Redox-active Disulfide Research Articles

Self-assembled silibinin-containing phosphazene/cystamine hybrid nanospheres as biodagradable dual-drug carriers with improved anticancer activity on a breast cancer cell line

In this study, pH and redox sensitive biodegradable PPZs@Silibinin polymeric nanospheres were synthesized by the polymerization reaction of Hexachlorocyclotriphosphazene (HCCP), Cystamine dihydrochloride (CA) and the anticancer drug Silibinin as monomers. Later designed as a therapeutic system, this nano drug-carrier was also loaded with Doxorubicin (Dox), a type of cancer drug. As a result, a drug self-delivery system (DSDS) was developed, which had the property of biodegradation in tumor tissues thanks to the redox active disulfide bonds in its structure from pH-sensitive nanospheres containing two anticancer drugs. Biodegradation-associated in vitro release of cancer drug (Dox) from the Dox loaded PPZs@Silibinin nano drug-carrier was performed in the presence of glutathione (GSH) at pH 6.5 and pH 7.4. While 93.06% Dox release was observed in pH 6.5 medium containing 10 mM GSH in 24 h, this value was 56.60% in pH 7.4 + 10 mM GSH medium. In addition, clonogenic analysis and cytotoxicity studies were performed on the MDA-MB-231 cell line to determine the anticancer properties of PPZs@Silibinin loaded with Dox. It was determined that PPZs@Silibinin@Dox nanospheres containing 20.5 nM Dox for 0.75 µL are highly effective in MDA-MB-231 cells than free Dox.

Targeting the Cysteine Redox Proteome in Parkinson's Disease: The Role of Glutathione Precursors and Beyond.

Encouraging recent data on the molecular pathways underlying aging have identified variants and expansions of genes associated with DNA replication and repair, telomere and stem cell maintenance, regulation of the redox microenvironment, and intercellular communication. In addition, cell rejuvenation requires silencing some transcription factors and the activation of pluripotency, indicating that hidden molecular networks must integrate and synchronize all these cellular mechanisms. Therefore, in addition to gene sequence expansions and variations associated with senescence, the optimization of transcriptional regulation and protein crosstalk is essential. The protein cysteinome is crucial in cellular regulation and plays unexpected roles in the aging of complex organisms, which show cumulative somatic mutations, telomere attrition, epigenetic modifications, and oxidative dysregulation, culminating in cellular senescence. The cysteine thiol groups are highly redox-active, allowing high functional versatility as structural disulfides, redox-active disulfides, active-site nucleophiles, proton donors, and metal ligands to participate in multiple regulatory sites in proteins. Also, antioxidant systems control diverse cellular functions, including the transcription machinery, which partially depends on the catalytically active cysteines that can reduce disulfide bonds in numerous target proteins, driving their biological integration. Since we have previously proposed a fundamental role of cysteine-mediated redox deregulation in neurodegeneration, we suggest that cellular rejuvenation of the cysteine redox proteome using GSH precursors, like N-acetyl-cysteine, is an underestimated multitarget therapeutic approach that would be particularly beneficial in Parkinson's disease.

Looking for the mechanism of arsenate respiration of Fusibacter sp. strain 3D3, independent of ArrAB.

The literature has reported the isolation of arsenate-dependent growing microorganisms which lack a canonical homolog for respiratory arsenate reductase, ArrAB. We recently isolated an arsenate-dependent growing bacterium from volcanic arsenic-bearing environments in Northern Chile, Fusibacter sp. strain 3D3 (Fas) and studied the arsenic metabolism in this Gram-positive isolate. Features of Fas deduced from genome analysis and comparative analysis with other arsenate-reducing microorganisms revealed the lack of ArrAB coding genes and the occurrence of two arsC genes encoding for putative cytoplasmic arsenate reductases named ArsC-1 and ArsC-2. Interestingly, ArsC-1 and ArsC-2 belong to the thioredoxin-coupled family (because of the redox-active disulfide protein used as reductant), but they conferred differential arsenate resistance to the E. coli WC3110 ΔarsC strain. PCR experiments confirmed the absence of arrAB genes and results obtained using uncouplers revealed that Fas growth is linked to the proton gradient. In addition, Fas harbors ferredoxin-NAD+ oxidoreductase (Rnf) and electron transfer flavoprotein (etf) coding genes. These are key molecular markers of a recently discovered flavin-based electron bifurcation mechanism involved in energy conservation, mainly in anaerobic metabolisms regulated by the cellular redox state and mostly associated with cytoplasmic enzyme complexes. At least three electron-bifurcating flavoenzyme complexes were evidenced in Fas, some of them shared in conserved genomic regions by other members of the Fusibacter genus. These physiological and genomic findings permit us to hypothesize the existence of an uncharacterized arsenate-dependent growth metabolism regulated by the cellular redox state in the Fusibacter genus.

