Redox sensitive epidithiodioxopiperazines in biological mechanisms of toxicity

Epidithiodioxopiperazines (ETPs) are a class of secondary metabolites characterized by a bridged disulfide linkage across the alpha,alpha'-positions of the dioxopiperazine ring. This class of compounds displays a range of biological activities, attributed to the sulfur moiety in the oxidized disulfide form and/or the reduced dithiol form. The underlying mechanisms of toxicity of the ETP metabolites are still a matter of debate and this review presents an overview of the evidence for the possible pathways of toxicity.     

Characterization and analysis of scFv-IgG bispecific antibody size variants

ABSTRACT

Bispecific antibodies are an emergent class of biologics that is of increasing interest for therapeutic applications. In one bispecific antibody format, single-chain variable fragments (scFv) are linked to or inserted in different locations of an intact immunoglobulin G (IgG) molecule to confer dual epitope binding. To improve biochemical stability, cysteine residues are often engineered on the heavy- and light-chain regions of the scFv to form an intrachain disulfide bond. Although this disulfide bond often improves stability, it can also introduce unexpected challenges to manufacturing or development. We report size variants that were observed for an appended scFv-IgG bispecific antibody. Structural characterization studies showed that the size variants resulted from the engineered disulfide bond on the scFv, whereby the engineered disulfide was found to be either open or unable to form an intrachain disulfide bond due to cysteinylation or glutathionylation of the cysteines. Furthermore, the scFv engineered cysteines also formed intermolecular disulfide bonds, leading to the formation of highly stable dimers and aggregates. Because both the monomer variants and dimers showed lower bioactivity, they were considered to be product-related impurities that must be monitored and controlled. To this end, we developed and optimized a robust, precise, and accurate high-resolution size-exclusion chromatographic method, using a statistical design-of-experiments methodology.     

Oxidative Folding of Conopeptides Modified by <Emphasis Type="Italic">Conus</Emphasis> Protein Disulfide Isomerase

Protein disulfide isomerase is a type of enzyme that catalyses the oxidation, isomerization and reduction of disulfide bonds. Conotoxins that containing disulfide bonds are likely substrates of protein disulfide isomerise. Here, we cloned 12 protein disulfide isomerise genes from 12 different cone snail species that inhabited the sea near Sanya in China. The full-length amino acid sequences of these protein disulfide isomerase genes share a high degree of homology, including the same -CGHC- active site sequence and -RDEL- endoplasmic reticulum retention signal. To obtain enough conus protein disulfide isomerase for functional studies, we constructed the expression vector pET28a-sPDI. Conus protein disulfide isomerase was successfully expressed using Escherichia coli expression system and purified using chromatography method of affinity chromatography. The recombinant conus protein disulfide isomerase showed the ability to catalyse disulfide bond formation and rearrangement in the lysozyme enzyme activity assay. The role of conus protein disulfide isomerase in the in vitro oxidative folding of conotoxins was investigated using synthetic linear conotoxin lt14a, a peptide composed of 13 amino acids. It was confirmed by high performance liquid chromatography and mass spectrometry analysis that conus protein disulfide isomerase can catalyse the disulfide bond formation of linear lt14a. Then, conus protein disulfide isomerase was acted as a fusion partner during the production of engineered peptidyl-prolyl cis–trans isomerase and lt14a derived from cone snails. It was shown that peptidyl-prolyl cis–trans isomerase and conotoxin lt14a are successfully expressed in a highly soluble form by fusion with conus protein disulfide isomerase. Thus, conus protein disulfide isomerase functions not only as an enzyme that catalyses oxidative process but also a fusion partner in recombinant conotoxin expression.     

PGRL1 Is the Elusive Ferredoxin-Plastoquinone Reductase in Photosynthetic Cyclic Electron Flow

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Highlights? PGRL1 is the elusive FQR which has been sought for almost three decades ? PGR5 is required for the transfer of electrons from ferredoxin to PGRL1 ? The six redox-active cysteine residues of PGRL1 can form intra- and intermolecular disulfide bridges ? Thioredoxins destabilize PGRL1 homodimers     

Molecular Dynamics Simulations on Pars Intercerebralis Major Peptide-C (PMP-C) Reveal the Role of Glycosylation and Disulfide Bonds in its Enhanced Structural Stability and Function

