A New Scaffold of an Old Protein Fold Ensures Binding to the Bisintercalator Thiocoraline

Thiocoraline is a thiodepsipeptide with potent antitumor activity. TioX, a protein with an unidentified function, is encoded by a gene of the thiocoraline biosynthetic gene cluster. The crystal structure of the full-length TioX protein at 2.15 Å resolution reveals that TioX protomer shares an ancient βαβββ fold motif with glyoxalase I and bleomycin resistance protein families, despite a very low sequence homology. Intriguingly, four TioX monomers form a unique 2-fold symmetric tetrameric assembly that is stabilized by four intermolecular disulfide bonds formed cyclically between Cys60 and Cys66 of adjacent monomers. The arrangement of two of the four monomers in the TioX tetramer is analogous to that in dimeric bleomycin resistance proteins. This analogy indicates that this novel higher-order structural scaffold of TioX may have evolved to bind thiocoraline. Our equilibrium titration studies demonstrate the binding of a thiocoraline chromophore analog, quinaldic acid, to TioX, thereby substantiating this model. Furthermore, a strain of Streptomyces albus containing an exogenous thiocoraline gene cluster devoid of functional tioX maintains thiocoraline production, albeit with a lower yield. Taken together, these observations rule out a direct enzymatic function of TioX and suggest that TioX is involved in thiocoraline resistance or secretion.     

Cutting edge: abortive proliferation of CD46-induced Tr1-like cells due to a defective Akt/Survivin signaling pathway

T regulatory cell 1 (Tr1) are low proliferating peripherally induced suppressive T cells. Engaging CD3 and CD46 on human CD4+ T cells induces a Tr1-like phenotype. In this study, we report that human Tr1-like cells do not sustain proliferation over time. The weak proliferation of these cells results first from their inability to sustain expression of various cell cycle-associated proteins, to efficiently degrade the inhibitor of cell cycle progression p27/Kip1 and, as a consequence, in their accumulation in the G0-G1 phase. Also, the reduced proliferation of Tr1-like cells results from their increased sensitivity to death as they divide, through a mechanism that is neither Fas-mediated nor Bcl2/Bcl-xL related. Both properties, impaired cell cycle and death sensitivity, are explained by a specific defective activation of Akt that impairs the expression of Survivin. Thus, our results show that CD3/CD46-induced Tr1-like cells die through a process of abortive proliferation.     

Proapoptotic effects of P. aeruginosa involve inhibition of surfactant phosphatidylcholine synthesis

Pseudomonas aeruginosa causes sepsis-induced acute lung injury, a disorder associated with deficiency of surfactant phosphatidylcholine (PtdCho). P. aeruginosa (PA103) utilizes a type III secretion system (TTSS) to induce programmed cell death. Herein, we observed that PA103 reduced alveolar PtdCho levels, resulting in impaired lung biophysical activity, an effect partly attributed to caspase-dependent cleavage of the key PtdCho biosynthetic enzyme, CTP:phosphocholine cytidylyltransferase-alpha (CCTalpha). Expression of recombinant CCTalpha variants harboring point mutations at putative caspase cleavage sites in murine lung epithelia resulted in partial proteolytic resistance of CCTalpha to PA103. Further, caspase-directed CCTalpha degradation, decreased PtdCho levels, and cell death in murine lung epithelia were lessened after exposure of cells to bacterial strains lacking the TTSS gene product, exotoxin U (ExoU), but not ExoT. These observations suggest that during the proapoptotic program driven by P. aeruginosa, deleterious effects on phospholipid metabolism are mediated by a TTSS in concert with caspase activation, resulting in proteolysis of a key surfactant biosynthetic enzyme.     