Dual Stimuli-Responsive Dynamic Covalent Peptide Tags: Toward Sequence-Controlled Release in Tumor-like Microenvironments.

Dynamic covalent chemistry (DCvC) has emerged as a versatile synthetic tool for devising stable, stimuli-responsive linkers or conjugates. The interplay of binding affinity, association and dissociation constants exhibits a strong influence on the selectivity of the reaction, the conversion rate, as well as the stability in aqueous solutions. Nevertheless, dynamic covalent interactions often exhibit fast binding and fast dissociation events or vice versa, affecting their conversion rates or stabilities. To overcome the limitation in linker design, we reported herein dual responsive dynamic covalent peptide tags combining a pH responsive boronate ester with fast association and dissociation rates, and a redox-active disulfide with slow formation and dissociation rate. Precoordination by boronic acid–catechol interaction improves self-sorting and selectivity in disulfide formation into heterodimers. The resulting bis-peptide conjugate exhibited improved complex stability in aqueous solution and acidic tumor-like extracellular microenvironment. Furthermore, the conjugate responds to pH changes within the physiological range as well as to redox conditions found inside cancer cells. Such tags hold great promise, through cooperative effects, for controlling the stability of bioconjugates under dilution in aqueous media, as well as designing intelligent pharmaceutics that react to distinct biological stimuli in cells.

Atomic Force Microscopy to Elicit Conformational Transitions of Ferredoxin-Dependent Flavin Thioredoxin Reductases.

Flavin and redox-active disulfide domains of ferredoxin-dependent flavin thioredoxin reductase (FFTR) homodimers should pivot between flavin-oxidizing (FO) and flavin-reducing (FR) conformations during catalysis, but only FR conformations have been detected by X-ray diffraction and scattering techniques. Atomic force microscopy (AFM) is a single-molecule technique that allows the observation of individual biomolecules with sub-nm resolution in near-native conditions in real-time, providing sampling of molecular properties distributions and identification of existing subpopulations. Here, we show that AFM is suitable to evaluate FR and FO conformations. In agreement with imaging under oxidizing condition, only FR conformations are observed for Gloeobacter violaceus FFTR (GvFFTR) and isoform 2 of Clostridium acetobutylicum FFTR (CaFFTR2). Nonetheless, different relative dispositions of the redox-active disulfide and FAD-binding domains are detected for FR homodimers, indicating a dynamic disposition of disulfide domains regarding the central protein core in solution. This study also shows that AFM can detect morphological changes upon the interaction of FFTRs with their protein partners. In conclusion, this study paves way for using AFM to provide complementary insight into the FFTR catalytic cycle at pseudo-physiological conditions. However, future approaches for imaging of FO conformations will require technical developments with the capability of maintaining the FAD-reduced state within the protein during AFM scanning.

Disulfide-Linked Allosteric Modulators for Multi-cycle Kinetic Control of DNA-Based Nanodevices.

Nature employs sulfur switches, that is, redox-active disulfides, to kinetically control biological pathways in a highly efficient and reversible way. Inspired by this mechanism, we describe herein a DNA-based synthetic nanodevice that acts as a sulfur switch and can be temporally controlled though redox regulation. To do this, we rationally designed disulfide DNA strands (modulators) that hybridize to a ligand-binding DNA nanodevice and act as redox-active allosteric regulators inducing the nanodevice to release or load its ligand. Upon reduction, the allosteric modulator spontaneously de-hybridizes from the nanodevice and, as a result, its effect is transient. The system is reversible and has an unprecedented high tolerance to waste products and displays transient behavior for over 40 cycles without significant loss of efficiency. Kinetic control of DNA-based ligand-binding nanodevices through purely chemical reactions paves the way for temporal regulation of more complex chemical pathways.

