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1.  The conformational bases for the two functionalities of 2-cysteine peroxiredoxins as peroxidase and chaperone 
Journal of Experimental Botany  2013;64(11):3483-3497.
2-Cysteine peroxiredoxins (2-CysPrxs) are ubiquitous and highly abundant proteins that serve multiple functions as peroxidases, chaperones, and thiol oxidases and in redox-dependent cell signalling. The chloroplast protein plays a role in seedling development and protection of the photosynthetic apparatus. This study aimed to unequivocally link conformation and function. To this end, a set of non-tagged site-directed mutagenized At2-CysPrx variants was engineered, which mimicked the conformational states and their specific functions: hyperoxidized form (C54D), reduced form (C54S, C176S), oxidized form (C54DC176K), phosphorylated form (T92D), reduced ability for oligomerization by interfering with the dimer–dimer interface (F84R) and a C-terminally truncated form [ΔC (–20 aa)]. These variants were fully or partly fixed in their quaternary structure and function, respectively, and were analysed for their conformational state and peroxidase and chaperone activity, as well as for their sensitivity to hyperoxidation. The presence of a His6-tag strongly influenced the properties of the protein. The ΔC variant became insensitive to hyperoxidation, while T92D and F84R became more sensitive. The C54D variant revealed the highest chaperone activity. The highest peroxidase activity was observed for the F84R and ΔC variants. Efficient interaction with NADP-dependent thioredoxin reductase C depended on the presence of Cys residues and the C-terminal tail. The results suggest that the structural flexibility is important for the switch between peroxidase and chaperone function and that evolution has conserved the functional switch instead of maximizing a single function. These variants are ideal tools for future conformation-specific studies in vivo and in vitro.
doi:10.1093/jxb/ert184
PMCID: PMC3733160  PMID: 23828546
Chaperone; chloroplast; conformation; peroxidase; peroxiredoxin; thiol switch.
2.  A comparative study of type I and type II tryparedoxin peroxidases in Leishmania major 
The FEBS journal  2007;274(21):5643-5658.
Summary
The genome of Leishmania major, the causative agent of cutaneous Leishmaniasis, contains three nearly identical genes encoding putative glutathione peroxidases, which only differ at their N- and C-termini. Since the gene homologues are essential in trypanosomes, they may also represent potential drug targets in leishmania. Recombinant protein for the shortest of these showed negligible peroxidase activity with glutathione as electron donor indicating that it is not a bone fide glutathione peroxidase. In contrast, high peroxidase activity was obtained with tryparedoxin indicating that these proteins belong to a new class of monomeric tryparedoxin-dependent peroxidases (TDPX) distinct from the classical decameric 2-Cys peroxiredoxins (TryP). Mass spectrometry studies revealed that oxidation of TDPX1 with peroxides results in formation of an intramolecular disulphide bridge between Cys35 and Cys83. Site-directed mutagenesis and kinetic studies showed that Cys35 is essential for peroxidase activity whereas Cys83 is essential for reduction by tryparedoxin. Detailed kinetic studies comparing TDPX1 and TryP1 showed that both enzymes obey saturation ping pong kinetics with respect to tryparedoxin and peroxide. Both enzymes show high affinity for tryparedoxin and broad substrate specificity for hydroperoxides. TDPX1 shows higher affinity towards hydrogen peroxide and cumene hydroperoxide than to t-butyl hydroperoxide whereas no specific substrate preference could be detected for TryP1. TDPX1 exhibits rate constants up to 8 × 104 M−1s−1 whereas TryP1 exhibits higher rate constants ~106 M−1s−1. All three TDPX proteins together constitute about 0.05 % of the Leishmania major promastigote protein content whereas the TryPs are ~40 times more abundant. Possible specific functions of TDPXs are discussed.
doi:10.1111/j.1742-4658.2007.06087.x
PMCID: PMC3430366  PMID: 17922848
Glutathione peroxidase; tryparedoxin peroxidase; peroxiredoxin; trypanothione; Leishmania
3.  Antitumor Quinol PMX464 Is a Cytocidal Anti-trypanosomal Inhibitor Targeting Trypanothione Metabolism* 
The Journal of Biological Chemistry  2011;286(10):8523-8533.
