Lateral roots (LR) are essential components of the plant edaphic interface; contributing to water and nutrient uptake, biotic and abiotic interactions, stress survival, and plant anchorage. We have identified the TETRATRICOPEPTIDE-REPEAT THIOREDOXIN-LIKE 3 (TTL3) gene as being related to LR emergence and later development. Loss of function of TTL3 leads to a reduced number of emerged LR due to delayed development of lateral root primordia (LRP). This trait is further enhanced in the triple mutant ttl1ttl3ttl4. TTL3 interacts with microtubules and endomembranes, and is known to participate in the brassinosteroid (BR) signaling pathway. Both ttl3 and ttl1ttl3ttl4 mutants are less sensitive to BR treatment in terms of LR formation and primary root growth. The ability of TTL3 to modulate biophysical properties of the cell wall was established under restrictive conditions of hyperosmotic stress and loss of root growth recovery, which was enhanced in ttl1ttl3ttl4. Timing and spatial distribution of TTL3 expression is consistent with its role in development of LRP before their emergence and subsequent growth of LR. TTL3 emerged as a component of the root system morphogenesis regulatory network.

• Klíčová slova
• TETRATRICOPEPTIDE-REPEAT THIOREDOXIN-LIKE 3, TTL3, brassinosteroids, cytoskeleton, lateral root, osmotic stress, tubulin,
• MeSH
• Arabidopsis * metabolismus   MeSH
• brassinosteroidy metabolismus   MeSH
• buněčná stěna metabolismus   MeSH
• cytoskelet metabolismus   MeSH
• kořeny rostlin metabolismus   MeSH
• membránové proteiny metabolismus   MeSH
• mikrotubuly metabolismus   MeSH
• proteiny huseníčku * genetika   metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• thioredoxiny metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• brassinosteroidy MeSH
• membránové proteiny MeSH
• proteiny huseníčku * MeSH
• thioredoxiny MeSH
• TTL protein, Arabidopsis MeSH Prohlížeč

The mitochondrial protein import machinery of trypanosomatids is highly divergent from that of the well-studied models such as baker's yeast. A notable example is that the central catalyst of the mitochondrial intermembrane space import and assembly pathway (MIA), named Mia40, is missing in trypanosomatids. Mia40 works in a two-step process. First it recognizes by direct binding reduced MIA substrate proteins and then catalyzes their oxidative folding to produce intramolecular disulfide bridges. It was recently proposed that a thioredoxin-like subunit of the trypanosomal mitochondrial contact site and cristae organizing system (MICOS) called TbMic20 may be the Mia40 replacement. Our study performed on procyclic stage of the parasite revealed that each of the two cysteines in TbMic20's active site is essential for the stability of MIA substrate proteins although they do not form a disulfide bridge in vivo. The two cysteines of Mia40's active site form an intramolecular disulfide bridge at steady state, which is a prerequisite for its oxidative folding of MIA substrates. Thus, we conclude that TbMic20 is unlikely to represent a bona fide Mia40 replacement and plays a still unresolved role in the stability and/or import of MIA substrates in trypanosomatids. Despite this, the effect of TbMic20 depletion and mutation indicates that the trypanosomal MICOS complex still plays a vital role in the maturation and/or stability of proteins imported by the MIA pathway.

• Klíčová slova
• Intermembrane space, MICOS, Mitochondrion, Oxidative folding, Protein import, Trypanosoma,
• MeSH
• chlorprofam metabolismus   MeSH
• cystein metabolismus   MeSH
• disulfidy MeSH
• mitochondriální importní komplex MeSH
• mitochondriální proteiny metabolismus   MeSH
• oxidace-redukce MeSH
• Saccharomyces cerevisiae - proteiny * genetika   MeSH
• Saccharomyces cerevisiae metabolismus   MeSH
• sbalování proteinů MeSH
• thioredoxiny genetika   metabolismus   MeSH
• transport proteinů MeSH
• transportní proteiny mitochondriální membrány genetika   MeSH
• transportní proteiny metabolismus   MeSH
• Trypanosoma brucei brucei * genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chlorprofam MeSH
• cystein MeSH
• disulfidy MeSH
• MIA40 protein, S cerevisiae MeSH Prohlížeč
• mitochondriální importní komplex MeSH
• mitochondriální proteiny MeSH
• Saccharomyces cerevisiae - proteiny * MeSH
• thioredoxiny MeSH
• transportní proteiny mitochondriální membrány MeSH
• transportní proteiny MeSH

