Disulphide reduction in lysosomes. The role of cysteine.
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ABSTRACT:
SUBMITTER: Lloyd JB
PROVIDER: S-EPMC1146975 | biostudies-other | 1986 Jul
REPOSITORIES: biostudies-other
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Project description:Dozens of genes contribute to the wide variation in human pigmentation. Many of these genes encode proteins that localize to the melanosome-the organelle, related to the lysosome, that synthesizes pigment-but have unclear functions1,2. Here we describe MelanoIP, a method for rapidly isolating melanosomes and profiling their labile metabolite contents. We use this method to study MFSD12, a transmembrane protein of unknown molecular function that, when suppressed, causes darker pigmentation in mice and humans3,4. We find that MFSD12 is required to maintain normal levels of cystine-the oxidized dimer of cysteine-in melanosomes, and to produce cysteinyldopas, the precursors of pheomelanin synthesis made in melanosomes via cysteine oxidation5,6. Tracing and biochemical analyses show that MFSD12 is necessary for the import of cysteine into melanosomes and, in non-pigmented cells, lysosomes. Indeed, loss of MFSD12 reduced the accumulation of cystine in lysosomes of fibroblasts from patients with cystinosis, a lysosomal-storage disease caused by inactivation of the lysosomal cystine exporter cystinosin7-9. Thus, MFSD12 is an essential component of the cysteine importer for melanosomes and lysosomes.
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Project description:TDP-43 is the major disease protein in ubiquitin-positive inclusions of amyotrophic lateral sclerosis and frontotemporal lobar degeneration (FTLD) characterized by TDP-43 pathology (FTLD-TDP). Accumulation of insoluble TDP-43 aggregates could impair normal TDP-43 functions and initiate disease progression. Thus, it is critical to define the signalling mechanisms regulating TDP-43 since this could open up new avenues for therapeutic interventions. Here, we have identified a redox-mediated signalling mechanism directly regulating TDP-43. Using in vitro and cell-based studies, we demonstrate that oxidative stress promotes TDP-43 cross-linking via cysteine oxidation and disulphide bond formation leading to decreased TDP-43 solubility. Biochemical analysis identified several cysteine residues located within and adjacent to the second RNA-recognition motif that contribute to both intra- and inter-molecular interactions, supporting TDP-43 as a target of redox signalling. Moreover, increased levels of cross-linked TDP-43 species are found in FTLD-TDP brains, indicating that aberrant TDP-43 cross-linking is a prominent pathological feature of this disease. Thus, TDP-43 is dynamically regulated by a redox regulatory switch that links oxidative stress to the modulation of TDP-43 and its downstream targets.
| S-EPMC3297986 | biostudies-literature
Project description:Disulphide bonds between cysteine residues in proteins play a key role in protein folding, stability, and function. Loss of a disulphide bond is often associated with functional differentiation of the protein. The evolution of disulphide bonds is still actively debated; analysis of naturally occurring variants can promote understanding of the protein evolutionary process. One of the disulphide bond-containing protein families is the potato proteinase inhibitor II (PI-II, or Pin2, for short) superfamily, which is found in most solanaceous plants and participates in plant development, stress response, and defence. Each PI-II domain contains eight cysteine residues (8C), and two similar PI-II domains form a functional protein that has eight disulphide bonds and two non-identical reaction centres. It is still unclear which patterns and processes affect cysteine residue loss in PI-II. Through cDNA sequencing and data mining, we found six natural variants missing cysteine residues involved in one or two disulphide bonds at the first reaction centre. We named these variants Pi7C and Pi6C for the proteins missing one or two pairs of cysteine residues, respectively. This PI-II-7C/6C family was found exclusively in potato. The missing cysteine residues were in bonding pairs but distant from one another at the nucleotide/protein sequence level. The non-synonymous/synonymous substitution (Ka/Ks) ratio analysis suggested a positive evolutionary gene selection for Pi6C and various Pi7C. The selective deletion of the first reaction centre cysteine residues that are structure-level-paired but sequence-level-distant in PI-II illustrates the flexibility of PI-II domains and suggests the functionality of their transient gene versions during evolution.