Abstract

Fucosylation of Thr 9 in pars intercerebralis major peptide-C (PMP-C) enhances its structural stability and functional ability as a serine protease inhibitor. In order to understand the role of disulfide bonds and glycosylation on the structure and function of PMP-C, we have carried out multiple explicit solvent molecular dynamics (MD) simulations on fucosylated and non-fucosylated forms of PMP-C, both in the presence and absence of the disulfide bonds. Our simulations revealed that there were no significant structural changes in the native disulfide bonded forms of PMP-C due to fucosylation. On the other hand, the non-fucosylated form of PMP-C without disulfide bonds showed larger deviations from the starting structure than the fucosylated form. However, the structural deviations were restricted to the terminal regions while core β-sheet retained its hydrogen bonded structure even in absence of disulfide bonds as well as fucosylation. Interestingly, fucosylation of disulfide bonded native PMP-C led to a decreased thermal flexibility in the residue stretch 29–32 which is known to interact with the active site of the target proteases. Our analysis revealed that disulfide bonds covalently connect the residue stretch 29–32 to the central β-sheet of PMP-C and using a novel network of side chain interactions and disulfide bonds fucosylation at Thr 9 is altering the flexibility of the stretch 29–32 located at a distal site. Thus, our simulations explain for the first time, how presence of disulfide bonds between conserved cysteines and fucosylation enhance the function of PMP-C as a protease inhibitor.     

Conformations of cysteine disulfides of peptide toxins: Advantage of differentiating forward and reverse asymmetric disulfide conformers

Conformations of cysteine disulfides were analyzed in X-ray, nuclear magnetic resonance (NMR), and co-crystal structures of peptide toxins retrieved from Protein Data Bank. The parameters side chain torsional angles, disulfide strain energy, interatomic Cα/Cβ distances, and Ramachandran angles were used as probes to derive conformational features of cysteine disulfides. Schmidt, Ho, and Hogg (2006 Schmidt, B., Ho, L., &; Hogg, P. J. (2006). Allosteric disulfide bonds. Biochemistry, 45, 7429–7433.[Crossref], [PubMed], [Web of Science ®] , [Google Scholar]) Allosteric disulfide bonds. Biochemistry, 45, 7429–7433 scheme was adapted to classify the disulfide conformations of peptide toxins. Anomalies were observed while treating “forward” and “reverse” asymmetric disulfide conformers as same disulfide conformation in peptide toxins. Thus, new scheme was proposed to classify “forward” and “reverse” asymmetric disulfide conformers separately. Total available conformers space for classification of toxins disulfides is 32. Interestingly, all 32 disulfide conformations are observed in peptide toxins. –LHSpiral is predominant disulfide conformation of peptide toxins. Significant variations were observed in population of occurrence of disulfide conformers, disulfide strain energy, and distribution of D_Cα-Cα and D_Cβ-Cβ values between X-ray, NMR, and co-crystal structures of peptide toxins. The observed differences in conformations of disulfides of same peptide toxins between different states were used as platform to demonstrate advantage of differentiating forward and reverse asymmetric disulfide conformers. Newly proposed scheme allows accurate representation of true conformational diversity of disulfides between X-ray and NMR structures of same peptide toxins. Newly proposed scheme also permits to derive additional structural information from nomenclature which was illustrated by comparing conformations of disulfides between unbound and bound form of toxin with channel/receptor. The results will be of interest for growing field of structural venomics and conformational analysis of peptide/protein disulfides.

Communicated by Ramaswamy H. Sarma     

Absorption,distribution, metabolism and excretion (ADME) of oral selenium from organic and inorganic sources: A review

BackgroundSelenium is a trace element traditionally ingested either in its organic form via food or in its inorganic form through nutritional supplements, while selenium formulated as nanoparticles is a putative long-acting alternative. To understand the physiology and toxicology of the different selenium formulations, it is important to determine how their selenium content is absorbed, distributed, metabolised and excreted; therefore, we reviewed their biokinetics following oral exposure.MethodsWe retrieved and reviewed the literature on the absorption, distribution, metabolism, and excretion of oral exposure to different forms of selenium.ResultsSelenium in both the organic form (containing carbon to selenium chemical bonds) and the inorganic form is absorbed into the blood in humans. The mean normal blood level of many studies was 139 μg/L. There are indications that selenium from organic sources is more bioavailable than selenium from inorganic sources. Selenium is distributed throughout the body, including in breast milk. The elimination of selenium mainly involves the faecal and urinary pathways, whereas breath, saliva and hair are minor contributors. Urinary metabolites include trimethylselenium ions, selenosugars and Se-methylselenoneine.ConclusionSelenium is absorbed to a high extent, and selenium from organic sources is more bioavailable than from inorganic sources. Selenium, as expected as an essential trace element, is distributed throughout the body. Selenium is extensively metabolised, and various excretion metabolites have been identified in both urine and breath, while some selenium is also excreted via faeces.     