Apoptosis signal-regulating kinase 1 (ASK1) is a ubiquitously expressed mitogen-activated protein kinase kinase kinase 5, which mediates various stress signals including oxidative stress. The catalytic activity of ASK1 is tightly controlled by oligomerization and binding of several cofactors. Among these cofactors, thioredoxin stands out as the most important ASK1 inhibitor, but only the reduced form of thioredoxin inhibits ASK1 by direct binding to its N-terminal domain. In addition, oxidation-driven thioredoxin dissociation is the key event in oxidative stress-mediated ASK1 activation. However, the structural mechanism of ASK1 regulation by thioredoxin remains unknown. Here, we report the characterization of the ASK1 domain responsible for thioredoxin binding and its complex using NMR spectroscopy and chemical cross-linking, thus providing the molecular basis for ASK1: thioredoxin complex dissociation under oxidative stress conditions. Our data reveal that the N-terminal domain of ASK1 adopts a fold resembling the thioredoxin structure while retaining substantial conformational plasticity at the thioredoxin-binding interface. Although oxidative stress induces relatively moderate structural changes in thioredoxin, the formation of intramolecular disulfide bridges leads to a considerable conformational rearrangement of the thioredoxin-binding interface on ASK1. Moreover, the cysteine residue at position 250 of ASK1 is the key element of this molecular switch. Finally, our results show that the redox-active site of thioredoxin is directly involved in ASK1 binding that is modulated by oxidative stress, thereby identifying a key target for the structure-based drug design.

• Klíčová slova
• apoptosis signal-regulating kinase 1, mitogen-activated protein kinase kinase kinase, oxidative stress, protein-protein interaction, thioredoxin,
• MeSH
• apoptóza * MeSH
• inhibitory proteinkinas farmakologie   MeSH
• lidé MeSH
• magnetická rezonanční spektroskopie MeSH
• MAP kinasa-kinasa-kinasa 5 antagonisté a inhibitory   metabolismus   MeSH
• molekulární modely MeSH
• oxidace-redukce MeSH
• oxidační stres * MeSH
• thioredoxiny chemie   metabolismus   MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• inhibitory proteinkinas MeSH
• MAP kinasa-kinasa-kinasa 5 MeSH
• MAP3K5 protein, human MeSH Prohlížeč
• thioredoxiny MeSH

Apoptosis signal-regulating kinase 1 (ASK1, also known as MAP3K5), a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, activates the p38 mitogen-activated protein kinase and the c-Jun N-terminal kinase (JNK) signaling cascades in response to various stressors. ASK1 activity is tightly regulated through phosphorylation and interaction with various binding partners. However, the mechanistic details underlying the ASK1 regulation are still not fully understood. This review focuses on recent advances in structural studies of protein kinase ASK1 and on the insights they provide into its mechanism of regulation. In addition, we also discuss protein-protein interactions between ASK1 and its binding partners thioredoxin (TRX) and 14-3-3 protein.

• MeSH
• biologické modely MeSH
• fosforylace MeSH
• lidé MeSH
• MAP kinasa-kinasa-kinasa 5 genetika   metabolismus   MeSH
• mitogenem aktivované proteinkinasy p38 genetika   metabolismus   MeSH
• proteiny 14-3-3 genetika   metabolismus   MeSH
• thioredoxiny genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• MAP kinasa-kinasa-kinasa 5 MeSH
• mitogenem aktivované proteinkinasy p38 MeSH
• proteiny 14-3-3 MeSH
• thioredoxiny MeSH

Mitochondrial electron transport chain (ETC) targeting shows a great promise in cancer therapy. It is particularly effective in tumors with high ETC activity where ETC-derived reactive oxygen species (ROS) are efficiently induced. Why modern ETC-targeted compounds are tolerated on the organismal level remains unclear. As most somatic cells are in non-proliferative state, the features associated with the ETC in quiescence could account for some of the specificity observed. Here we report that quiescent cells, despite increased utilization of the ETC and enhanced supercomplex assembly, are less susceptible to cell death induced by ETC disruption when glucose is not limiting. Mechanistically, this is mediated by the increased detoxification of ETC-derived ROS by mitochondrial antioxidant defense, principally by the superoxide dismutase 2 - thioredoxin axis. In contrast, under conditions of glucose limitation, cell death is induced preferentially in quiescent cells and is correlated with intracellular ATP depletion but not with ROS. This is related to the inability of quiescent cells to compensate for the lost mitochondrial ATP production by the upregulation of glucose uptake. Hence, elevated ROS, not the loss of mitochondrially-generated ATP, are responsible for cell death induction by ETC disruption in ample nutrients condition, e.g. in well perfused healthy tissues, where antioxidant defense imparts specificity. However, in conditions of limited glucose, e.g. in poorly perfused tumors, ETC disruption causes rapid depletion of cellular ATP, optimizing impact towards tumor-associated dormant cells. In summary, we propose that antioxidant defense in quiescent cells is aided by local glucose limitations to ensure selectivity of ETC inhibition-induced cell death.

