RESUMO
Cystinosis is an autosomal recessive metabolic disease that belongs to the family of lysosomal storage disorders. It is caused by a defect in the lysosomal cystine transporter, cystinosin, which results in an accumulation of cystine in all organs. Despite the ubiquitous expression of cystinosin, a renal Fanconi syndrome is often the first manifestation of cystinosis, usually presenting within the first year of life and characterized by the early and severe dysfunction of proximal tubule cells, highlighting the unique vulnerability of this cell type. The current therapy for cystinosis, cysteamine, facilitates lysosomal cystine clearance and greatly delays progression to kidney failure but is unable to correct the Fanconi syndrome. This Review summarizes decades of studies that have fostered a better understanding of the pathogenesis of the renal Fanconi syndrome associated with cystinosis. These studies have unraveled some of the early molecular changes that occur before the onset of tubular atrophy and identified a role for cystinosin beyond cystine transport, in endolysosomal trafficking and proteolysis, lysosomal clearance, autophagy and the regulation of energy balance. These studies have also led to the identification of new potential therapeutic targets and here, we outline the potential role of stem cell therapy for cystinosis and provide insights into the mechanism of haematopoietic stem cell-mediated kidney protection.
Assuntos
Cistinose/complicações , Cistinose/terapia , Síndrome de Fanconi/etiologia , Síndrome de Fanconi/terapia , Cristalização , Cistina/fisiologia , Cistinose/metabolismo , Previsões , Humanos , Estresse OxidativoRESUMO
KEY POINTS: 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. ABSTRACT: 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.
Assuntos
Sistemas de Transporte de Aminoácidos Neutros/genética , Cistina/fisiologia , Túbulos Renais Proximais/citologia , Lisossomos/fisiologia , Mitocôndrias/fisiologia , Trifosfato de Adenosina/metabolismo , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cisteamina/farmacologia , Cistina/metabolismo , Inativação Gênica , Glutationa/metabolismo , Humanos , Lisossomos/metabolismo , Potencial da Membrana Mitocondrial , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismoRESUMO
We recently reported that a trans-dimer, homotypic disulfide bond involving Cys367 in keratin 14 (K14) occurs in an atomic-resolution structure of the interacting K5/K14 2B domains and in keratinocyte cell lines. Here we show that a sizable fraction of the K14 and K5 protein pools participates in interkeratin disulfide bonding in primary cultures of mouse skin keratinocytes. By comparing the properties of wild-type K14 with a completely cysteine-free variant thereof, we found that K14-dependent disulfide bonding limited filament elongation during polymerization in vitro but was necessary for the genesis of a perinuclear-concentrated network of keratin filaments, normal keratin cycling, and the sessile behavior of the nucleus and whole cell in keratinocytes studied by live imaging. Many of these phenotypes were rescued when analyzing a K14 variant harboring a single Cys residue at position 367. These findings establish disulfide bonding as a novel and important mechanism regulating the assembly, intracellular organization, and dynamics of K14-containing intermediate filaments in skin keratinocytes.
Assuntos
Cistina/fisiologia , Filamentos Intermediários/ultraestrutura , Queratina-14/ultraestrutura , Queratinócitos/ultraestrutura , Sequência de Aminoácidos , Animais , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Células Cultivadas , Filamentos Intermediários/metabolismo , Queratina-14/metabolismo , Queratinócitos/metabolismo , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Pele/citologiaRESUMO
Measurement of the changes in intracellular Ca2+ levels is an important assay for drug discovery. In this report, we describe a novel Ca2+ indicator, dCys-GCaMP, based on the green fluorescent protein and the development of a rapid and simple cell-based functional assay using this new Ca2+ indicator. We demonstrated the sensitivity and reliability of the assay by measuring the cellular responses to the agonists, antagonists, channel blockers, and modulators of the ionotropic N-methyl-D-aspartate (NMDA) subtype of glutamate receptors. HEK293 cells coexpressing the NMDA receptor and dCys-GCaMP displayed a strong increase in fluorescence intensity when stimulated with the agonist glutamate. This increase in the fluorescence signal was agonist concentration dependent and could be blocked by NMDAR antagonists and channel blockers. The pharmacological parameters measured with the dCys-GCaMP assay are in close agreement with those derived from conventional assays with synthetic dye fluo-4 and literature values. In addition, we showed that this assay could be used on G protein-coupled receptors as well, as exemplified by studies on the α1A adrenergic receptor. A limited scale evaluation of the assay performance in a 96-well compound screening format suggests that the dCys-GCaMP assay could be easily adapted to a high-throughput screening environment. The most important advantage of this new assay over the conventional fluo-4 and aequorin assays is the elimination of the dye-loading or substrate-loading process.