Oxygen enhances lethal effect of high-intensity, ultrashort electrical pulses

The study explored the effect of ambient oxygen on mammalian cell survival after exposure to 10 ns duration, high voltage electrical pulses (nsEP, 80-90 or 120-130 kV/cm; 200-400 pulses per exposure). Cell samples were equilibrated with pure nitrogen, atmospheric air, or pure oxygen prior to the nsEP treatment and were returned to the incubator (air + 5% CO2) shortly after the exposure. The experiments established that survival of hypoxic Jurkat and U937 cells exceeded that of air-equilibrated controls about twofold (P < .01). Conversely, saturation of the medium with oxygen prior to exposure decreased Jurkat cell survival about 1.5 times, P < .01. Attenuation of the cytotoxic effect under hypoxic conditions resembled a well-known effect of oxygen on cell killing by sparsely ionizing radiations and may be indicative of the similarity of underlying cell damage mechanisms.     

Acetohydroxyacid synthase isozyme I from Escherichia coli has unique catalytic and regulatory properties

AHAS I is an isozyme of acetohydroxyacid synthase which is apparently unique to enterobacteria. It has been known for over 20 years that it has many properties which are quite different from those of the other two enterobacterial AHASs isozymes, as well as from those of “typical” AHASs which are single enzymes in a given organism. These include a unique mechanism for regulation of expression and the absence of a preference for forming acetohydroxybutyrate. We have cloned the two subunits, ilvB and ilvN, of this Escherichia coli isoenzyme and examined the enzymatic properties of the purified holoenzyme and the enzyme reconstituted from purified subunits. Unlike other AHASs, AHAS I demonstrates cooperative feedback inhibition by valine, and the kinetics fit closely to an exclusive binding model. The formation of acetolactate by AHAS I is readily reversible and acetolactate can act as substrate for alternative AHAS I-catalyzed reactions.     

Karyological evidence for meiosis in the three different types of life cycles existing in Agaricus bisporus

In Agaricus bisporus all cytological studies performed until now concerned the pseudohomothallic and bisporic var. bisporus. In the past 12 y two tetrasporic varieties have been described, the heterothallic var. burnettii and the homothallic var. eurotetrasporus. Our aim was to compare the behavior of the nuclei in the vegetative and reproductive cells of the three varieties with light microscopy (Feulgen and DAPI staining) and transmission electron microscopy. Most of the vegetative cells contained 3-5 nuclei in the three varieties. Nuclear migrations through the septum were detected. In the basidia relative locations of nuclei and vacuoles, meiotic spindle alignments, relative content of nuclear DNA and synaptonemal complexes were measured or observed. From the observation of numerous asynchronous second division of meiosis within basidia of var. bisporus and var. burnettii a new hypothesis emerges to explain the nonrandom distribution of the four meiotic products in the two spores of the bisporic basidia. Karyogamy and meiosis similarly occurred in the three varieties. In the case of A. bisporus var. eurotetrasporus this implies that the reproductive mode is sexual and therefore homothallic in the strict sense. The three different types of life cycles are described.     

Detection and quantification of unbound phytochelatin 2 in plant extracts of Brassica napus grown with different levels of mercury

The mercury (Hg) accumulation mechanism was studied in rape (Brassica napus) plants grown under a Hg concentration gradient (0 microm-1,000 microm). Hg mainly accumulated in roots. Therefore, the presence of phytochelatins (PCs) was studied in the roots of the plants. The high stability of the PC-Hg multicomplexes (mPC-nHg) seems to be the main reason for the lack of previous Hg-PC characterization studies. We propose a modification of the method to detect and quantify unbound PC of Hg in plant extracts via high-performance liquid chromatography coupled to electrospray tandem mass spectrometry and inductively coupled plasma mass spectrometry in parallel. We separated the PC from the Hg by adding the chelating agent sodium 2,3-dimercaptopropanesulfonate monohydrate. We only detected the presence of PC after the addition of the chelating agent. Some multicomplexes mPC-nHg could be formed but, due to their large sizes, could not be detected. In this study, only PC(2) was observed in plant samples. Hg accumulation was correlated with PC(2) concentration (r(2) = 0.98).