• Klíčová slova
• Antioxidant defense, Electron transport chain, SOD2, Supercomplexes, Thioredoxin,
• MeSH
• adenosintrifosfát metabolismus   MeSH
• buněčná smrt genetika   MeSH
• buněčné dýchání MeSH
• buněčný cyklus genetika   MeSH
• elektronový transportní řetězec genetika   metabolismus   MeSH
• endoteliální buňky cytologie   metabolismus   MeSH
• epitelové buňky cytologie   metabolismus   MeSH
• exprese genu MeSH
• glukosa metabolismus   MeSH
• lidé MeSH
• mitochondrie metabolismus   MeSH
• nádorové buněčné linie MeSH
• oxidace-redukce MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• superoxiddismutasa genetika   metabolismus   MeSH
• thioredoxiny genetika   metabolismus   MeSH
• transformované buněčné linie MeSH
• transport elektronů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfát MeSH
• elektronový transportní řetězec MeSH
• glukosa MeSH
• reaktivní formy kyslíku MeSH
• superoxiddismutasa MeSH
• superoxide dismutase 2 MeSH Prohlížeč
• thioredoxiny MeSH
• TXN protein, human MeSH Prohlížeč

We describe the production of a highly-active mutant VEGF variant, α2-PI1-8-VEGF121, which contains a substrate sequence for factor XIIIa at the aminoterminus designed for incorporation into a fibrin gel. The α2-PI1-8-VEGF121 gene was synthesized, cloned into a pET-32a(+) vector and expressed in Escherichia coli Origami B (DE3) host cells. To increase the protein folding and the solubility, the resulting thioredoxin-α2-PI1-8-VEGF121 fusion protein was co-expressed with recombinant molecular chaperones GroES/EL encoded by independent plasmid pGro7. The fusion protein was purified from the soluble fraction of cytoplasmic proteins using affinity chromatography. After cleavage of the thioredoxin fusion part with thrombin, the target protein was purified by a second round of affinity chromatography. The yield of purified α2-PI1-8-VEGF121 was 1.4 mg per liter of the cell culture. The α2-PI1-8-VEGF121 expressed in this work increased the proliferation of endothelial cells 3.9-8.7 times in comparison with commercially-available recombinant VEGF121. This very high mitogenic activity may be caused by co-expression of the growth factor with molecular chaperones not previously used in VEGF production. At the same time, α2-PI1-8-VEGF121 did not elicit considerable inflammatory activation of human endothelial HUVEC cells and human monocyte-like THP-1 cells.

• MeSH
• chromatografie afinitní metody   MeSH
• endoteliální buňky pupečníkové žíly (lidské) MeSH
• Escherichia coli metabolismus   MeSH
• fibrin metabolismus   MeSH
• klonování DNA MeSH
• kultivované buňky MeSH
• lidé MeSH
• molekulární chaperony metabolismus   MeSH
• plazmidy metabolismus   MeSH
• rekombinantní fúzní proteiny metabolismus   MeSH
• rozpustnost MeSH
• sbalování proteinů MeSH
• sekvence aminokyselin MeSH
• thioredoxiny metabolismus   MeSH
• vaskulární endoteliální růstový faktor A metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fibrin MeSH
• molekulární chaperony MeSH
• rekombinantní fúzní proteiny MeSH
• thioredoxiny MeSH
• vaskulární endoteliální růstový faktor A MeSH
• VEGFA protein, human MeSH Prohlížeč

