are frameshifts, and truncating mutations that affect product function result in relatively mild symptoms of cystinosis. The present finding confirms previous research and proves the importance of the association of this gene rare mutations with cystinosis. Since reported mutations are rare, their previous reports in Iranian patients indicate the high frequency of these mutations in our region., (© 2022. The Author(s).)

Cystinuria is an autosomal recessive disorder characterized by a decrease in the reabsorption of cystine and dibasic amino acids (lysine, ornithine, and arginine) in the renal proximal tubule. It presents with recurrent urolithiasis. Cystinuria accounts for 6-8% of all pediatric urolithiasis. The age of onset is typically 10-30 years. Here, we report a case of early-onset cystinuria. A 4-month-old girl presented with hematuria. We noticed multiple renal calculi in ultrasonography and abdominal computerized tomography scans. The diagnosis was cystinuria with urinary calculus analysis and urinary amino acid analysis. The patient was treated with urine alkalinization and cystine chelating drugs. Gene analysis showed a P482L heterozygous mutation from her mother, and an A70V heterozygous mutation from her father, in the SLC7A9 gene. This gene encodes a putative subunit of the neutral and basic amino acid transport protein, BAT1. Although cystinuria is an autosomal recessive disease, there have been previous reports of P482L heterozygous mutations greatly suppressing cystine reabsorption and causing cystinuria symptoms. Therefore, the highly influential P482L mutation of the SLC7A9 gene may have contributed to the onset of this autosomal recessive disease at an extremely young age., (© 2021. Japanese Society of Nephrology.)

Alcohol misuse is common in many societies worldwide and is associated with extensive morbidity and mortality, often leading to alcohol use disorders (AUD) and alcohol-related end-organ damage. The underlying mechanisms contributing to the development of AUD are largely unknown; however, growing evidence suggests that alcohol consumption is strongly associated with alterations in DNA methylation. Identification of alcohol-associated methylomic variation might provide novel insights into pathophysiology and novel treatment targets for AUD. Here we performed the largest single-cohort epigenome-wide association study (EWAS) of alcohol consumption to date (N = 8161) and cross-validated findings in AUD populations with relevant endophenotypes, as well as alcohol-related animal models. Results showed 2504 CpGs significantly associated with alcohol consumption (Bonferroni p value < 6.8 × 10 -8 ) with the five leading probes located in SLC7A11 (p = 7.75 × 10 -108 ), JDP2 (p = 1.44 × 10 -56 ), GAS5 (p = 2.71 × 10 -47 ), TRA2B (p = 3.54 × 10 -42 ), and SLC43A1 (p = 1.18 × 10 -40 ). Genes annotated to associated CpG sites are implicated in liver and brain function, the cellular response to alcohol and alcohol-associated diseases, including hypertension and Alzheimer's disease. Two-sample Mendelian randomization confirmed the causal relationship of consumption on AUD risk (inverse variance weighted (IVW) p = 5.37 × 10 -09 ). A methylation-based predictor of alcohol consumption was able to discriminate AUD cases in two independent cohorts (p = 6.32 × 10 -38 and p = 5.41 × 10 -14 ). The top EWAS probe cg06690548, located in the cystine/glutamate transporter SLC7A11, was replicated in an independent cohort of AUD and control participants (N = 615) and showed strong hypomethylation in AUD (p < 10 -17 ). Decreased CpG methylation at this probe was consistently associated with clinical measures including increased heavy drinking days (p < 10 -4 ), increased liver function enzymes (GGT (p = 1.03 × 10 -21 ), ALT (p = 1.29 × 10 -6 ), and AST (p = 1.97 × 10 -8 )) in individuals with AUD. Postmortem brain analyses documented increased SLC7A11 expression in the frontal cortex of individuals with AUD and animal models showed marked increased expression in liver, suggesting a mechanism by which alcohol leads to hypomethylation-induced overexpression of SLC7A11. Taken together, our EWAS discovery sample and subsequent validation of the top probe in AUD suggest a strong role of abnormal glutamate signaling mediated by methylomic variation in SLC7A11. Our data are intriguing given the prominent role of glutamate signaling in brain and liver and might provide an important target for therapeutic intervention., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Attachment of sugars to nitrogen and oxygen in peptides is ubiquitous in biology, but glycosylation of sulfur atoms has only been recently described. Here, we characterize two S-glycosyltransferases SunS and ThuS that selectively glycosylate one of five Cys residues in their substrate peptides; substitution of this Cys with Ser results in a strong decrease in glycosylation activity. Crystal structures of SunS and ThuS in complex with UDP-glucose or a derivative reveal an unusual architecture in which a glycosyltransferase type A (GTA) fold is decorated with additional domains to support homodimerization. Dimer formation creates an extended cavity for the substrate peptide, drawing functional analogy with O-glycosyltransferases involved in cell wall biosynthesis. This extended cavity contains a sharp bend that may explain the site selectivity of the glycosylation because the target Cys is in a Gly-rich stretch that can accommodate the bend. These studies establish a molecular framework for understanding the unusual S-glycosyltransferases., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The impenetrability of the blood-brain barrier (BBB) to most conventional drugs impedes the treatment of central nervous system (CNS) disorders. Interventions for diseases like brain cancer, neurodegeneration, or age-associated inflammatory processes require varied approaches to CNS drug delivery. Cystine-dense peptides (CDPs) have drawn recent interest as drugs or drug-delivery vehicles. Found throughout the phylogenetic tree, often in drug-like roles, their size, stability, and protein interaction capabilities make CDPs an attractive mid-size biologic scaffold to complement conventional antibody-based drugs. Here, we describe the identification, maturation, characterization, and utilization of a CDP that binds to the transferrin receptor (TfR), a native receptor and BBB transporter for the iron chaperone transferrin. We developed variants with varying binding affinities (K D as low as 216 pM), co-crystallized it with the receptor, and confirmed murine cross-reactivity. It accumulates in the mouse CNS at ~25% of blood levels (CNS blood content is only ~1%-6%) and delivers neurotensin, an otherwise non-BBB-penetrant neuropeptide, at levels capable of modulating CREB signaling in the mouse brain. Our work highlights the utility of CDPs as a diverse, easy-to-screen scaffold family worthy of inclusion in modern drug discovery strategies, demonstrated by the discovery of a candidate CNS drug delivery vehicle ready for further optimization and preclinical development., Competing Interests: Competing Interests Z.R.C., E.J.G., C.Y., G.H., A.D.S., A.J.M., R.K.S., and J.M.O. are listed as authors on patent filings related to technology described in this work. Z.R.C. and G.P.S. have a paid consulting relationship with Blaze Bioscience, Inc. J.M.O. is a founder and shareholder of Blaze Bioscience, Inc. The remaining authors declare no competing financial interests., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Rust fungi are plant pathogens that secrete an arsenal of effector proteins interfering with plant functions and promoting parasitic infection. Effectors are often species-specific, evolve rapidly, and display low sequence similarities with known proteins. How rust fungal effectors function in host cells remains elusive, and biochemical and structural approaches have been scarcely used to tackle this question. In this study, we produced recombinant proteins of eleven candidate effectors of the leaf rust fungus Melampsora larici-populina in Escherichia coli. We successfully purified and solved the three-dimensional structure of two proteins, MLP124266 and MLP124017, using NMR spectroscopy. Although both MLP124266 and MLP124017 show no sequence similarity with known proteins, they exhibit structural similarities to knottins, which are disulfide-rich small proteins characterized by intricate disulfide bridges, and to nuclear transport factor 2-like proteins, which are molecular containers involved in a wide range of functions, respectively. Interestingly, such structural folds have not been reported so far in pathogen effectors, indicating that MLP124266 and MLP124017 may bear novel functions related to pathogenicity. Our findings show that sequence-unrelated effectors can adopt folds similar to known proteins, and encourage the use of biochemical and structural approaches to functionally characterize effector candidates.