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Project description:ObjectiveRedox status and inflammation are important in the pathophysiology of numerous chronic diseases. Epidemiological studies have linked vitamin D status to a number of chronic diseases. We aimed to examine the relationships between serum 25-hydroxyvitamin D [25(OH)D] and circulating thiol/disulphide redox status and biomarkers of inflammation.DesignThis was a cross-sectional study of N = 693 adults (449 females, 244 males) in an apparently healthy, working cohort in Atlanta, GA. Plasma glutathione (GSH), cysteine (Cys) and their associated disulphides were determined with high-performance liquid chromatography, and their redox potentials (Eh GSSG and Eh CySS) were calculated using the Nernst equation. Serum inflammatory markers included interleukin-6 (IL-6), interleukin-8 (IL-8) and tumour necrosis factor-α, assayed on a multiplex platform, and C-reactive protein (CRP), assayed commercially. Relationships were assessed with multiple linear regression analyses.ResultsSerum 25(OH)D was positively associated with plasma GSH (β ± SE: 0·002 ± 0·0004) and negatively associated with plasma Eh GSSG (β ± SE: -0·06 ± 0·01) and Cys (β ± SE: -0·01 ± 0·003) (P < 0·001 for all); statistical significance remained after adjusting for age, gender, race, percentage body fat and traditional cardiovascular risk factors (P = 0·01-0·02). The inverse relationship between serum 25(OH)D and CRP was confounded by percentage body fat, and full adjustment for covariates attenuated serum 25(OH)D relationships with other inflammatory markers to nonstatistical significance.ConclusionsSerum 25(OH)D concentrations were independently associated with major plasma thiol/disulphide redox systems, suggesting that vitamin D status may be involved in redox-mediated pathophysiology.
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Project description:Inhibitors of cysteine proteinases have been used extensively to dissect the roles of these proteinases in cells. Surprisingly though, little work has been performed to demonstrate unequivocally that the inhibitors reach and inactivate their target proteinases in cell culture or in vivo. In the present study, the permeability of lysosomes and whole cells has been studied. Benzyloxycarbonyl (Z)-[125I]iodo-Tyr-Ala-diazomethane (CHN2), an inhibitor of cathepsins L and B, has been shown to label active forms of these enzymes in lysosomes and whole cells. The ability of other cysteine proteinase inhibitors to block this labelling has been used to indicate the permeation of these compounds. All the inhibitors were able to block labelling by Z-[125I]iodo-Tyr-Ala-CHN2 in lysosomal extracts. In intact lysosomes or cells, however, only N-[N-(L-3-trans-ethoxycarbonyloxirane-2-carbonyl)-L-leucyl]-3- methylbutylamine ('E-64d') Z-Tyr-Ala-CHN2, Z-Phe-Ala-CHN2 and Z-Phe-Phe-CHN2 were able to block labelling by Z-[125I]iodo-Tyr-Ala-CHN2. N-[N-(L-3-trans-Carboxyoxirane-2-carbonyl)-L-leucyl]amino-4-gua nidinobutane (E-64) and leupeptin were unable to block labelling by Z-[125I]iodo-Tyr-Ala-CHN2 in lysosomes or in cells. The ability to block labelling in lysosomes is an indication of the ability of the inhibitor to diffuse across membranes. Thus E-64 and leupeptin do not readily permeate membranes and therefore their uptake into cells probably only occurs via pinocytosis.
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Project description:The importance of disulphide bond in mediating viral peptide entry into host cells is well known. In the present work, we elucidate the role of disulphide (SS) bond in partitioning mechanism of membrane-active Hepatitis A Virus-2B (HAV-2B) peptide, which harbours three cysteine residues promoting formation of multiple SS-bonded states. The inclusion of SS-bond not only results in a compact conformation but also induces distorted α-helical hairpin geometry in comparison to SS-free state. Owing to these, the hydrophobic residues get buried, restricting the insertion of SS-bonded HAV-2B peptide into lipid packing defects and thus the partitioning of the peptide is completely or partly abolished. In this way, the disulphide bond can potentially regulate the partitioning of HAV-2B peptide such that the membrane remodelling effects of this viral peptide are significantly reduced. The current findings may have potential implications in drug designing, targeting the HAV-2B protein by promoting disulphide bond formation within its membrane-active region.