Assuntos
Cálcio/química , Cistina/química , Proteínas de Fluorescência Verde/química , Cistina/fisiologia , Proteínas de Fluorescência Verde/fisiologia , Células HEK293 , HumanosRESUMO
Redox homeostasis is especially important in the brain where high oxygen consumption produces an abundance of harmful oxidative by-products. Glutathione (GSH) is a tripeptide non-protein thiol. It is the central nervous system's most abundant antioxidant and the master controller of brain redox homeostasis. The glutamate transporters, System xc(-) (SXC) and the Excitatory Amino Acid Transporters (EAAT), play important, synergistic roles in the synthesis of GSH. In glial cells, SXC mediates the uptake of cystine, which after intracellular reduction to cysteine, reacts with glutamate during the rate-limiting step of GSH synthesis. EAAT3 mediates direct cysteine uptake for neuronal GSH synthesis. SXC and EAAT work in concert in glial cells to provide two intracellular substrates for GSH synthesis, cystine and glutamate. Their cyclical basal function also prevents a buildup of extracellular glutamate, which SXC releases extracellularly in exchange for cystine uptake. Maintaining extracellular glutamate homeostasis is critical to prevent neuronal toxicity, as well as glutamate-mediated SXC inhibition, which could lead to a depletion of intracellular GSH and loss of cellular redox control. Many neurological diseases show evidence of GSH dysfunction, and increased GSH has been widely associated with chemotherapy and radiotherapy resistance of gliomas. We present evidence suggesting that gliomas expressing elevated levels of SXC are more reliant on GSH for growth and survival. They have an increased inherent radiation resistance, however, inhibition of SXC can increase tumor sensitivity at low radiation doses. GSH depletion through SXC inhibition may be a viable mechanism to enhance current glioma treatment strategies and make tumors more sensitive to radiation and chemotherapy protocols.
Assuntos
Sistema X-AG de Transporte de Aminoácidos/metabolismo , Encéfalo/fisiologia , Homeostase/fisiologia , Animais , Química Encefálica/fisiologia , Cistina/fisiologia , Glioma/patologia , Glutationa/fisiologia , Humanos , Camundongos , Doenças do Sistema Nervoso/metabolismo , Doenças do Sistema Nervoso/fisiopatologia , Oxirredução , Ensaios Antitumorais Modelo de XenoenxertoAssuntos
Cisteína/uso terapêutico , Cistina/uso terapêutico , Citocromos c/uso terapêutico , Desidroepiandrosterona/uso terapêutico , Idoso , Desempenho Atlético , Cisteína/fisiologia , Cistina/fisiologia , Citocromos c/fisiologia , Suplementos Nutricionais , Fosfato de Di-Hidroxiacetona/uso terapêutico , Exercício Físico/fisiologia , Humanos , Imunocompetência/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Ácido Pirúvico/uso terapêutico , Sarcosina/análogos & derivados , Sarcosina/uso terapêutico , Testosterona/metabolismoRESUMO
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.
Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Camptotecina/análogos & derivados , Cistina/fisiologia , Regulação para Baixo/fisiologia , Neoplasias de Cabeça e Pescoço/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Sistemas de Transporte de Aminoácidos/antagonistas & inibidores , Sistemas de Transporte de Aminoácidos/biossíntese , Animais , Camptotecina/farmacologia , Linhagem Celular Tumoral , Cistina/antagonistas & inibidores , Cistina/metabolismo , Regulação para Baixo/efeitos dos fármacos , Feminino , Humanos , Irinotecano , Camundongos , Camundongos Nus , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Seminal studies by Richardson and Thornton defined the constraints imposed by protein structure on disulfide formation and flagged forbidden regions of primary or secondary structure seemingly incapable of forming disulfide bonds between resident cysteine pairs. With respect to secondary structure, disulfide bonds were not found between cysteine pairs: A. on adjacent beta-stands; B. in a single helix or strand; C. on non-adjacent strands of the same beta-sheet. In primary structure, disulfide bonds were not found between cysteine pairs: D. adjacent in the sequence. In the intervening years it has become apparent that all these forbidden regions are indeed occupied by disulfide-bonded cysteines, albeit rather strained ones. It has been observed that sources of strain in a protein structure, such as residues in forbidden regions of the Ramachandran plot and cis-peptide bonds, are found in functionally important regions of the protein and warrant further investigation. Like the Ramachandran plot, the earlier studies by Richardson and Thornton have identified a fundamental truth in protein stereochemistry: "forbidden" disulfides adopt strained conformations, but there is likely a functional reason for this. Emerging evidence supports a role for forbidden disulfides in redox-regulation of proteins.
Assuntos
Cistina/fisiologia , Proteínas/metabolismo , Transdução de Sinais/fisiologia , Animais , Cistina/química , Dissulfetos/química , Humanos , Modelos Moleculares , Oxirredução , Estrutura Secundária de Proteína , Proteínas/química , Receptores de Citocinas/química , Receptores de Citocinas/metabolismoRESUMO
P2X receptors are ATP-gated nonselective cation channels. Functional receptors are assembled as homotrimers or heterotrimers of seven cloned subunits. Each subunit contains two transmembrane domains linked by a large extracellular loop that is required for agonist binding. So far, there is no direct evidence indicating whether the agonist binding site is formed within one subunit or at the interface of two neighboring subunits. Here we used a disulfide cross-linking approach to identify pairs of residues that are in close proximity within the ATP binding site of the P2X1 homotrimer. Eight amino acid residues that have previously been shown to be essential for high ATP potency (K68, K70, F185, K190, F291, R292, R305, and K309) were substituted by cysteine residues, and the respective mutant subunits were pairwise expressed in Xenopus laevis oocytes. Nonreducing SDS-PAGE analysis of the purified receptors revealed a spontaneous and specific dimer formation between the K68C and F291C mutants. An almost complete cross-link into trimers was achieved with the K68C/F291C double mutant, consistent with the formation of intersubunit disulfide bridges. In support of this interpretation, two-electrode voltage-clamp analysis of the K68C/F291C mutations introduced into a nondesensitizing P2X(2-1) chimera showed only small ATP-activated currents that, however, increased approximately 60-fold after extracellular application of the reducing agent dithiothreitol. In addition, we show that a K68C/K309C double mutant is nonfunctional and can be functionally rescued by coexpression with nonmutated subunits. Our data are consistent with loops from neighboring P2X subunits forming the ATP-binding site in P2X receptors.
Assuntos
Trifosfato de Adenosina/metabolismo , Cistina/fisiologia , Receptores Purinérgicos P2/química , Trifosfato de Adenosina/farmacologia , Substituição de Aminoácidos , Animais , Sítios de Ligação , Cisteína/química , Dimerização , Ditiotreitol/farmacologia , Feminino , Glicosilação , Modelos Moleculares , Mutagênese Sítio-Dirigida , Oócitos , Oxirredução , Técnicas de Patch-Clamp , Conformação Proteica , Dobramento de Proteína , Processamento de Proteína Pós-Traducional , Subunidades Proteicas , Ratos , Receptores Purinérgicos P2X , Proteínas Recombinantes de Fusão/química , Xenopus laevisRESUMO
Milk protein gene expression varies during the pregnancy/lactation cycle under the influence of lactogenic hormones which induce the activation of several transcription factors. Beyond this activation modifying the binding properties of these factors to their consensus sequences, their interactions with DNA is regulated by variations of the chromatin structure. In the nuclei of the mammary epithelial cell, the three dimensional organisation of the chromatin loops, located between matrix attachment regions, is now being studied. The main milk components are organised in supramolecular structures. Milk fat globules are made of a triglyceride core enwrapped by a tripartite membrane originating from various intracellular compartments. The caseins, the main milk proteins, form aggregates: the casein micelles. Their gradual aggregation in the secretory pathway is initiated as soon as from the endoplasmic reticulum. The mesostructures of the milk fat globule and of the casein micelle remain to be elucidated. Our goal is to make some progress into the understanding of the molecular and cellular mechanisms involved in the formation of these milk products.