Disulfide Bond Formation in the Mammalian Endoplasmic Reticulum

The formation of disulfide bonds between cysteine residues occurs during the folding of many proteins that enter the secretory pathway. As the polypeptide chain collapses, cysteines brought into proximity can form covalent linkages during a process catalyzed by members of the protein disulfide isomerase family. There are multiple pathways in mammalian cells to ensure disulfides are introduced into proteins. Common requirements for this process include a disulfide exchange protein and a protein oxidase capable of forming disulfides de novo. In addition, any incorrect disulfides formed during the normal folding pathway are removed in a process involving disulfide exchange. The pathway for the reduction of disulfides remains poorly characterized. This work will cover the current knowledge in the field and discuss areas for future investigation.One of the characteristics of proteins that enter the secretory pathway is that they frequently contain covalent linkages called disulfide bonds within and between constituent polypeptide chains. The presence of these linkages is thought to confer stability when secreted proteins are exposed to the extracellular milieu or when membrane proteins are recycled through acidic endocytic compartments. In addition to structural disulfides it is now clear that a number of proteins use the formation and breaking of disulfides as a mechanism for regulation of activity (Schwertassek et al. 2007). Hence, it is important that we have a clear understanding of how correct disulfides are formed within proteins both during the protein folding process and to regulate protein function. The focus of this article will be on how correct disulfides are introduced into proteins within the secretory pathway, specifically within the endoplasmic reticulum (ER) during folding and assembly.The formation of disulfides within polypeptides begins as the protein is being cotranslationally translocated into the ER (Chen et al. 1995). The initial collapse of the polypeptide and formation of secondary structure brings cysteine residues into close enough proximity for them to form disulfides. Correct disulfide formation requires enzymes to both introduce disulfides between proximal cysteines and to reduce disulfides that form during folding but that are not present in the final native structure (Jansens et al. 2002). In addition, proteins that do not fold correctly are targeted for degradation and may require their disulfides to be broken before dislocation across the ER membrane into the cytosol (Ushioda et al. 2008). Hence, there must be a reduction and oxidation pathway present in the ER to ensure that native disulfides form and nonnative disulfides are broken during protein folding.Central to both reduction and oxidation pathways is the protein disulfide isomerase (PDI) family of enzymes (Ellgaard and Ruddock 2005) that are capable of exchanging disulfides with their substrate proteins (Fig. 1). Whether disulfide exchange results in the formation or breaking of a disulfide depends on the relative stability of the disulfides in the enzyme and substrate. To drive the formation of disulfides, the PDI family member must itself be oxidized. It is now clear that there are a number of ways for the disulfide exchange proteins to be oxidized by specific oxidases. Importantly, these oxidases do not introduce disulfides into nascent polypeptide chains; rather, they specifically oxidize members of the PDI family. The exception to this rule is the enzyme quiescin sulfydryl oxidase (QSOX; see below). The pathway for disulfide reduction is not as well characterized. It is known that the PDI family members can be reduced by the low molecular mass thiol glutathione (GSH) (Chakravarthi and Bulleid 2004; Jessop and Bulleid 2004; Molteni et al. 2004) but no enzymatic process for reduction has been identified. The glutathione redox balance within the ER is significantly more oxidized than in the cytosol (Hwang et al. 1992; Dixon et al. 2008), indicating that GSH is actively oxidized to glutathione disulfide either during the reduction of PDI family members or by reducing disulfides in nascent polypeptides directly. However, there is currently no clear indication as to how glutathione disulfide is itself reduced.Open in a separate windowFigure 1.PDI family of enzymes catalyzes disulfide exchange reactions in the endoplasmic reticulum. Nascent polypeptide chains are cotranslationally translocated across the ER membrane whereupon cysteines in close proximity can form disulfides. The reaction is catalyzed by members of the PDI family (depicted as PDI) by a disulfide exchange reaction resulting in the reduction of the PDI active site. If nonnative disulfides are formed these can be reduced by the reverse disulfide exchange reaction, resulting in the oxidation of the PDI active site.Both the formation and breaking of disulfides can be thought of as electron transport pathways that require suitable electron acceptors or donors to drive the flow of electrons. For the purposes of this article the two pathways will be discussed separately, but it should be appreciated that each pathway occurs within the same organelle so the possibility of crossover between them is real. Whether futile redox reactions occur between the pathways is unclear but any kinetic segregation of the pathways will be highlighted where it is known to occur.     