Apoptosis signal-regulating kinase 1 (ASK1, MAP3K5) activates p38 mitogen-activated protein kinase and the c-Jun N-terminal kinase in response to proinflammatory and stress signals. In nonstress conditions, ASK1 is inhibited by association with thioredoxin (TRX) which binds to the TRX-binding domain (ASK1-TBD) at the N terminus of ASK1. TRX dissociates in response to oxidative stress allowing the ASK1 activation. However, the molecular basis for the ASK1:TRX1 complex dissociation is still not fully understood. Here, the role of cysteine residues on the interaction between TRX1 and ASK1-TBD in both reducing and oxidizing conditions was investigated. We show that from the two catalytic cysteines of TRX1 the residue C32 is responsible for the high-affinity binding of TRX1 to ASK1-TBD in reducing conditions. The disulfide bond formation between C32 and C35 within the active site of TRX1 is the main factor responsible for the TRX1 dissociation upon its oxidation as the formation of the second disulfide bond between noncatalytic cysteines C62 and C69 did not have any additional effect. ASK1-TBD contains seven conserved cysteine residues which differ in solvent accessibility with the residue C250 being the only cysteine which is both solvent exposed and essential for TRX1 binding in reducing conditions. Furthermore, our data show that the catalytic site of TRX1 interacts with ASK1-TBD region containing cysteine C200 and that the oxidative stress induces intramolecular disulfide bond formation within ASK1-TBD and affects its structure in regions directly involved and/or important for TRX1 binding.

• Klíčová slova
• TRX, ASK1, cysteine, disulfide bond, mass spectrometry,
• MeSH
• cystein chemie   MeSH
• kinetika MeSH
• konformace proteinů MeSH
• lidé MeSH
• MAP kinasa-kinasa-kinasa 5 chemie   genetika   metabolismus   MeSH
• molekulární modely MeSH
• mutageneze cílená MeSH
• oxidace-redukce MeSH
• oxidační stres MeSH
• peptidové fragmenty chemie   genetika   metabolismus   MeSH
• proteinové domény MeSH
• rekombinantní proteiny chemie   genetika   metabolismus   MeSH
• substituce aminokyselin MeSH
• thioredoxiny chemie   genetika   metabolismus   MeSH
• vazebná místa genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cystein MeSH
• MAP kinasa-kinasa-kinasa 5 MeSH
• MAP3K5 protein, human MeSH Prohlížeč
• peptidové fragmenty MeSH
• rekombinantní proteiny MeSH
• thioredoxiny MeSH
• TXN protein, human MeSH Prohlížeč

BACKGROUND: Oxidative stress plays an important role in the pathogenesis of diabetes and its complications. The aim of this study was to examine the possible association between seven single nucleotide polymorphisms (SNPs) of the Trx2/TXNIP and TrxR2 genes encoding proteins involved in the thioredoxin antioxidant defence system and the risk of diabetic retinopthy (DR). DESIGN: Cross-sectional case-control study. PARTICIPANTS: A total of 802 Slovenian patients with Type 2 diabetes mellitus; 277 patients with DR and 525 with no DR were enrolled. METHODS: Patients genotypes of the SNPs; including rs8140110, rs7211, rs7212, rs4755, rs1548357, rs4485648 and rs5748469 were determined by the competitive allele specific PCR method. MAIN OUTCOME MEASURES: Each genotype of examined SNPs was regressed in a logistic model, assuming the co-dominant, dominant and the recessive models of inheritance with covariates of duration of diabetes, HbA1c, insulin therapy, total cholesterol and LDL cholesterol levels. RESULTS: In the present study, for the first time we identified an association between the rs4485648 polymorphism of the TrxR2 gene and DR in Caucasians with Type 2 DM. The estimated ORs of adjusted logistic regression models were found to be as follows: 4.4 for CT heterozygotes, 4.3 for TT homozygotes (co-dominant genetic model) and 4.4 for CT+TT genotypes (dominant genetic model). CONCLUSIONS: In our case-control study we were not able to demonstrate any association between rs8140110, rs7211, rs7212, rs4755, rs1548357, and rs5748469 and DR, however, our findings provide evidence that the rs4485648 polymorphism of the TrxR2 gene might exert an independent effect on the development of DR.

• Klíčová slova
• Diabetic retinopathy, Oxidative stress, Single nucleotide polymorphism, Thioredoxin antioxidant system, Type 2 diabetes mellitus,
• MeSH
• diabetes mellitus 2. typu komplikace   genetika   MeSH
• diabetická retinopatie genetika   MeSH
• genetická predispozice k nemoci MeSH
• genetické asociační studie MeSH
• jednonukleotidový polymorfismus MeSH
• lidé středního věku MeSH
• lidé MeSH
• metabolické sítě a dráhy genetika   MeSH
• mitochondriální proteiny genetika   MeSH
• mitochondrie genetika   metabolismus   MeSH
• průřezové studie MeSH
• rizikové faktory MeSH
• senioři MeSH
• studie případů a kontrol MeSH
• thioredoxinreduktasa 2 genetika   MeSH
• thioredoxiny genetika   metabolismus   MeSH
• transportní proteiny genetika   MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• mitochondriální proteiny MeSH
• thioredoxinreduktasa 2 MeSH
• thioredoxiny MeSH
• transportní proteiny MeSH
• TXN2 protein, human MeSH Prohlížeč
• TXNIP protein, human MeSH Prohlížeč
• TXNRD2 protein, human MeSH Prohlížeč