Assuntos
Núcleo Celular/fisiologia , Regulação da Expressão Gênica/fisiologia , Lactação/fisiologia , Glândulas Mamárias Animais/metabolismo , Proteínas do Leite/genética , Animais , Mama/citologia , Mama/metabolismo , Caseínas/biossíntese , Caseínas/química , Caseínas/genética , Bovinos , Núcleo Celular/ultraestrutura , Cromatina/genética , Cromatina/ultraestrutura , Cistina/fisiologia , Células Epiteliais/metabolismo , Feminino , Genes Reguladores , Glicolipídeos/metabolismo , Glicoproteínas/metabolismo , Glicoproteínas/ultraestrutura , Hormônios/fisiologia , Humanos , Membranas Intracelulares/fisiologia , Membranas Intracelulares/ultraestrutura , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Lactação/genética , Gotículas Lipídicas , Glândulas Mamárias Animais/citologia , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Camundongos , Micelas , Proteínas do Leite/biossíntese , Matriz Nuclear/fisiologia , Matriz Nuclear/ultraestrutura , Coelhos , Proteínas Modificadoras da Atividade de Receptores , Fatores de Transcrição/fisiologia , Triglicerídeos/metabolismoRESUMO
DsbD is a redox-active protein of the inner Escherichia coli membrane possessing an N-terminal (nDsbD) and a C-terminal (cDsbD) periplasmic domain. nDsbD interacts with four different redox proteins involved in the periplasmic disulfide isomerization and in the cytochrome c maturation systems. We review here the studies that led to the structural characterization of all soluble DsbD domains involved and, most importantly, of trapped disulfide intermediate complexes of nDsbD with three of its four redox partners. These results revealed the structural features enabling nDsbD, a 'redox hub' with an immunoglobulin-like fold, to interact efficiently with its different thioredoxin-like partners.
Assuntos
Proteínas de Escherichia coli/fisiologia , Escherichia coli/metabolismo , Oxirredutases/fisiologia , Motivos de Aminoácidos , Cisteína/química , Cisteína/fisiologia , Cistina/química , Cistina/fisiologia , Dimerização , Transporte de Elétrons , Ativação Enzimática , Escherichia coli/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Ligação de Hidrogênio , Modelos Moleculares , NADP/metabolismo , Oxirredução , Oxirredutases/química , Periplasma/metabolismo , Proteínas Periplásmicas/fisiologia , Conformação Proteica , Proteína Dissulfeto Redutase (Glutationa)/metabolismo , Isomerases de Dissulfetos de Proteínas/fisiologia , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Tiorredoxinas/químicaRESUMO
An insertion of residues in the third extracellular loop and a disulfide bond linking this loop to the N-terminal domain were identified in a structural model of a G-protein coupled receptor specific to angiotensin II (AT1 receptor), built in homology to the seven-transmembrane-helix bundle of rhodopsin. Both the insertion and the disulfide bond were located close to an extracellular locus, flanked by the second extracellular loop (EC-2), the third extracellular loop (EC-3) and the N-terminal domain of the receptor; they contained residues identified by mutagenesis studies to bind the angiotensin II N-terminal segment (residues D1 and R2). It was postulated that the insertion and the disulfide bond, also found in other receptors such as those for bradykinin, endothelin, purine and other ligands, might play a role in regulating the function of the AT1 receptor. This possibility was investigated by assaying AT1 forms devoid of the insertion and with mutations to Ser on both positions of Cys residues forming the disulfide bond. Binding and activation experiments showed that abolition of this bond led to constitutive activation, decay of agonist binding and receptor activation levels. Furthermore, the receptors thus mutated were translocated to cytosolic environments including those in the nucleus. The receptor form with full deletion of the EC-3 loop residue insertion, displayed a wild type receptor behavior.