The incidence of symptomatic radiation induced lung pneumonitis (RILP), a major dose limiting side effect of thoracic radiotherapy, is in the range of 15-40%. Therapeutic options for the prevention and treatment of RILP are limited. Hence there is a need for developing novel radioprotectors to prevent RILP which can be patient compliant. This study sought to evaluate the efficacy of oral 3,3'-diselenodipropionic acid (DSePA), a novel selenocystine derivative to prevent RILP. C3H/HeJ (pneumonitis responding) mice received a single dose of 18 Gy, whole thorax irradiation and a subset were treated with DSePA orally (2.5 mg/kg), three times per week beginning 2 h post irradiation and continued till 6 months. DSePA delayed onset of grade ≥ 2 RILP by 45 days compared to radiation control (~105 versus ~60 days). It also reversed the severity of pneumonitis in 3/10 radiation treated mice leading to significant improvement in asymptomatic survival compared to radiation control (~180 versus ~102 days). DSePA significantly (p < 0.05) reduced the radiation-mediated infiltration of polymorphonuclear neutrophils (PMN) and elevation in levels of cytokines such as IL1-β, ICAM-1, E-selectin, IL-17 and TGF-β in the bronchoalveolar lavage fluid. Moreover DSePA lowered PMN-induced oxidants, maintained glutathione peroxidase activity and suppressed NF-kB/IL-17/G-CSF/neutrophil axis in the lung of irradiated mice. Additionally, this compound did not protect A549 (lung cancer) derived xenograft tumor from radiation exposure in SCID mice. DSePA offers protection to normal lung against RILP without affecting radiation sensitivity of tumors. It has the potential to be developed as an oral agent for preventing RILP., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Cystine-stabilized peptides have great utility as they naturally block ion channels, inhibit acetylcholine receptors, or inactivate microbes. However, only a tiny fraction of these peptides has been characterized. Exploration for novel peptides most efficiently starts with the identification of candidates from genome sequence data. Unfortunately, though cystine-stabilized peptides have shared structures, they have low DNA sequence similarity, restricting the utility of BLAST and even more powerful sequence alignment-based annotation algorithms, such as PSI-BLAST and HMMER. In contrast, a supervised machine learning approach may improve discovery and function assignment of these peptides. To this end, we employed our previously described m-NGSG algorithm, which utilizes hidden signatures embedded in peptide primary sequences that define and categorize structural or functional classes of peptides. From the generalized m-NGSG framework, we derived five specific models that categorize cystine-stabilized peptide sequences into specific functional classes. When compared with PSI-BLAST, HMMER and existing function-specific models, our novel approach (named CSPred) consistently demonstrates superior performance in discovery and function-assignment. We also report an interactive version of CSPred, available through download ( https://bitbucket.org/sm&#95;islam/cystine-stabilized-proteins/src ) or web interface (watson.ecs.baylor.edu/cspred), for the discovery of cystine-stabilized peptides of specific function from genomic datasets and for genome annotation. We fully describe, in the Availability section following the Discussion, the quick and simple usage of the CsPred website to automatically deliver function assignments for batch submissions of peptide sequences.