Apoptosis signal-regulating kinase 1 (ASK1), a mitogen-activated protein kinase kinase kinase, plays a key role in the pathogenesis of multiple diseases. Its activity is regulated by thioredoxin (TRX1) but the precise mechanism of this regulation is unclear due to the lack of structural data. Here, we performed biophysical and structural characterization of the TRX1-binding domain of ASK1 (ASK1-TBD) and its complex with reduced TRX1. ASK1-TBD is a monomeric and rigid domain that forms a stable complex with reduced TRX1 with 1:1 molar stoichiometry. The binding interaction does not involve the formation of intermolecular disulfide bonds. Residues from the catalytic WCGPC motif of TRX1 are essential for complex stability with Trp(31) being directly involved in the binding interaction as suggested by time-resolved fluorescence. Small-angle x-ray scattering data reveal a compact and slightly asymmetric shape of ASK1-TBD and suggest reduced TRX1 interacts with this domain through the large binding interface without inducing any dramatic conformational change.

• Klíčová slova
• Analytical Ultracentrifugation, Apoptosis Signal-regulating Kinase 1 (ASK1), Circular Dichroism (CD), Fluorescence, Small-angle X-ray Scattering (SAXS), Thioredoxin,
• MeSH
• biofyzika MeSH
• cirkulární dichroismus MeSH
• fluorescenční spektrometrie MeSH
• konformace proteinů MeSH
• MAP kinasa-kinasa-kinasa 5 metabolismus   MeSH
• oxidace-redukce MeSH
• thioredoxiny metabolismus   MeSH
• ultracentrifugace MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• MAP kinasa-kinasa-kinasa 5 MeSH
• thioredoxiny MeSH

Tuberculosis remains a major health concern worldwide. Eradication of its causative agent, the bacterial pathogen Mycobacterium tuberculosis, is particularly challenging due to a vast reservoir of latent carriers of the disease. Despite the misleading terminology of a so-called dormant state associated with latent infections, the bacteria have to maintain basic metabolic activities. Hypoxic conditions have been widely used as an in vitro system to study this dormancy. Such studies identified a rearrangement of central carbon metabolism to exploit fermentative processes caused by the lack of oxygen. Phosphoenolpyruvate carboxykinase (Pck; EC 4.1.1.32) is the enzyme at the center of these metabolic rearrangements. Although Pck is associated with gluconeogenesis under standard growth conditions, the enzyme can catalyze the reverse reaction, supporting anaplerosis of the tricarboxylic acid cycle, under conditions leading to slowed or stopped bacterial replication. To study the mechanisms that regulate the switch between two Pck functions, we systematically investigated factors influencing the gluconeogenic and anaplerotic reaction kinetics. We demonstrate that a reducing environment, as found under hypoxia-triggered non-replicating conditions, accelerates the reaction in the anaplerotic direction. Furthermore, we identified proteins that interact with Pck. The interaction between Pck and the reduced form of mycobacterial thioredoxin, gene expression of which is increased under hypoxic conditions, also increased the Pck anaplerotic activity. We thus propose that a reducing environment and the protein-protein interaction with thioredoxin in particular enable the Pck anaplerotic function under fermentative growth conditions.

• Klíčová slova
• Enzyme Kinetics, Hypoxia, Metabolism, Mycobacterium Tuberculosis, Oxidation-Reduction, Phosphoenolpyruvate Carboxykinase, Thioredoxin,
• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• citrátový cyklus fyziologie   MeSH
• fosfoenolpyruvátkarboxykinasa (závislá na ATP) genetika   metabolismus   MeSH
• Mycobacterium tuberculosis enzymologie   genetika   MeSH
• oxidace-redukce MeSH
• regulace genové exprese enzymů fyziologie   MeSH
• regulace genové exprese u bakterií fyziologie   MeSH
• thioredoxiny genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• fosfoenolpyruvátkarboxykinasa (závislá na ATP) MeSH
• thioredoxiny MeSH