Assuntos
Cistina/fisiologia , Dissulfetos/metabolismo , Receptor Tipo 1 de Angiotensina/fisiologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Angiotensina II/metabolismo , Animais , Compostos de Boro , Células CHO , Cricetinae , Cricetulus , Citometria de Fluxo , Proteínas de Fluorescência Verde/biossíntese , Fosfatos de Inositol/biossíntese , Microscopia Confocal , Modelos Moleculares , Estrutura Terciária de Proteína , Receptor Tipo 1 de Angiotensina/química , Receptor Tipo 1 de Angiotensina/genética , Proteínas Recombinantes de Fusão/biossíntese , Alinhamento de SequênciaRESUMO
The major platelet integrin alpha(IIb)beta(3), also known as the platelet glycoprotein (GP) IIb-IIIa complex, mediates platelet aggregation by serving as the receptor for fibrinogen and von Willebrand factor. In addition to its physiologic role, GPIIb-IIIa also bears a number of clinically important alloantigenic determinants. Previous studies have shown that disruption of the long-range Cys(5)-Cys(435) disulfide bond of the beta(3) subunit results in the production of isoforms that bind some, but not all, anti-Pl(A1) alloantibodies, suggesting that mutations in this so-called long-range disulfide bond can alter the conformation of GPIIIa. The purpose of this study was to examine the effects of either the Cys5Ala or Cys435Ala substitution of GPIIIa on the adhesive properties of the GPIIb-IIIa complex. We found that both Ala5GPIIIa and Ala435GPIIIa were capable of associating with GPIIb and were expressed normally on the cell surface when cotransfected into Chinese hamster ovary (CHO) cells. CHO cells expressing GPIIb-Ala5GPIIIa or GPIIb-Ala435IIIa bound well-characterized, conformationally sensitive ligand-induced binding site (LIBS) antibodies, and were capable of constitutively binding the fibrinogen-mimetic monoclonal antibodies Pl-55 and PAC-1, as well as soluble fibrinogen. Both GPIIb-Ala5IIIa- and GPIIb-Ala435IIIa-transfected CHO cells also bound more avidly to immobilized fibrinogen and were capable of mediating the tyrosine phosphorylation of pp125(FAK) on cell adhesion. These data are consistent with the notion that these regions of GPIIIa participate in the conformational change associated with receptor activation. Additionally, these studies may provide a molecular explanation for the previously reported ability of mild reducing agents to activate the GPIIb-IIIa complex and promote platelet aggregation.