Structure and Electron-Transfer Pathway of the Human Methionine Sulfoxide Reductase MsrB3.

Aims: The post-translational oxidation of methionine to methionine sulfoxide (MetSO) is a reversible process, enabling the repair of oxidative damage to proteins and the use of sulfoxidation as a regulatory switch. MetSO reductases catalyze the stereospecific reduction of MetSO. One of the mammalian MetSO reductases, MsrB3, has a signal sequence for entry into the endoplasmic reticulum (ER). In the ER, MsrB3 is expected to encounter a distinct redox environment compared with its paralogs in the cytosol, nucleus, and mitochondria. We sought to determine the location and arrangement of MsrB3 redox-active cysteines, which may couple MsrB3 activity to other redox events in the ER.Results: We determined the human MsrB3 structure by using X-ray crystallography. The structure revealed that a disulfide bond near the protein amino terminus is distant in space from the active site. Nevertheless, biochemical assays showed that these amino-terminal cysteines are oxidized by the MsrB3 active site after its reaction with MetSO.Innovation: This study reveals a mechanism to shuttle oxidizing equivalents from the primary MsrB3 active site toward the enzyme surface, where they would be available for further dithiol-disulfide exchange reactions.Conclusion: Conformational changes must occur during the MsrB3 catalytic cycle to transfer oxidizing equivalents from the active site to the amino-terminal redox-active disulfide. The accessibility of this exposed disulfide may help couple MsrB3 activity to other dithiol-disulfide redox events in the secretory pathway.

Functional properties and the oligomeric state of alkyl hydroperoxide reductase subunit F (AhpF) in Pseudomonas aeruginosa

Alkyl hydroperoxide reductase subunit F (AhpF) is a well-known flavoprotein that transfers electrons from pyridine nucleotides to the peroxidase protein AhpC via redox-active disulfide centers to detoxify hydrogen peroxide. However, study of AhpF has historically been limited to particular eubacteria, and the connection between the functional and structural properties of AhpF remains unknown. The present study demonstrates the dual function of Pseudomonas aeruginosa AhpF (PaAhpF) as a reductase and a molecular chaperone. It was observed that the functions of PaAhpF are closely linked with its structural status. The reductase and foldase chaperone function of PaAhpF predominated for its low-molecular-weight (LMW) form, whereas the holdase chaperone function of PaAhpF was found associated with its high-molecular-weight (HMW) complex. Further, the present study also demonstrates the multiple function of PaAhpF in controlling oxidative and heat stresses in P. aeruginosa resistance to oxidative and heat stress.

SARS-CoV2 infectivity is potentially modulated by host redox status

The current coronavirus disease (COVID-19) outbreak caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV2) has emerged as a threat to global social and economic systems. Disparity in the infection of SARS-CoV2 among host population and species is an established fact without any clear explanation. To initiate infection, viral S-protein binds to the Angiotensin-Converting Enzyme 2 (ACE2) receptor of the host cell. Our analysis of retrieved amino acid sequences deposited in data bases shows that S-proteins and ACE2 are rich in cysteine (Cys) residues, many of which are conserved in various SARS-related coronaviruses and participate in intra-molecular disulfide bonds. High-resolution protein structures of S-proteins and ACE2 receptors highlighted the probability that two of these disulfide bonds are potentially redox-active, facilitating the primal interaction between the receptor and the spike protein. Presence of redox-active disulfides in the interacting parts of S-protein, ACE2, and a ferredoxin-like fold domain in ACE2, strongly indicate the role of redox in COVID-19 pathogenesis and severity. Resistant animals lack a redox-active disulfide (Cys133-Cys141) in ACE2 sequences, further strengthening the redox hypothesis for infectivity. ACE2 is a known regulator of oxidative stress. Augmentation of cellular oxidation with aging and illness is the most likely explanation of increased vulnerability of the elderly and persons with underlying health conditions to COVID-19.