Natural antifoulants from the marine cyanobacterium Lyngbya majuscula

Filamentous benthic marine cyanobacteria are a prolific source of structurally unique bioactive secondary metabolites. A total of 12 secondary metabolites, belonging to the mixed polyketide–polypeptide structural class, were isolated from the marine cyanobacterium, Lyngbya majuscula, and were tested to determine if they showed activity against barnacle larval settlement. The assays revealed four compounds, dolastatin 16 (1), hantupeptin C (4), majusculamide A (10), and isomalyngamide A (12), that showed moderate to potent anti-larval settlement activities, with EC₅₀ values ranging from 0.003 to 10.6 μg ml^?1. In addition, field testing conducted over a period of 28 days (using the modified Phytagel? method) based on the cyanobacterial compound, dolastatin 16, showed significantly reduced barnacle settlement as compared to controls at all the concentrations tested. The results of this study highlight the importance of marine cyanobacteria as an underexplored source of potential environmentally friendly antifoulants.     

Metabolite coupling in genome-scale metabolic networks

Background  

Biochemically detailed stoichiometric matrices have now been reconstructed for various bacteria, yeast, and for the human cardiac mitochondrion based on genomic and proteomic data. These networks have been manually curated based on legacy data and elementally and charge balanced. Comparative analysis of these well curated networks is now possible. Pairs of metabolites often appear together in several network reactions, linking them topologically. This co-occurrence of pairs of metabolites in metabolic reactions is termed herein "metabolite coupling." These metabolite pairs can be directly computed from the stoichiometric matrix, S. Metabolite coupling is derived from the matrix ŜŜ ^T, whose off-diagonal elements indicate the number of reactions in which any two metabolites participate together, where Ŝ is the binary form of S.     

S-glutathionylation of human glyceraldehyde-3-phosphate dehydrogenase and possible role of Cys152-Cys156 disulfide bridge in the active site of the protein

BackgroundWe previously showed that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is S-glutathionylated in the presence of H₂O₂ and GSH. S-glutathionylation was shown to result in the formation of a disulfide bridge in the active site of the protein. In the present work, the possible biological significance of the disulfide bridge was investigated.MethodsHuman recombinant GAPDH with the mutation C156S (hGAPDH_C156S) was obtained to prevent the formation of the disulfide bridge. Properties of S-glutathionylated hGAPDH_C156S were studied in comparison with those of the wild-type protein hGAPDH.ResultsS-glutathionylation of hGAPDH and hGAPDH_C156S results in the reversible inactivation of the proteins. In both cases, the modification results in corresponding mixed disulfides between the catalytic Cys152 and GSH. In the case of hGAPDH, the mixed disulfide breaks down yielding Cys152-Cys156 disulfide bridge in the active site. In hGAPDH_C156S, the mixed disulfide is stable. Differential scanning calorimetry method showed that S-glutathionylation leads to destabilization of hGAPDH molecule, but does not affect significantly hGAPDH_C156S. Reactivation of S-glutathionylated hGAPDH in the presence of GSH and glutaredoxin 1 is approximately two-fold more efficient compared to that of hGAPDH_C156S.ConclusionsS-glutathionylation induces the formation of Cys152-Cys156 disulfide bond in the active site of hGAPDH, which results in structural changes of the protein molecule. Cys156 is important for reactivation of S-glutathionylated GAPDH by glutaredoxin 1.General significanceThe described mechanism may be important for interaction between GAPDH and other proteins and ligands, involved in cell signaling.     

Versatility of the Endoplasmic Reticulum Protein Folding Factory

ABSTRACT

The endoplasmic reticulum (ER) is dedicated to import, folding and assembly of all proteins that travel along or reside in the secretory pathway of eukaryotic cells. Folding in the ER is special. For instance, newly synthesized proteins are N-glycosylated and by default form disulfide bonds in the ER, but not elsewhere in the cell. In this review, we discuss which features distinguish the ER as an efficient folding factory, how the ER monitors its output and how it disposes of folding failures.     

1H, 13C and 15N resonance assignments of the bb′ domains of human protein disulfide isomerase

Protein disulfide isomerase (PDI) participates in protein folding and catalyses formation of disulfide bonds. The b′ domain of human PDI contributes to binding unfolded proteins; its structure is stabilized by the b domain. Here, we report NMR chemical shift assignments for the bb′ fragment.     