Assuntos
Antígenos CD/metabolismo , Cistina/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Substituição de Aminoácidos , Animais , Antígenos CD/genética , Antígenos CD/fisiologia , Células CHO , Adesão Celular , Cricetinae , Cistina/fisiologia , Fibrinogênio/metabolismo , Humanos , Integrina beta3 , Mutagênese Sítio-Dirigida , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/fisiologia , Glicoproteínas da Membrana de Plaquetas/genética , Glicoproteínas da Membrana de Plaquetas/fisiologia , Ligação Proteica , Conformação Proteica , Transdução de Sinais , TransfecçãoRESUMO
PURPOSE: Many variables are known to be associated with the formation of calcium oxalate stones. We noted that on analysis a number of patients with calcium oxalate calculi also had cystine in the stones. Some but not all of these patients showed urinary cystine excretion slightly above the normal limits, resembling heterozygous carriers. This finding raised the question of whether some recurrent stone formers may be at risk for calcium oxalate calculi when they excrete cystine in above normal concentrations. MATERIALS AND METHODS: Pooled urine obtained from 3 pairs of age and sex matched controls was independently analyzed. Each urine sample was divided into spun and filtered, and ultrafiltered urine. A Multisizer II (Coulter Electronics Ltd, Beds, England) was used to measure particle number, diameter and volume. The metastable limit of each specimen was determined. Promotion activity was measured in spun and filtered, and ultrafiltered urine using 3 concentrations of cystine (80, 160 and 320 micromol./l.). Results were confirmed by measuring the incorporation of (14)C-oxalate into the crystals. Scanning electron microscopy was performed to study further the agglomerates as well as exclude cystine crystals. Each experiment was repeated 6 times. Crystalline material was collected for x-ray powder diffraction analysis. RESULTS: The urine metastable limit did not change with increasing cystine concentrations. Particle diameter increased significantly from 10.6 microm in ultrafiltered urine alone to 11.6 and 13.5 microm (p < 0.05) at 160 and 320 micromol/l. cystine, respectively. In addition, particle volume also increased proportionally in a dose response manner to cystine concentration. The dose of 320 micromol/l. cystine increased the crystal growth rate 52%. 14C-oxalate experiments confirmed these results. Scanning electron microscopy at 500x magnification revealed no cystine crystals in any experiments performed. Furthermore, x-ray powder diffraction analysis of samples revealed that experimentally determined parameters matched reference values for calcium oxalate trihydrate but not for cystine, again confirming absent cystine in the samples. CONCLUSIONS: Adding cystine to undiluted human urine resulted in the marked enhancement of calcium oxalate crystal precipitation. When considered with the finding of cystine in calcium oxalate stones in the noncystinuric population, this result implies that urinary cystine may be a risk factor for calcium oxalate calculi. Cystine was not observed in any calcium oxalate crystals, suggesting that the mechanism of crystal formation was a salting out effect.
Assuntos
Oxalato de Cálcio/urina , Cistina/análise , Cistina/fisiologia , Cristalização , Cistinúria/urina , Humanos , Fatores de Risco , Cálculos Urinários/etiologia , Difração de Raios XRESUMO
The chaperonin GroEL binds nonnative substrate protein in the central cavity of an open ring through exposed hydrophobic residues at the inside aspect of the apical domains and then mediates productive folding upon binding ATP and the cochaperonin GroES. Whether nonnative proteins bind to more than one of the seven apical domains of a GroEL ring is unknown. We have addressed this using rings with various combinations of wild-type and binding-defective mutant apical domains, enabled by their production as single polypeptides. A wild-type extent of binary complex formation with two stringent substrate proteins, malate dehydrogenase or Rubisco, required a minimum of three consecutive binding-proficient apical domains. Rhodanese, a less-stringent substrate, required only two wild-type domains and was insensitive to their arrangement. As a physical correlate, multivalent binding of Rubisco was directly observed in an oxidative cross-linking experiment.