Galectin-13/placental protein 13: redox-active disulfides as switches for regulating structure, function and cellular distribution.

Galectin-13 (Gal-13) plays numerous roles in regulating the relationship between maternal and fetal tissues. Low expression levels or mutations of the lectin can result in pre-eclampsia. The previous crystal structure and gel filtration data show that Gal-13 dimerizes via formation of two disulfide bonds formed by Cys136 and Cys138. In the present study, we mutated them to serine (C136S, C138S and C136S/C138S), crystalized the variants and solved their crystal structures. All variants crystallized as monomers. In the C136S structure, Cys138 formed a disulfide bond with Cys19, indicating that Cys19 is important for regulation of reversible disulfide bond formation in this lectin. Hemagglutination assays demonstrated that all variants are inactive at inducing erythrocyte agglutination, even though gel filtration profiles indicate that C136S and C138S could still form dimers, suggesting that these dimers do not exhibit the same activity as wild-type (WT) Gal-13. In HeLa cells, the three variants were found to be distributed the same as with WT Gal-13. However, a Gal-13 variant (delT221) truncated at T221 could not be transported into the nucleus, possibly explaining why women having this variant get pre-eclampsia. Considering the normally high concentration of glutathione in cells, WT Gal-13 should exist mostly as a monomer in cytoplasm, consistent with the monomeric variant C136S/C138S, which has a similar ability to interact with HOXA1 as WT Gal-13.

Cross-strand disulfides in the hydrogen bonding site of antiparallel β-sheet (aCSDhs): Forbidden disulfides that are highly strained, easily broken.

Some disulfide bonds perform important structural roles in proteins, but another group has functional roles via redox reactions. Forbidden disulfides are stressed disulfides found in recognizable protein contexts, which currently constitute more than 10% of all disulfides in the PDB. They likely have functional redox roles and constitute a major subset of all redox-active disulfides. The torsional strain of forbidden disulfides is typically higher than for structural disulfides, but not so high as to render them immediately susceptible to reduction under physionormal conditions. Previously we characterized the most abundant forbidden disulfide in the Protein Data Bank, the aCSDn: a canonical motif in which disulfide-bonded cysteine residues are positioned directly opposite each other on adjacent anti-parallel β-strands such that the backbone hydrogen-bonded moieties are directed away from each other. Here we perform a similar analysis for the aCSDh, a less common motif in which the opposed cysteine residues are backbone hydrogen bonded. Oxidation of two Cys in this context places significant strain on the protein system, with the β-chains tilting toward each other to allow disulfide formation. Only left-handed aCSDh conformations are compatible with the inherent right-handed twist of β-sheets. aCSDhs tend to be more highly strained than aCSDns, particularly when both hydrogen bonds are formed. We discuss characterized roles of aCSDh motifs in proteins of the dataset, which include catalytic disulfides in ribonucleotide reductase and ahpC peroxidase as well as a redox-active disulfide in P1 lysozyme, involved in a major conformation change. The dataset also includes many binding proteins.

Expression of OsTPX Gene Improves Cellular Redox Homeostasis and Photosynthesis Efficiency in Synechococcus elongatus PCC 7942.

Cyanobacterial 2-Cys peroxiredoxin (thioredoxin peroxidase, TPX) comprises a family of thiol antioxidant enzymes critically involved in cell survival under oxidative stress. In our previous study, a putative TPX was identified using a proteomics analysis of rice (Oryza sativa L. japonica, OsTPX) seedlings exposed to oxidative stress. This OsTPX gene is structurally similar to the Synechococcus elongatus TPX gene in the highly conserved redox-active disulfide bridge (Cys114, Cys236) and other highly conserved regions. In the present study, the OsTPX gene was cloned into rice plants and S. elongatus PCC 7942 strain to study hydrogen peroxide (H2O2) stress responses. The OsTPX gene expression was confirmed using semi-quantitative RT-PCR and western blot analysis. The OsTPX gene expression increased growth under oxidative stress by decreasing reactive oxygen species and malondialdehyde level. Additionally, the OsTPX gene expression in S. elongatus PCC 7942 (OT) strain exhibited a reduced loss of chlorophyll and enhanced photosynthesis efficiency under H2O2 stress, thereby increasing biomass yields twofold compared with that of the control wild type (WT) strain. Furthermore, redox balance, ion homeostasis, molecular chaperone, and photosynthetic systems showed upregulation of some genes in the OT strain than in the WT strain by RNA-Seq analysis. Thus, OsTPX gene expression enhances oxidative stress tolerance by increasing cell defense regulatory networks through the cellular redox homeostasis in the rice plants and S. elongatus PCC 7942.