Modulation of metabotropic glutamate 2 (mGlu2) receptor function has huge potential for treating psychiatric and neurological diseases. Development of drugs acting on mGlu2 receptors depends on the development and use of translatable animal models of disease. We report here a stop codon mutation at cysteine 407 in Grm2 (cys407*) that is common in some Wistar rats. Therefore, researchers in this field need to be aware of strains with this mutation. Our genotypic survey found widespread prevalence of the mutation in commercial Wistar strains, particularly those known as Han Wistar. Such Han Wistar rats are ideal for research into the separate roles of mGlu2 and mGlu3 receptors in CNS function. Previous investigations, unknowingly using such mGlu2 receptor-lacking rats, provide insights into the role of mGlu2 receptors in behaviour. The Grm2 mutant rats, which dominate some selectively bred lines, display characteristics of altered emotionality, impulsivity and risk-related behaviours and increased voluntary alcohol intake compared with their mGlu2 receptor-competent counterparts. In addition, the data further emphasize the potential therapeutic role of mGlu2 receptors in psychiatric and neurological disease, and indicate novel methods of studying the role of mGlu2 and mGlu3 receptors. This article is part of the Special Issue entitled 'Metabotropic Glutamate Receptors, 5 years on'., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Voltage-dependent potassium (Kv) channels allow for the selective permeability of potassium ions in a membrane potential dependent manner, playing crucial roles in neurotransmission and muscle contraction. Kv channel is a tetramer, in which each subunit possesses a voltage-sensing domain (VSD) and a pore domain (PD). Although several lines of evidence indicated that membrane depolarization is sensed as the movement of helix S4 of the VSD, the detailed voltage-sensing mechanism remained elusive, due to the difficulty of structural analyses at resting potential. In this study, we conducted a comprehensive disulfide locking analysis of the VSD using 36 double Cys mutants, in order to identify the proximal residue pairs of the VSD in the presence or absence of a membrane potential. An intramolecular SS-bond was formed between 6 Cys pairs under both polarized and depolarized environment, and one pair only under depolarized environment. The multiple conformations captured by the SS-bond can be divided by two states, up and down, where S4 lies on the extracellular and intracellular sides of the membrane, respectively, with axial rotation of 180°. The transition between these two states is caused by the S4 translocation of 12 Å, enabling allosteric regulation of the gating at the PD.

Familial British dementia (FBD) is an inherited neurodegenerative disease believed to result from a mutation in the BRI2 gene. Post-translational processing of wild type BRI2 and FBD-BRI2 result in the production of a 23-residue long Bri peptide and a 34-amino acid long ABri peptide, respectively, and ABri is found deposited in the brains of individuals with FBD. Similarities in the neuropathology and clinical presentation shared by FBD and Alzheimer disease (AD) have led some to suggest that ABri and the AD-associated amyloid β-protein (Aβ) are molecular equivalents that trigger analogous pathogenic cascades. But the sequences and innate properties of ABri and Aβ are quite different, notably ABri contains two cysteine residues that can form disulfide bonds. Thus we sought to determine whether ABri was neurotoxic and if this activity was regulated by oxidation and/or aggregation. Crucially, the type of oxidative cross-linking dramatically influenced both ABri aggregation and toxicity. Cyclization of Bri and ABri resulted in production of biologically inert monomers that showed no propensity to assemble, whereas reduced ABri and reduced Bri aggregated forming thioflavin T-positive amyloid fibrils that lacked significant toxic activity. ABri was more prone to form inter-molecular disulfide bonds than Bri and the formation of covalently stabilized ABri oligomers was associated with toxicity. These results suggest that extension of the C-terminal of Bri causes a shift in the type of disulfide bonds formed and that structures built from covalently cross-linked oligomers can interact with neurons and compromise their function and viability., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Crouzon is an autosomal dominant craniosynostosis syndrome caused by mutation in the fibroblast growth factor receptor (FGFR)-2 gene. Recent findings from animal studies imply a critical role for FGFs in the regulation of mineralization. Here, we presented a 5-year-old girl with severe meningeal calcification. Subsequently, we analyzed FGFR2 mutation and identified a mutation of Cys342Tyr. The findings suggest that abnormal calcification was atypical phenotype of Crouzon patients with Cys342Tyr mutation in FGFR2.