Assuntos
Proteínas de Bactérias/fisiologia , Chaperonina 10/fisiologia , Chaperonina 60/fisiologia , Malato Desidrogenase/química , Peptídeos/química , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Ribulose-Bifosfato Carboxilase/química , Tiossulfato Sulfurtransferase/química , Trifosfato de Adenosina/metabolismo , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/ultraestrutura , Sítios de Ligação , Bovinos , Chaperonina 10/química , Chaperonina 10/ultraestrutura , Chaperonina 60/química , Chaperonina 60/ultraestrutura , Fenômenos Químicos , Físico-Química , Microscopia Crioeletrônica , Cistina/fisiologia , Escherichia coli/metabolismo , Etilmaleimida/farmacologia , Processamento de Imagem Assistida por Computador , Substâncias Macromoleculares , Modelos Moleculares , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeAssuntos
Sinalização do Cálcio/fisiologia , Coração/fisiologia , Proteínas Musculares/fisiologia , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia , Retículo Sarcoplasmático/fisiologia , Animais , Cistina/fisiologia , Radicais Livres , Humanos , Peróxido de Hidrogênio/farmacologia , Peso Molecular , Contração Muscular/fisiologia , Proteínas Musculares/química , Músculo Esquelético/ultraestrutura , Contração Miocárdica/fisiologia , Miocárdio/ultraestrutura , Oxirredução , Canal de Liberação de Cálcio do Receptor de Rianodina/químicaRESUMO
RyRs contain 80-100 cysteine residues per subunit, of which approximately 25% are free for covalent modification, while the remainder are either modified or form intraprotein disulfides. Oxidizing and nitrosylating reagents have several effects on single RyR channel activity, which depend on the type of modifying reagent, the isoform of the RyR, and ligands bound to the channel. We present evidence here for four major classes of functional cysteine residues associated with RyR channels, i.e., two classes with free -SH groups that either activate or inhibit channels when covalently modified and two classes, with endogenous modification, that either inhibit or activate. Single-channel characteristics provide evidence for four discrete responses within the first activating class, two responses within the second inhibiting class and two types of response within the third endogenously modified class. All but one of these changes in channel properties depend on residues located on the cytoplasmic or membrane-associated domains of the RyR; the remaining response is confined to the luminal domain. If it is assumed that each type of response depends on a separate subclass of cysteine residue and that each subclass contains a minimum of one cysteine per subunit, our results suggest that there are at least nine cysteine residues per subunit with functional connections to the gating mechanism of RyR channels. These cysteine residues may be selectively modified under physiological and pathological conditions to regulate Ca2+ release from the sarcoplasmic reticulum and contraction.
Assuntos
Sinalização do Cálcio/fisiologia , Cisteína/fisiologia , Proteínas Musculares/química , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Animais , Sinalização do Cálcio/efeitos dos fármacos , Cistina/fisiologia , Dissulfetos/farmacologia , Ácido Ditionitrobenzoico/farmacologia , Ditiotreitol/farmacologia , Glutationa/farmacologia , Coração/efeitos dos fármacos , Transporte de Íons/efeitos dos fármacos , Contração Muscular/efeitos dos fármacos , Proteínas Musculares/efeitos dos fármacos , Proteínas Musculares/fisiologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestrutura , Contração Miocárdica/efeitos dos fármacos , Miocárdio/metabolismo , Miocárdio/ultraestrutura , Oxidantes/farmacologia , Oxirredução , Conformação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Subunidades Proteicas , Piridinas/farmacologia , Substâncias Redutoras/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/efeitos dos fármacos , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia , Retículo Sarcoplasmático/efeitos dos fármacos , Retículo Sarcoplasmático/metabolismo , Relação Estrutura-Atividade , Compostos de Sulfidrila/farmacologia , Timerosal/farmacologiaRESUMO
Viral glycoproteins fold and oligomerize in the endoplasmic reticulum of the host cell. They employ the cellular machinery and receive assistance from cellular folding factors. During the folding process, they are retained in the compartment and their structural quality is checked by the quality control system of the endoplasmic reticulum. A special characteristic that distinguishes viral fusion proteins from most cellular proteins is the extensive conformational change they undergo during fusion of the viral and cellular membrane. Many viral proteins fold in conjunction with and dependent on a viral partner protein, sometimes even synthesized from the same mRNA. Relevant for folding is that viral glycoproteins from the same or related virus families may consist of overlapping sets of domain modules. The consequences of these features for viral protein folding are at the heart of this review.