Crystal Structure of the Apo-Form of NADPH-Dependent Thioredoxin Reductase from a Methane-Producing Archaeon.

The redox regulation of proteins via reversible dithiol/disulfide exchange reactions involves the thioredoxin system, which is composed of a reductant, a thioredoxin reductase (TR), and thioredoxin (Trx). In the pyridine nucleotide-dependent Trx reduction pathway, reducing equivalents, typically from reduced nicotinamide adenine dinucleotide phosphate (NADPH), are transferred from NADPH-TR (NTR) to Trx and, in turn, to target proteins, thus resulting in the reversible modification of the structural and functional properties of the targets. NTR enzymes contain three functional sites: an NADPH binding pocket, a non-covalently bound flavin cofactor, and a redox-active disulfide in the form of CxxC. With the aim of increasing our knowledge of the thioredoxin system in archaea, we here report the high-resolution crystal structure of NTR from the methane-generating organism Methanosarcina mazei strain Gö1 (MmNTR) at 2.6 Å resolution. Based on the crystals presently described, MmNTR assumes an overall fold that is nearly identical to the archetypal fold of authentic NTRs; however, surprisingly, we observed no electron density for flavin adenine dinucleotide (FAD) despite the well-defined and conserved FAD-binding cavity in the folded module. Remarkably, the dimers of the apo-protein within the crystal were different from those observed by small angle X-ray scattering (SAXS) for the holo-protein, suggesting that the binding of the flavin cofactor does not require major protein structural rearrangements. Rather, binding results in the stabilization of essential parts of the structure, such as those involved in dimer stabilization. Altogether, this structure represents the example of an apo-form of an NTR that yields important insight into the effects of the cofactor on protein folding.

Mechanistic and structural insights into the in vitro inhibitory action of hypericin on glutathione reductase purified from baker's yeast.

This work aims at studying the interaction between glutathione reductase (GR) and hypericin. The type of inhibition was determined by measuring changes in GR activity at increasing concentrations of hypericin as well as at varying concentrations of glutathione disulfide (GSSG) and nicotinamide adenine dinucleotide phosphate (NADPH), and the binding pose of hypericin was predicted by molecular docking. Accordingly, hypericin emerges as an effective inhibitor of GR. When the variable substrate is GSSG, the type of inhibition is competitive. When the variable substrate is NADPH, however, the type of inhibition appears to be linear mixed-type competitive. Our computational analyses suggest that hypericin binds in the large intermonomer cavity of GR, and that it may interfere with the normal positioning/functioning of the redox-active disulfide center at the enzyme's active site. Overall, besides its contributory role in promoting oxidative stress via the formation of reactive oxygen species in photodynamic therapy, hypericin can also weaken cancer cells through inhibiting GR.

Allosteric control of human cystathionine β-synthase activity by a redox active disulfide bond

Cystathionine β-synthase (CBS) is the central enzyme in the trans-sulfuration pathway that converts homocysteine to cysteine. It is also one of the three major enzymes involved in the biogenesis of H2S. CBS is a complex protein with a modular three-domain architecture, the central domain of which contains a 272CXXC275 motif whose function has yet to be determined. In the present study, we demonstrated that the CXXC motif exists in oxidized and reduced states in the recombinant enzyme by mass spectroscopic analysis and a thiol labeling assay. The activity of reduced CBS is ∼2-3-fold greater than that of the oxidized enzyme, and substitution of either cysteine in CXXC motif leads to a loss of redox sensitivity. The Cys272-Cys275 disulfide bond in CBS has a midpoint potential of -314 mV at pH 7.4. Additionally, the CXXC motif also exists in oxidized and reduced states in HEK293 cells under oxidative and reductive conditions, and stressing these cells with DTT results in more reduced enzyme and a concomitant increase in H2S production in live HEK293 cells as determined using a H2S fluorescent probe. By contrast, incubation of cells with aminooxyacetic acid, an inhibitor of CBS and cystathionine γ-lyase, eliminated the increase of H2S production after the cells were exposed to DTT. These findings indicate that CBS is post-translationally regulated by a redox-active disulfide bond in the CXXC motif. The results also demonstrate that CBS-derived H2S production is increased in cells under reductive stress conditions.