Previously, we reported that human β-defensin (hBD)-3 can both antagonize CXCR4 function on T cells and promote receptor internalization in the absence of activation. In the present study, we explored the important structural elements of hBD-3 that are involved in blocking CXCR4 activation by its natural ligand, stromal-derived factor 1α (SDF-1α; CXCL12). Results from site-directed mutagenesis studies suggest that the ability of hBD-3 to inhibit SDF-1α-CXCR4 interaction, as assayed either by blocking SDF-1 binding to CXCR4 or antagonizing SDF-1-induced Ca(2+) mobilization, is correlated with the presence of hBD-3 cysteine residues, specific surface-distributed cationic residues, and the electrostatic properties and availability of both hBD-3 termini. Specifically, hBD-3 activity against CXCR4 is reduced by: (a) replacing all six cysteines; (b) replacing the cationic residues with acidic ones in the N-terminus and C- terminus; (c) removal of the first 10 N-terminal residues; and (d) replacing the surface-exposed basic residues Lys8, Lys32 and Arg36 with neutral ones. The hBD-3-CXCR4 interaction has potentially wide-ranging implications for HIV-related biology, as well as for a host of CXCR4-dependent activities, including hematopoiesis, neurogenesis, angiogenesis, carcinogenesis, and immune cell trafficking. CXCR4 is highly expressed on T cells, monocytes, and epithelial cells. Therefore, understanding the structure-function relationship between hBD-3 and CXCR4 that accounts for the antagonistic interaction between the two molecules may provide new insights into HIV/highly active antiretroviral therapy-related pathology, as well as novel insights into the interaction between innate and adaptive immunity at mucosal sites., (© 2013 FEBS.)

The ICK (inhibitor cystine knot) defines a large superfamily of polypeptides with high structural stability and functional diversity. Here, we describe a new scorpion venom-derived K+ channel toxin (named λ-MeuKTx-1) with an ICK fold through gene cloning, chemical synthesis, nuclear magnetic resonance spectroscopy, Ca2+ release measurements and electrophysiological recordings. λ-MeuKTx-1 was found to adopt an ICK fold that contains a three-strand anti-parallel β-sheet and a 310-helix. Functionally, this peptide selectively inhibits the Drosophila Shaker K+ channel but is not capable of activating skeletal-type Ca2+ release channels/ryanodine receptors, which is remarkably different from the previously known scorpion venom ICK peptides. The removal of two C-terminal residues of λ-MeuKTx-1 led to the loss of the inhibitory activity on the channel, whereas the C-terminal amidation resulted in the emergence of activity on four mammalian K+ channels accompanied by the loss of activity on the Shaker channel. A combination of structural and pharmacological data allows the recognition of three putative functional sites involved in channel blockade of λ-MeuKTx-1. The presence of a functional dyad in λ-MeuKTx-1 supports functional convergence among scorpion venom peptides with different folds. Furthermore, similarities in precursor organization, exon-intron structure, 3D-fold and function suggest that scorpion venom ICK-type K+ channel inhibitors and Ca2+ release channel activators share a common ancestor and their divergence occurs after speciation between buthidae and non-buthids. The structural and functional characterizations of the first scorpion venom ICK toxin with K+ channel-blocking activity sheds light on functionally divergent and convergent evolution of this conserved scaffold of ancient origin.

θ-Defensins are ribosomally synthesized cyclic peptides found in the leukocytes of some primate species and have promising applications as antimicrobial agents and scaffolds for peptide drugs. The cyclic cystine ladder motif, comprising a cyclic peptide backbone and three parallel disulfide bonds, is characteristic of θ-defensins. In this study, we explore the role of the cyclic peptide backbone and cystine ladder in the structure, stability, and activity of θ-defensins. θ-Defensin analogues with different numbers and combinations of disulfide bonds were synthesized and characterized in terms of their NMR solution structures, serum and thermal stabilities, and their antibacterial and membrane-binding activities. Whereas the structures and stabilities of the peptides were primarily dependent on the number and position of the disulfide bonds, their antibacterial and membrane-binding properties were dependent on the cyclic backbone. The results provide insights into the mechanism of action of θ-defensins and illustrate the potential of θ-defensin analogues as scaffolds for peptide drug design.

Antibodies are important tools for a broad range of applications due to their high specificity and ability to recognize virtually any target molecule. However, in order to be practically useful, antibodies must be highly stable and bind their target antigens with high affinity. We present a combinatorial approach to generate high-affinity, highly stable antibodies through the design of stable frameworks, specificity grafting and maturation via somatic hypermutation in vitro. By collectively employing these methods, we have engineered a highly stable, high-affinity, full-length antibody with a T(m) over 90°C that retains significant activity after heating to 90°C for 1 h, and has ~95-fold improved antigen-binding affinity. The stabilized IgG framework is compatible with affinity maturation, and should provide a broadly useful scaffold for grafting a variety of complementarity-determining region loops for the development of stable antibodies with desired specificities.