A Relaxed Specificity in Interchain Disulfide Bond Formation Characterizes the Assembly of a Low-Molecular-Weight Glutenin Subunit in the Endoplasmic Reticulum

Wheat (Triticum spp.) grains contain large protein polymers constituted by two main classes of polypeptides: the high-molecular-weight glutenin subunits and the low-molecular-weight glutenin subunits (LMW-GS). These polymers are among the largest protein molecules known in nature and are the main determinants of the superior technological properties of wheat flours. However, little is known about the mechanisms controlling the assembly of the different subunits and the way they are arranged in the final polymer. Here, we have addressed these issues by analyzing the formation of interchain disulfide bonds between identical and different LMW-GS and by studying the assembly of mutants lacking individual intrachain disulfides. Our results indicate that individual cysteine residues that remain available for disulfide bond formation in the folded monomer can form interchain disulfide bonds with a variety of different cysteine residues present in a companion subunit. These results imply that the coordinated expression of many different LMW-GS in wheat endosperm cells can potentially lead to the formation of a large set of distinct polymeric structures, in which subunits can be arranged in different configurations. In addition, we show that not all intrachain disulfide bonds are necessary for the generation of an assembly-competent structure and that the retention of a LMW-GS in the early secretory pathway is not dependent on polymer formation.The unique ability of wheat (Triticum spp.) flour to form a dough that has the rheological properties required for the production of leavened bread and other foods is largely due to the characteristics of the proteins that accumulate in wheat endosperm cells during seed development (Gianibelli et al., 2001). Among these endosperm proteins, a major role is played by prolamines, a large group of structurally different proteins sharing the characteristic of being particularly high in Pro and Gln.On the basis of their polymerization status, wheat prolamines can be subdivided into two groups, the gliadins and the glutenins. While gliadins are monomeric, glutenins are heterogeneous mixtures of polymers where individual subunits are held together by interchain disulfide bonds (Galili et al., 1996; Tatham and Shewry, 1998). The subunits participating to the formation of these large polymers have been classified into four groups according to their electrophoretic mobility (Gianibelli et al., 2001). The A group is constituted by the so-called high-molecular-weight glutenin subunits (HMW-GS), while polypeptides in groups B, C, and D are collectively termed low-molecular-weight glutenin subunits (LMW-GS). While only three to five HMW-GS are expressed in common wheat endosperm, LMW-GS include a very large number of different polypeptides.Different models of glutenin assembly have been proposed (see Gianibelli et al., 2001 for a review), but the determination of their precise structure and M_r distribution has been hampered by their large size and complex subunit composition. Crucially, because disulfide bonds appear to be the major factor affecting polymer stability, it would be very useful to know whether the pairing between specific Cys residues, rather than random assembly, controls glutenin polymer formation. Indeed, data obtained with HMW-GS indicate that the formation of certain types of intermolecular disulfide bonds is particularly favored (Tao et al., 1992; Shimoni et al., 1997). In the case of LMW-GS, at least two functionally distinct types of subunits can be distinguished. Subunits of the first type, to which the majority of B-type subunits belong, would act as chain extenders, because they contain two Cys residues that remain available for the formation of interchain disulfide bonds. Subunits of the second type, containing a single Cys residue able to form an interchain disulfide bond, would instead act as chain terminators (Kasarda, 1989). Most of the members of this second group are indeed modified gliadins that participate to polymer formation thanks to the presence of extra Cys residues (D''Ovidio and Masci, 2004). Given the complexity of the situation found in wheat endosperm, where many different subunits are synthesized at the same time and can participate in the formation of complex high-M_r polymers, the study of glutenin polymer formation can take advantage of the use of heterologous expression systems where the behavior of individual subunits can be more easily monitored. For instance, the expression of HMW-GS in transgenic tobacco (Nicotiana tabacum) has provided insights into the rules governing the assembly of some of the subunits belonging to this class (Shani et al., 1994; Shimoni et al., 1997). In this work, we have used heterologous expression of wild-type and modified LMW-GS in tobacco protoplasts to study the assembly of this class of gluten polypeptides. Our results confirm that disulfide bonds are crucial for the assembly of these proteins and indicate that a relaxed specificity in Cys pairing from different subunits can drive the formation of complex glutenin polymers.     