Unprecedented pathway of reducing equivalents in a diflavin-linked disulfide oxidoreductase

Flavoproteins participate in a wide variety of physiologically relevant processes that typically involve redox reactions. Within this protein superfamily, there exists a group that is able to transfer reducing equivalents from FAD to a redox-active disulfide bridge, which further reduces disulfide bridges in target proteins to regulate their structure and function. We have identified a previously undescribed type of flavin enzyme that is exclusive to oxygenic photosynthetic prokaryotes and that is based on the primary sequence that had been assigned as an NADPH-dependent thioredoxin reductase (NTR). However, our experimental data show that the protein does not transfer reducing equivalents from flavins to disulfides as in NTRs but functions in the opposite direction. High-resolution structures of the protein from Gloeobacter violaceus and Synechocystis sp. PCC6803 obtained by X-ray crystallography showed two juxtaposed FAD molecules per monomer in redox communication with an active disulfide bridge in a variant of the fold adopted by NTRs. We have tentatively named the flavoprotein "DDOR" (diflavin-linked disulfide oxidoreductase) and propose that its activity is linked to a thiol-based transfer of reducing equivalents in bacterial membranes. These findings expand the structural and mechanistic repertoire of flavoenzymes with oxidoreductase activity and pave the way to explore new protein engineering approaches aimed at designing redox-active proteins for diverse biotechnological applications.

Essential role of the flexible linker on the conformational equilibrium of bacterial peroxiredoxin reductase for effective regeneration of peroxiredoxin

Reactive oxygen species (ROS) can damage DNA, proteins, and lipids, so cells have antioxidant systems that regulate ROS. In many bacteria, a dedicated peroxiredoxin reductase, alkyl hydroperoxide reductase subunit F (AhpF), catalyzes the rapid reduction of the redox-active disulfide center of the antioxidant protein peroxiredoxin (AhpC) to detoxify ROS such as hydrogen peroxide, organic hydroperoxide, and peroxynitrite. AhpF is a flexible multidomain protein that enables a series of electron transfers among the redox centers by accepting reducing equivalents from NADH. A flexible linker connecting the N-terminal domain (NTD) and C-terminal domain (CTD) of AhpF suggests that the enzyme adopts a large-scale domain motion that alternates between the closed and open states to shuttle electrons from the CTD via the NTD to AhpC. Here, we conducted comprehensive mutational, biochemical, and biophysical analyses to gain insights into the role of the flexible linker and the residues critical for the domain motions of Escherichia coli AhpF (EcAhpF) during electron transfer. Small-angle X-ray scattering studies of linker mutants revealed that a group of charged residues, 200EKR202, is crucial for the swiveling motion of the NTD. Moreover, NADH binding significantly affected EcAhpF flexibility and the movement of the NTD relative to the CTD. The mutants also exhibited a decrease in H2O2 reduction by the AhpF-AhpC ensemble. We propose that a concerted movement involving the NTD, C-terminal NADH, and FAD domains, and the flexible linker between them is essential for optimal intra-domain cross-talk and for efficient electron transfer to the redox partner AhpC required for peroxidation.

From structure to redox: The diverse functional roles of disulfides and implications in disease.