Tyrosine-kinase-based signal transduction mediated by modular protein domains is critical for cellular function. The Src homology (SH)2 domain is an important conductor of intracellular signaling that binds to phosphorylated tyrosines on acceptor proteins, producing molecular complexes responsible for signal relay. Cortactin is a cytoskeletal protein and tyrosine kinase substrate that regulates actin-based motility through interactions with SH2-domain-containing proteins. The Src kinase SH2 domain mediates cortactin binding and tyrosine phosphorylation, but how Src interacts with cortactin is unknown. Here we demonstrate that Src binds cortactin through cystine bonding between Src C185 in the SH2 domain within the phosphotyrosine binding pocket and cortactin C112/246 in the cortactin repeats domain, independent of tyrosine phosphorylation. Interaction studies show that the presence of reducing agents ablates Src-cortactin binding, eliminates cortactin phosphorylation by Src, and prevents Src SH2 domain binding to cortactin. Tandem MS/MS sequencing demonstrates cystine bond formation between Src C185 and cortactin C112/246. Mutational studies indicate that an intact cystine binding interface is required for Src-mediated cortactin phosphorylation, cell migration, and pre-invadopodia formation. Our results identify a novel phosphotyrosine-independent binding mode between the Src SH2 domain and cortactin. Besides Src, one quarter of all SH2 domains contain cysteines at or near the analogous Src C185 position. This provides a potential alternative mechanism to tyrosine phosphorylation for cysteine-containing SH2 domains to bind cognate ligands that may be widespread in propagating signals regulating diverse cellular functions.

The presence of lipopolysaccharide (LPS) on the cell surface of Gram-negative bacteria is critical for viability. A conserved β-barrel membrane protein LptD (lipopolysaccharide transport protein D) translocates LPS from the periplasm across the outer membrane (OM). In Escherichia coli, this protein contains two disulfide bonds and forms the OM LPS translocon with the lipoprotein LptE. Here, we identified seven in vivo states on the oxidative-folding pathway of LptD. Proper assembly involved a nonfunctional intermediate containing non-native disulfides. Intermediate formation required the oxidase DsbA, and subsequent maturation to the active form with native disulfides was triggered by LptE. Thus, disulfide bond-dependent protein folding of LptD requires the proper assembly of a two-protein complex to promote disulfide bond rearrangement.

The progranulin gene (GRN) g.10325&#95;10331delCTGCTGT (relative to nt1 in NG&#95;007886.1), alias Cys157LysfsX97, has been so far reported only once in a patient with frontotemporal dementia. Here, we describe a 63-year old patient carrying the same mutation, presenting with a 3-year history of language disorder, and diagnosed clinically with nonfluent variant of primary progressive aphasia according to current criteria. This patient's description expands the spectrum of clinical presentations of frontotemporal lobar degeneration caused by the GRN Cys157LysfsX97 mutation.

Malignant glioma represents one of the most aggressive and lethal human neoplasias. A hallmark of gliomas is their rapid proliferation and destruction of vital brain tissue, a process in which excessive glutamate release by glioma cells takes center stage. Pharmacologic antagonism with glutamate signaling through ionotropic glutamate receptors attenuates glioma progression in vivo, indicating that glutamate release by glioma cells is a prerequisite for rapid glioma growth. Glutamate has been suggested to promote glioma cell proliferation in an autocrine or paracrine manner, in particular by activation of the (RS)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrate (AMPA) subtype of glutamate receptors. Here, we dissect the effects of glutamate secretion on glioma progression. Glioma cells release glutamate through the amino-acid antiporter system X(c)(-), a process that is mechanistically linked with cystine incorporation. We show that disrupting glutamate secretion by interfering with the system X(c)(-) activity attenuates glioma cell proliferation solely cystine dependently, whereas glutamate itself does not augment glioma cell growth in vitro. Neither AMPA receptor agonism nor antagonism affects glioma growth in vitro. On a molecular level, AMPA insensitivity is concordant with a pronounced transcriptional downregulation of AMPA receptor subunits or overexpression of the fully edited GluR2 subunit, both of which block receptor activity. Strikingly, AMPA receptor inhibition in tumor-implanted brain slices resulted in markedly reduced tumor progression associated with alleviated neuronal cell death, suggesting that the ability of glutamate to promote glioma progression strictly requires the tumor microenvironment. Concerning a potential pharmacotherapy, targeting system X(c)(-) activity disrupts two major pathophysiological properties of glioma cells, that is, the induction of excitotoxic neuronal cell death and incorporation of cystine required for rapid proliferation.