Assuntos
Retículo Endoplasmático/fisiologia , Dobramento de Proteína , Proteínas do Envelope Viral/química , Animais , Proteínas de Ligação ao Cálcio/fisiologia , Calnexina , Calreticulina , Cistina/fisiologia , Evolução Molecular , Glicosilação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/fisiologia , Humanos , Chaperonas Moleculares/fisiologia , Conformação Proteica , Processamento de Proteína Pós-Traducional , Ribonucleoproteínas/fisiologia , Relação Estrutura-Atividade , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/fisiologia , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/fisiologiaRESUMO
Death of oligodendrocyte (OL) precursors can be triggered in vitro by cystine deprivation, a form of oxidative stress that involves depletion of intracellular glutathione. We report here that OLs demonstrate maturation-dependent differences in survival when subjected to free radical-mediated injury induced by glutathione depletion. Using immunopanning to isolate rat preoligodendrocytes (preOLs), we generated highly enriched populations of preOLs and mature OLs under chemically defined conditions. Cystine deprivation caused a similar decrease in glutathione levels in OLs at both stages. However, preOLs were completely killed by cystine deprivation, whereas mature OLs remained viable. Although the glutathione-depleting agents buthionine sulfoximine and diethylmaleate were more potent in depleting glutathione in mature OLs, both agents were significantly more toxic to preOLs. Glutathione depletion markedly increased intracellular free radical generation in preOLs, but not in mature OLs, as indicated by oxidation of the redox-sensitive probe dihydrorhodamine 123. The antioxidants alpha-tocopherol, idebenone, and glutathione monoethylester prevented the oxidation of dihydrorhodamine in cystine-depleted preOLs and markedly protected against cell death. When the intracellular glutathione level was not manipulated, preOLs were also more vulnerable than mature OLs to exogenous free radical toxicity generated by a xanthine-xanthine oxidase system. Ultrastructural features of free radical-mediated injury in glutathione-depleted preOLs included nuclear condensation, margination of chromatin, and mitochondrial swelling. These observations indicate that preOLs are significantly more sensitive to the toxic effects of glutathione depletion and that oligodendroglial maturation is associated with decreased susceptibility to oxidative stress.
Assuntos
Glutationa/deficiência , Oligodendroglia/fisiologia , Estresse Oxidativo/fisiologia , Animais , Butionina Sulfoximina/farmacologia , Contagem de Células , Morte Celular/fisiologia , Divisão Celular/fisiologia , Células Cultivadas , Senescência Celular/fisiologia , Cistina/deficiência , Cistina/fisiologia , Sequestradores de Radicais Livres/farmacologia , Radicais Livres/metabolismo , Glutationa/análogos & derivados , Glutationa/farmacologia , Substâncias de Crescimento/farmacologia , Oligodendroglia/citologia , Oligodendroglia/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Células-Tronco/citologiaRESUMO
FSH is a member of the pituitary/placental glycoprotein hormone family including luteinizing hormone, thyroid-stimulating hormone, and chorionic gonadotropin. These heterodimeric hormones share a common alpha-subunit and a highly homologous but distinct beta-subunit. The determinant loop of the FSH beta-subunit acts both as a specificity discriminator and as an essential receptor-binding site. The three-dimensional structure of hCG illustrates the proximity of the determinant loop to the carboxyl-terminal residues of the common alpha-subunit. Thus, site-directed mutagenesis was used to mak high-resolution substitutions at this carboxyl-terminal locus. The effects of those substitutions were studied. Twelve single mutations and one composite mutation were made of the region of hFSH alpha 74-92, each residue substituted by alanine. Side chain replacement in this region of FSH proved to be detrimental to binding. hFSH with mutations of either alpha S85A, alpha T86A, alpha K91A, or alpha S92A only retained 10% or less of the hFSH receptor-binding activity, while compared to these, mutants alpha H79A, alpha Y88A, and alpha Y89A retained slightly more binding activity. The single mutant alpha F74A and composite mutant alpha V76A/E77A binding activity was reduced to half of that of wild-type (WT) hFSH. In contrast, mutations of either alpha K75A, alpha T80A, alpha H83A, or alpha H90A did not adversely affect receptor binding, demonstrating the specificity of observed effects. The hFSH and mutant hormones were tested in an in vitro bioassay for stimulation of progesterone production. Those mutations that did not affect receptor binding (alpha K75A, alpha T80A, alpha H83A, and alpha H90A) did not affect signal transduction, measured by progesterone responses. After comparison of wild-type and mutant hFSH activities determined in radioreceptor assays (ID50) and in vitro bioassays (ED50), it became evident that signal transduction correlated with receptor binding.