… -ome sweet -ome

Abstract

Arsenic-induced tissue damage is a major concern to the human population. An impaired antioxidant defense mechanism followed by oxidative stress is the major cause of arsenic-induced toxicity, which can lead to reproductive failure. The present study was carried out to investigate the preventive role of arjunolic acid, a triterpenoid saponin isolated from the bark of Terminalia arjuna, against arsenic-induced testicular damage in mice. Administration of arsenic (in the form of sodium arsenite, NaAsO₂, at a dose of 10 mg/kg body weight) for 2 days significantly decreased the intracellular antioxidant power, the activities of the antioxidant enzymes, as well as the levels of cellular metabolites. In addition, arsenic intoxication enhanced testicular arsenic content, lipid peroxidation, protein carbonylation and the level of glutathione disulfide (GSSG). Exposure to arsenic also caused significant degeneration of the seminiferous tubules with necrosis and defoliation of spermatocytes. Pretreatment with arjunolic acid at a dose of 20 mg/kg body weight for 4 days could prevent the arsenic-induced testicular oxidative stress and injury to the histological structures of the testes. Arjunolic acid had free radical scavenging activity in a cell-free system and antioxidant power in vivo. In summary, the results suggest that the chemopreventive role of arjunolic acid against arsenic-induced testicular toxicity may be due to its intrinsic antioxidant property.     

Redox-Dependent Control of FOXO/DAF-16 by Transportin-1

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Highlights? FOXO forms redox-sensitive, disulfide-dependent complexes with several proteins ? Transportin-1 binds to FOXO via a disulfide and regulates its nuclear localization ? Redox and insulin signaling govern FOXO nuclear localization via distinct pathways ? Redox control of longevity protein FOXO/DAF-16 is evolutionarily conserved     

A new bisabolane-type sesquiterpenoid from Curcuma domestica

Biochemical Systematics and Ecology, Volume x, Issue x, Pages x–x (dd mm yyyy). A new bisabolane-type sesquiterpenoid from Curcuma domestica. Takahiro Ishii, Hiroshi Matsuura, Kunimitsu Kaya, Charles Santhanaraju Vairappan.

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Highlights? Chemical constituents of lowland and highland Curcuma domestica of Borneo are reported. ? Three sesquiterpenes and three curcuminoids secondary metabolites were found in common. ? Highland populations contained two additional bisabolane-type sesquiterpenoid metabolites. ? Curcuma domestica is reported as a new source for novel compound, bisacurol B.     

耐甲氧西林金黄色葡萄球菌MRSA252辅酶A二硫化物还原酶缺失延缓苯唑西林的次级损伤效应

【目的】耐甲氧西林金黄色葡萄球菌在苯唑西林作用下,其辅酶A二硫化物还原酶表达上调2.3倍。本文研究苯唑西林对该酶缺失的金黄色葡萄球菌的杀菌效应。【方法】利用同源重组双交换技术对金黄色葡萄球菌进行基因敲除,并用质粒p OS1构建回补株;采用分光光度法检测菌株体外增殖能力;以时间-杀菌法考察苯唑西林对菌株杀菌效应;以2’,7’-二氯荧光黄双乙酸盐为探针检测胞内活性氧水平。【结果】辅酶A二硫化物还原酶基因敲除株较亲株生长缓慢(P0.05);20倍MIC浓度苯唑西林下敲除株的时间-杀菌曲线及胞内活性氧水平与亲株无显著性差异,5倍MIC浓度下敲除株的致死速率及胞内活性氧水平均较亲株下降。【结论】在较低浓度苯唑西林作用下,辅酶A二硫化物还原酶基因缺失降低胞内活性氧水平,减小杀菌速率,延缓次级损伤效应。     

Biological regulation through protein disulfide bond cleavage

Abstract

It is thought that disulfide bonds in secreted proteins are inert because of the oxidizing nature of the extracellular milieu. We have suggested that this is not necessarily the case and that certain secreted proteins contain one or more disulfide bonds that can be cleaved and that this cleavage is central to the protein's function. This review discusses disulfide bond cleavage in the secreted soluble protein, plasmin. Cleavage of plasmin disulfide bond(s) triggers peptide bond cleavage and formation of the tumour angiogenesis inhibitor, angiostatin. Tumour cells secrete phosphoglycerate kinase which facilitates cleavage of the plasmin disulfide bond(s). Phosphoglycerate kinase is not a conventional disulfide bond reductase. We propose that phosphoglycerate kinase facilitates cleavage of a particular plasmin disulfide bond by hydroxide ion, which results in formation of a sulfenic acid and a free thiol. The free thiol is then available to exchange with another nearby disulfide bond resulting in formation of a new disulfide and a new free thiol. The reduced plasmin is then susceptible to discreet proteolysis which results in release of angiostatin.