Protein crystallization continues to be a major bottleneck in X-ray crystallography. Previous studies suggest that symmetric proteins, such as homodimers, might crystallize more readily than monomeric proteins or asymmetric complexes. Proteins that are naturally monomeric can be made homodimeric artificially. Our approach is to create homodimeric proteins by introducing single cysteines into the protein of interest, which are then oxidized to form a disulfide bond between the two monomers. By introducing the single cysteine at different sequence positions, one can produce a variety of synthetically dimerized versions of a protein, with each construct expected to exhibit its own crystallization behavior. In earlier work, we demonstrated the potential utility of the approach using T4 lysozyme as a model system. Here we report the successful application of the method to Thermotoga maritima CelA, a thermophilic endoglucanase enzyme with low sequence identity to proteins with structures previously reported in the Protein Data Bank. This protein had resisted crystallization in its natural monomeric form, despite a broad survey of crystallization conditions. The synthetic dimerization of the CelA mutant D188C yielded well-diffracting crystals with molecules in a packing arrangement that would not have occurred with native, monomeric CelA. A 2.4 Å crystal structure was determined by single anomalous dispersion using a seleno-methionine derivatized protein. The results support the notion that synthetic symmetrization can be a useful approach for enlarging the search space for crystallizing monomeric proteins or asymmetric complexes.

Disulfide bonds play critical roles in protein stability and function. They are generally considered to be strongly conserved among species. Although there is compelling evidence in the literature for this conservation on a case-by-case basis, comparative genomic analyses of disulfide conservation have in the past been limited. By analyzing the conservation of all structurally validated disulfide bonds from the Protein Data Bank across 29 completely sequenced eukaryotic genomes, we observe elevated conservation of disulfide-bonded cysteines (half-cystines) compared with unpaired cysteines and other amino acids. Remarkably, half-cystines are even more conserved than tryptophan--the most conserved amino acid. Overall, once disulfide bonds are acquired in proteins, they are rarely lost. Moreover, the acquisition of disulfide bonds shows a strong positive correlation (R(2) = 0.74) with organismal complexity. Although the correlation weakens (R(2) = 0.59) when yeast is excluded from the analysis, this trend is still apparent when compared with the slightly negative correlation of unpaired cysteine acquisition with organismal complexity. The accrual of disulfide bonds is likely to reflect the demand for greater sophistication in protein function in complex species. Our findings provide further support for the increasing usage of cysteines in modern proteomes and suggest that there has been positive selection for disulfide bonds through eukaryotic evolution. Finally, we show that the acquisition of the functionally relevant disulfide bond in domain 2 of the CD4 protein occurred independently in primates and rodents.

Antibody conjugates have broad utility in basic, preclinical, and clinical applications. Conventional antibody conjugation through the amine group of lysine or the thiol group of cysteine residues yields heterogeneous products of undefined stoichiometry and considerable batch-to-batch variability. To preserve the two hallmarks of the antibody molecule, precision and predictability, methods that enable site-specific antibody conjugation are in high demand. On the basis of a mammalian cell expression system, we describe the utilization of the 21st natural amino acid selenocysteine for the generation of IgG and Fab molecules with unique nucleophilic reactivity that affords site-specific conjugation to electrophilic derivatives of biotin, fluorescein, and poly(ethylene glycol). The resulting antibody conjugates were found to fully retain their antigen binding capability and, in the case of IgG, the ability to mediate effector functions. Gain of function was demonstrated in vitro and in vivo. While these antibody conjugates are relevant for a variety of proteomic, diagnostic, and therapeutic applications, they also constitute a proof of principle for the generation of molecularly defined antibody-drug conjugates and radioimmunoconjugates. Compared to other site-specific antibody conjugation methods, selenocysteine interface technology (i) only involves a minor modification at the C-terminus that does not interfere with disulfide bridges, (ii) does not require activation, and (iii) generates unique 1:1 stoichiometries of biological and chemical components. Collectively, our method affords the generation of highly defined antibody conjugates with broad utility from proteomic applications to therapeutic intervention.

There is a critical need for compounds that target cell surface integrin receptors for applications in cancer therapy and diagnosis. We used directed evolution to engineer the Ecballium elaterium trypsin inhibitor (EETI-II), a knottin peptide from the squash family of protease inhibitors, as a new class of integrin-binding agents. We generated yeast-displayed libraries of EETI-II by substituting its 6-amino acid trypsin binding loop with 11-amino acid loops containing the Arg-Gly-Asp integrin binding motif and randomized flanking residues. These libraries were screened in a high-throughput manner by fluorescence-activated cell sorting to identify mutants that bound to alpha(v)beta(3) integrin. Select peptides were synthesized and were shown to compete for natural ligand binding to integrin receptors expressed on the surface of U87MG glioblastoma cells with half-maximal inhibitory concentration values of 10-30 nM. Receptor specificity assays demonstrated that engineered knottin peptides bind to both alpha(v)beta(3) and alpha(v)beta(5) integrins with high affinity. Interestingly, we also discovered a peptide that binds with high affinity to alpha(v)beta(3), alpha(v)beta(5), and alpha(5)beta(1) integrins. This finding has important clinical implications because all three of these receptors can be coexpressed on tumors. In addition, we showed that engineered knottin peptides inhibit tumor cell adhesion to the extracellular matrix protein vitronectin, and in some cases fibronectin, depending on their integrin binding specificity. Collectively, these data validate EETI-II as a scaffold for protein engineering, and highlight the development of unique integrin-binding peptides with potential for translational applications in cancer.