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Project description:1. Cystamine was reduced to the corresponding thiol by rat liver mitochondria, even in the presence of rotenone or antimycin A. 2. The reduction of disulphides was stimulated by the accumulation of NADH or by the addition of NADH to osmotically ;shocked' mitochondria. 3. Energy made available by oxidative phosphorylation was not essential for the reduction of disulphides. 4. Cystamine was not reduced during the oxidation of NADH by ultrasonically treated particles, which had lost their capacity for oxidation of alpha-oxo acids. 5. In intact mitochondria, arsenite and other inhibitors of vicinal dithiols caused a decrease in the capacity for reduction of disulphides concomitantly with an inhibition of the oxidation of alpha-oxo acids. 6. Isolated lipoamide dehydrogenase reduced cystamine at the expense of NADH, provided that lipoic acid was also present. 7. It is concluded that in mitochondria the reduction of cystamine and related disulphides is probably brought about by interaction with reduced lipoic acid, generated by the alpha-oxo acid dehydrogenase complexes during the oxidation of alpha-oxo acids or by reaction of lipoamide dehydrogenase with NADH.
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Project description:Spores of Bacillus species have novel properties, which allow them to lie dormant for years and then germinate under favourable conditions. In the current work, the role of a key metabolic integrator, coenzyme A (CoA), in redox regulation of growing cells and during spore formation in Bacillus megaterium and Bacillus subtilis is studied. Exposing these growing cells to oxidising agents or carbon deprivation resulted in extensive covalent protein modification by CoA (termed protein CoAlation), through disulphide bond formation between the CoA thiol group and a protein cysteine. Significant protein CoAlation was observed during sporulation of B. megaterium, and increased largely in parallel with loss of metabolism in spores. Mass spectrometric analysis identified four CoAlated proteins in B. subtilis spores as well as one CoAlated protein in growing B. megaterium cells. All five of these proteins have been identified as moderately abundant in spores. Based on these findings and published studies, protein CoAlation might be involved in facilitating establishment of spores' metabolic dormancy, and/or protecting sensitive sulfhydryl groups of spore enzymes.
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Project description:Long known as digestive organelles, lysosomes have now emerged as multifaceted centers responsible for degradation, nutrient sensing, and immunity. Growing evidence also implicates role of lysosome-related mechanisms in pathologic process. In this review, we discuss physiological function of lysosomes and, more importantly, how the homeostasis of lysosomes is disrupted in several diseases, including atherosclerosis, neurodegenerative diseases, autoimmune disorders, pancreatitis, lysosomal storage disorders, and malignant tumors. In atherosclerosis and Gaucher disease, dysfunction of lysosomes changes cytokine secretion from macrophages, partially through inflammasome activation. In neurodegenerative diseases, defect autophagy facilitates accumulation of toxic protein and dysfunctional organelles leading to neuron death. Lysosomal dysfunction has been demonstrated in pathology of pancreatitis. Abnormal autophagy activation or inhibition has been revealed in autoimmune disorders. In tumor microenvironment, malignant phenotypes, including tumorigenesis, growth regulation, invasion, drug resistance, and radiotherapy resistance, of tumor cells and behaviors of tumor-associated macrophages, fibroblasts, dendritic cells, and T cells are also mediated by lysosomes. Based on these findings, a series of therapeutic methods targeting lysosomal proteins and processes have been developed from bench to bedside. In a word, present researches corroborate lysosomes to be pivotal organelles for understanding pathology of atherosclerosis, neurodegenerative diseases, autoimmune disorders, pancreatitis, and lysosomal storage disorders, and malignant tumors and developing novel therapeutic strategies.
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