This review provides a comprehensive overview of the functional roles of disulfide bonds and their relevance to human disease.The critical roles of disulfide bonds in protein structure stabilization and redox regulation of protein activity are addressed.Disulfide bonds are essential to the structural stability of many proteins within the secretory pathway and can exist as intramolecular or inter-domain disulfides. The proper formation of these bonds often relies on folding chaperones and oxidases such as members of the protein disulfide isomerase (PDI) family. Many of the PDI family members catalyze disulfide-bond formation, reduction, and isomerization through redox-active disulfides and perturbed PDI activity is characteristic of carcinomas and neurodegenerative diseases. In addition to catalytic function in oxidoreductases, redox-active disulfides are also found on a diverse array of cellular proteins and act to regulate protein activity and localization in response to oxidative changes in the local environment. These redox-active disulfides are either dynamic intramolecular protein disulfides or mixed disulfides with small-molecule thiols generating glutathionylation and cysteinylation adducts. The oxidation and reduction of redox-active disulfides are mediated by cellular reactive oxygen species and activity of reductases, such as glutaredoxin and thioredoxin. Dysregulation of cellular redox conditions and resulting changes in mixed disulfide formation are directly linked to diseases such as cardiovascular disease and Parkinson's disease.

Chloroplast FBPase and SBPase are thioredoxin-linked enzymes with similar architecture but different evolutionary histories

The Calvin-Benson cycle of carbon dioxide fixation in chloroplasts is controlled by light-dependent redox reactions that target specific enzymes. Of the regulatory members of the cycle, our knowledge of sedoheptulose-1,7-bisphosphatase (SBPase) is particularly scanty, despite growing evidence for its importance and link to plant productivity. To help fill this gap, we have purified, crystallized, and characterized the recombinant form of the enzyme together with the better studied fructose-1,6-bisphosphatase (FBPase), in both cases from the moss Physcomitrella patens (Pp). Overall, the moss enzymes resembled their counterparts from seed plants, including oligomeric organization-PpSBPase is a dimer, and PpFBPase is a tetramer. The two phosphatases showed striking structural homology to each other, differing primarily in their solvent-exposed surface areas in a manner accounting for their specificity for seven-carbon (sedoheptulose) and six-carbon (fructose) sugar bisphosphate substrates. The two enzymes had a similar redox potential for their regulatory redox-active disulfides (-310 mV for PpSBPase vs. -290 mV for PpFBPase), requirement for Mg(2+) and thioredoxin (TRX) specificity (TRX f > TRX m). Previously known to differ in the position and sequence of their regulatory cysteines, the enzymes unexpectedly showed unique evolutionary histories. The FBPase gene originated in bacteria in conjunction with the endosymbiotic event giving rise to mitochondria, whereas SBPase arose from an archaeal gene resident in the eukaryotic host. These findings raise the question of how enzymes with such different evolutionary origins achieved structural similarity and adapted to control by the same light-dependent photosynthetic mechanism-namely ferredoxin, ferredoxin-thioredoxin reductase, and thioredoxin.

New Approach for Inhibition of HIV Entry: Modifying CD4 Binding Sites by Thiolated Pyrimidine Derivatives.

Thiolated pyrimidine derivatives have been synthetized and their antiretroviral effect against human immunodeficiency virus type 1 (HIV-1IIIB) and HIV-1 chimeric pseudovirions have been quantitatively determined in cell-based viral infectivity assays including syncytium inhibition assay as well as a single-cycle viral infection assay on HeLaCD4-LTR/ß-gal cells. Pseudotype virions prepared bearing HIV-1 envelope preference for CCR5 coreceptor, CXCR4 coreceptor or for both, respectively, with a HIV-1 core containing luciferase reporter gene were able to infect susceptible cells but are replication defective so unable to replicate in the cells . Data indicate that thiolated pyrimidine derivatives inhibited effectively virally induced cell fusion in vitro as well as infectivity of primary HIV-1IIIB strain and HIV-1 pseudovirions using chemokine receptors CCR5 or CXCR4 or both for virus entry a dose dependent manner. Inhibition was selective, depended on the pseudovirus coreceptor preference. Our results suggest that some of these sulfur containing pyrimidines interact with redoxactive -SH groups required for successful HIV entry, including a redox active disulfide in the CD4 molecule as well as -SH groups in HIV viral envelope gp120. This mode of action is unique representing a new class of potential HIV entry inhibitors.