Background: Pharmacogenetics of tardive dyskinesia and dopamine D3 (DRD3), serotonin 2A (HTR2A), and 2C (HTR2C) receptors has been examined in various populations, but not in Russians., Purpose: To investigate the association between orofaciolingual (TDof) and limb-truncal dyskinesias (TDlt) and Ser9Gly (DRD3), -1438G>A (HTR2A), and Cys23Ser (HTR2C) polymorphisms in Russian psychiatric inpatients from Tomsk, Siberia., Methods: In total, 146 subjects were included. Standard protocols were applied for genotyping. TDof and TDlt were assessed with AIMS items 1-4 and 5-7, respectively. Two-part model, logistic and log-normal regression analyses were applied to assess different variables (e.g., allele-carriership status, age, gender, and medication use)., Results: TDlt, but not TDof, exhibited an association with Ser9Gly and Cys23Ser (with 9Gly and 23Ser alleles exhibiting opposite effects). However, -1438G>A was not associated with TDof and Dlt., Conclusions: This is the first pharmacogenetic report on tardive dyskinesia in Russians. Subject to further replication, our findings extend and support the available data.

Ligand-gated ion channels respond to specific neurotransmitters by transiently opening an integral membrane ion-selective pore, allowing ions to move down their electrochemical gradient. A distinguishing feature of all members of the ligand-gated ion channel superfamily is the presence of a 13-amino acid disulfide loop (Cys-loop) in the extracellular ligand-binding domain. Structural data derived from the acetylcholine receptor place this loop at the interface between the ligand-binding domain and the transmembrane pore-forming domain where it is ideally located to participate in coupling ligand binding to channel opening. We have introduced specific mutations into a conserved motif at the mid-point of the Cys-loop of the GABA A receptor subunits alpha1, beta2 and gamma2S where the sequence reads aromatic, proline, aliphatic (ArProAl motif). Receptors carrying a mutation in the Cys-loop of one of their subunits were expressed in L929 cells and responses to both GABA and drugs were assessed using the whole-cell patch clamp technique. Drug potentiation and direct activation were significantly enhanced by mutations in this Cys-loop but these effects were subunit-dependent. Currents in response to agonists were larger when mutations were carried in the alpha and beta subunits but not in the gamma subunit. In contrast, potentiation of current responses by diazepam, etomidate and pentobarbital were all enhanced when mutations were carried in the alpha and gamma subunits, but not the beta subunit. Since the disruption of interactions mediated through the ArProAl motif enhances the mutant receptor's response to both agonist and drugs we suggest that this motif in the Cys-loop of the wild-type receptor participates in interactions that create activation barriers to conformational changes during channel gating.

Background & Aims: T-cell receptor reactivity of intestinal lamina propria T cells (LP-T) critically depends on the capacity of local accessory cells to secrete cysteine. For T cells, cysteine is the limiting precursor for glutathione synthesis, a prerequisite for antigen-dependent proliferation. We aimed to determine the role of the redoxactive microenvironment for hyporeactivity of LP-T in normal human gut vs hyperreactivity of LP-T in inflammatory bowel disease., Methods: Parameters relevant to cysteine production, determined as acid-soluble thiol, by intestinal lamina propria macrophages (LP-MO) vs peripheral blood monocytes were investigated (L-[(35)S]cystine uptake via system x(c)(-), messenger RNA, and protein expression of the cystine transporter subunit xCT). Glutathione levels in LP-T and peripheral blood T cells were analyzed both spectrophotometrically and by immunofluorescent staining in situ and in vitro., Results: LP-MO from normal gut, unlike peripheral blood monocytes, are unable to take up cystine, which is due to a deficient expression of the transporter xCT in situ and in vitro. As a consequence, LP-MO do not secrete cysteine. The glutathione content in LP-T from normal gut is <50% of that in autologous peripheral blood T cells. In contrast, in inflammatory bowel disease, CD14(+)CD68(+) LP-MO express xCT and secrete substantial amounts of cysteine upon stimulation, which results in high glutathione levels and full T-cell receptor reactivity in LP-T., Conclusions: The antioxidative microenvironment of LP-T in inflammatory bowel disease and the prooxidative microenvironment in normal gut explain the differential T-cell receptor reactivities.