Better drugs are urgently needed for the treatment of African sleeping sickness. We tested a series of promising anticancer agents belonging to the 4-substituted 4-hydroxycyclohexa-2,5-dienones class (“quinols”) and identified several with potent trypanocidal activity (EC50 < 100 nm). In mammalian cells, quinols are proposed to inhibit the thioredoxin/thioredoxin reductase system, which is absent from trypanosomes. Studies with the prototypical 4-benzothiazole-substituted quinol, PMX464, established that PMX464 is rapidly cytocidal, similar to the arsenical drug, melarsen oxide. Cell lysis by PMX464 was accelerated by addition of sublethal concentrations of glucose oxidase implicating oxidant defenses in the mechanism of action. Whole cells treated with PMX464 showed a loss of trypanothione (T(SH)2), a unique dithiol in trypanosomes, and tryparedoxin peroxidase (TryP), a 2-Cys peroxiredoxin similar to mammalian thioredoxin peroxidase. Enzyme assays revealed that T(SH)2, TryP, and a glutathione peroxidase-like tryparedoxin-dependent peroxidase were inhibited in time- and concentration-dependent manners. The inhibitory activities of various quinol analogues against these targets showed a good correlation with growth inhibition of Trypanosoma brucei. The monothiols glutathione and l-cysteine bound in a 2:1 ratio with PMX464 with Kd values of 6 and 27 μm, respectively, whereas T(SH)2 bound more tightly in a 1:1 ratio with a Kd value of 430 nm. Overexpression of trypanothione synthetase in T. brucei decreased sensitivity to PMX464 indicating that the key metabolite T(SH)2 is a target for quinols. Thus, the quinol pharmacophore represents a novel lead structure for the development of a new drug against African sleeping sickness.
doi:10.1074/jbc.M110.214833
PMCID: PMC3048735  PMID: 21212280
Drug Action; Metabolism; Peroxidase; Thiol; Trypanosome; Quinol; Trypanothione; Tryparedoxin Peroxidase
4.  Structural and mechanistic insights into type II trypanosomatid tryparedoxin-dependent peroxidases 
Biochemical Journal  2008;414(Pt 3):375-381.
TbTDPX (Trypanosoma brucei tryparedoxin-dependent peroxidase) is a genetically validated drug target in the fight against African sleeping sickness. Despite its similarity to members of the GPX (glutathione peroxidase) family, TbTDPX2 is functional as a monomer, lacks a selenocysteine residue and relies instead on peroxidatic and resolving cysteine residues for catalysis and uses tryparedoxin rather than glutathione as electron donor. Kinetic studies indicate a saturable Ping Pong mechanism, unlike selenium-dependent GPXs, which display infinite Km and Vmax values. The structure of the reduced enzyme at 2.1 Å (0.21 nm) resolution reveals that the catalytic thiol groups are widely separated [19 Å (0.19 nm)] and thus unable to form a disulphide bond without a large conformational change in the secondary-structure architecture, as reported for certain plant GPXs. A model of the oxidized enzyme structure is presented and the implications for small-molecule inhibition are discussed.
doi:10.1042/BJ20080889
PMCID: PMC2552391  PMID: 18522537
dithiol-dependent peroxidase; drug discovery; glutathione peroxidase; Leishmania; Trypanosoma; trypanothione; GPX, glutathione peroxidase; His6, hexahistidine; Lm, Leishmania major; PEG, poly(ethylene glycol); Pt, Populus trichocarpaxdeltoides (hybrid poplar); r.m.s.d., root mean square deviation; Tb, Trypanosoma brucei; TDPX, tryparedoxin-dependent peroxidase; TryX, tryparedoxin

Results 1-4 (4)