Dethiolation experiments of thiolated albumin with thionitrobenzoic acid and thiols (glutathione, cysteine, homocysteine) were carried out to understand the role of albumin in plasma distribution of thiols and disulfide species by thiol/disulfide (SH/SS) exchange reactions. During these experiments we observed that thiolated albumin underwent thiol substitution (Alb-SS-X+RSH<-->Alb-SS-R+XSH) or dethiolation (Alb-SS-X+XSH<-->Alb-SH+XSSX), depending on the different pK(a) values of thiols involved in protein-thiol mixed disulfides (Alb-SS-X). It appeared in these reactions that the compound with lower pK(a) in mixed disulfide was a good leaving group and that the pK(a) differences dictated the kind of reaction (substitution or dethiolation). Thionitrobenzoic acid, bound to albumin by mixed disulfide (Alb-TNB), underwent rapid substitution after thiol addition, forming the corresponding Alb-SS-X (peaks at 0.25-1 min). In turn, Alb-SS-X were dethiolated by the excess nonprotein SH groups because of the lower pK(a) value in mixed disulfide with respect to that of other thiols. Dethiolation of Alb-SS-X was accompanied by formation of XSSX and Alb-SH up to equilibrium levels at 35 min, which were different for each thiol. Structures by molecular simulation of thiolated albumin, carried out for understanding the role of sulfur exposure in mixed disulfides in dethiolation process, evidenced that the sulfur exposure is important for the rate but not for determining the kind of reaction (substitution or dethiolation). Our data underline the contribution of SH/SS exchanges to determine levels of various thiols as reduced and oxidized species in human plasma., ((c) 2007 Wiley-Liss, Inc.)

CADASIL is an autosomal dominant arteriopathy characterised by diffuse white matter lesions and small subcortical infarcts on neuroimaging and a variable combination of recurrent cerebral ischaemic episodes, cognitive deficits, migraine with aura and psychiatric symptoms. It is caused by mutations in the NOTCH3 gene encoding a NOTCH3 receptor protein. Here, we describe the genetical, clinical, neuropsychological and neuroimaging findings in an Italian CADASIL patient with a rare mutation in exon 10 leading to a Gly528Cys substitution.

Oxidative damage accumulates in the DNA of the human brain over time, and is supposed to play a critical role in the pathogenesis of Alzheimer's disease (AD). It has been suggested that the brain in AD might be subjected to the double insult of increased oxidative stress, as well as deficiencies in repair mechanisms responsible for the removal of oxidized bases. The type of damage that is most likely to occur in neuronal cells is oxidative DNA damage which is primarily removed by the base excision repair (BER) pathway, and a decrease in BER activity was observed in post-mortem brain regions of AD individuals, especially in the activity of 8-oxoguanine DNA glycosylase. There is evidence that the Ser326Cys polymorphism of the human 8-oxoguanine DNA glycosylase 1 (hOGG1) gene is associated with a reduced DNA repair activity. However, although a deficient BER was proposed in the etiology of AD by several authors, polymorphisms of BER genes have not been studied in AD yet. We performed a case-control study including 178 patients with sporadic AD (sAD) and 146 matched controls to evaluate the role of the Ser326Cys polymorphism as a risk factor for sAD. In the present study we failed to find any association between allele (chi2=0.03, p=0.86) or genotype (chi2=0.25, p=0.882) frequencies of hOGG1 Ser326Cys and the risk of sAD. Present results suggest that the Ser326Cys polymorphism of the hOGG1 gene is not an independent risk factor for sAD.

Cystine-knot microproteins exhibit several properties that make them highly interesting as scaffolds for oral peptide drug delivery. It was therefore the aim of the study to evaluate the novel clinically relevant cystine-knot microprotein McoEeTI regarding its potential for oral delivery. Additionally, based on the gained results, important features of McoEeTI were improved. Enzymatic degradation was caused by chymotrypsin, trypsin and porcine small intestinal juice whereas McoEeTI was stable towards elastase, membrane bound proteases, pepsin and porcine gastric juice. Only minor McoEeTI degradation was observed during a 24h incubation period in rat plasma. In the presence of various physiological ions about 50% of McoEeTI formed di- and/or trimers. P(app) value of McoEeTI was determined to be (7.4+/-0.4)x10(-6)cm/s. Sodium caprate and polycarbophil-cysteine (PCP-Cys) had no beneficial effect on McoEeTI permeation, whereas the utilization of a chitosan-thiobutylamidine (Chito-TBA) system improved McoEeTI permeation 3-fold. Enzymatic stability could be strongly improved by the utilization of Bowman-Birk-Inhibitor (BBI) as well as PCP-Cys. In conclusion, this study indicates that McoEeTI represents a promising candidate as a novel scaffold for oral peptide drug delivery.

Purpose: To describe a striking retinal finding that the authors have only seen in Norrie disease eyes and to determine if a particular genotype corresponds to this dramatic presentation., Methods: This is a retrospective, interventional case report of four patients seen in the clinic over a 1-year period. All patients had analysis of the Norrie gene by direct sequencing., Results: All patients presented with a similar retinal appearance of dense stalk tissue, globular dystrophic retina, and peripheral avascular retina with pigmentary changes. Each patient was found to have a mutation in the Norrie gene affecting a cystine residue in the cystine knot domain. The mutations are predicted to disrupt the structure of the protein product, norrin, which is required for activation of the Wnt receptor:beta-catenin pathway., Conclusions: No other vitreoretinopathy that the authors have seen demonstrates this characteristic retinal presentation of severe retinal dysplasia. All four patients were found to have mutations in the Norrie gene which alter the cystine knot motif. Mutations affecting this domain appear to have devastating effects on retinal development and indicate phenotype correlates with mutations affecting the cystine knot domain.