Procongopain from Trypanosoma congolense is processed at basic pH: an unusual feature among cathepsin L-like cysteine proteases

Congopain, the major cysteine protease from Trypanosoma congolense, is synthesized as an inactive zymogen, and further converted into its active form after removal of the proregion, most probably via an autocatalytic mechanism. Processing of recombinant procongopain occurs via an apparent one-step or a multistep mechanism depending on the ionic strength. The auto-activation is pH-dependent, with an optimum at pH 4.0, and no activation observed at pH 6.0. After addition of dextran sulfate (10 microg/ml), an approx. 20-fold increase of processing (expressed as enzymatic activity) is observed. Furthermore, in the presence of dextran sulfate, procongopain can be processed at pH 8.0, an unusual feature among papain-like enzymes. Detection of procongopain and trypanosomal enzymatic activity in the plasma of T. congolense-infected cattle, together with the capacity of procongopain to be activated at weakly basic pH, suggest that procongopain may be extracellularly processed in the presence of blood vessel glycosaminoglycans, supporting the hypothesis that congopain acts as a pathogenic factor in host-parasite relationships.     

HIV-1(Lai) genomic RNA: combined used of NMR and molecular dynamics simulation for studying the structure and internal dynamics of a mutated SL1 hairpin

The genome of all retroviruses consists of two identical copies of an RNA sequence associated in a non-covalent dimer. A region upstream from the splice donor (SL1) comprising a self-complementary sequence is responsible for the initiation of the dimerization. This region is able to dimerize in two conformations: a loop-loop complex or an extended duplex. Here, we solve by 2D NMR techniques the solution structure of a 23-nucleotide sequence corresponding to HIV-1 SL1(Lai) in which the mutation G12-->A12 is included to prevent dimerization. It is shown that this monomer adopts a stem-loop conformation with a seven base pairs stem and a nine nucleotide loop containing the G10 C11 A12 C13 G14 C15 sequence. The stem is well structured in an A-form duplex, while the loop is more flexible even though elements of structure are evident. We show that the structure adopted by the stem can be appreciably different from its relaxed structure when the adenines A8, A9 and A16 in the loop are mechanically constrained. This point could be important for the efficiency of the dimerization. This experimental study is complemented with a 10 ns molecular dynamics simulation in the presence of counterions and explicit water molecules. This simulation brings about information on the flexibility of the loop, such as a hinge motion between the stem and the loop and a labile lattice of hydrogen bonds in the loop. The bases of the nucleotides G10 to C15 were found outside of the loop during a part of the trajectory, which is certainly necessary to initiate the dimerization process of the genuine SL1(Lai) sequence.     

An xrcc4 defect or Wortmannin stimulates homologous recombination specifically induced by double-strand breaks in mammalian cells

Non-homologous end joining (NHEJ) and homologous recombination (HR) are two alternative/competitor pathways for the repair of DNA double-strand breaks (DSBs). To gain further insights into the regulation of DSB repair, we detail here the different HR pathways affected by (i) the inactivation of DNA-PK activity, by treatment with Wortmannin, and (ii) a mutation in the xrcc4 gene, involved in a late NHEJ step, using the XR-1 cell line. Here we have analyzed not only the impact of NHEJ inactivation on recombination induced by a single DSB targeted to the recombination substrate (using I-SceI endonuclease) but also on γ-ray- and UV-C-induced and spontaneous recombination and finally on Rad51 foci formation, i.e. on the assembly of the homologous recombination complex, at the molecular level. The results presented here show that in contrast to embryonic stem cells, the xrcc4 mutation strongly stimulates I-SceI-induced HR in adult hamster cells. More precisely, we show here that both single strand annealing and gene conversion are stimulated. In contrast, Wortmannin does not affect I-SceI-induced HR. In addition, γ-ray-induced recombination is stimulated by both xrcc4 mutation and Wortmannin treatment in an epistatic-like manner. In contrast, neither spontaneous nor UV-C-induced recombination was affected by xrcc4 mutation, showing that the channeling from NHEJ to HR is specific to DSBs. Finally, we show here that xrcc4 mutation or Wortmannin treatment results in a stimulation of Rad51 foci assembly, thus that a late NHEJ step is able to affect Rad51 recombination complex assembly. The present data suggest a model according to which NHEJ and HR do not simply compete for DSB repair but can act sequentially: a defect in a late NHEJ step is not a dead end and can make DSB available for subsequent Rad51 recombination complex assembly.     

Gene expression profile in human late radiation enteritis obtained by high-density cDNA array hybridization

Late radiation enteritis is a sequela of radiation therapy to the abdomen. The pathogenic process is poorly understood at the molecular level. cDNA array analysis was used to provide new insights into the pathogenesis of this disorder. Gene profiles of six samples of fibrotic bowel tissue from patients with radiation enteritis and six healthy bowel tissue samples from patients without radiation enteritis were compared using membrane-based arrays containing 1314 cDNAs. Results were confirmed with real-time RT-PCR and Western blot analysis. Array analysis identified many differentially expressed genes involved in fibrosis, stress response, inflammation, cell adhesion, intracellular and nuclear signaling, and metabolic pathways. Increased expression of genes coding for proteins involved in the composition and remodeling of the extracellular matrix, along with altered expression of genes involved in cell- to-cell and cell-to-matrix interactions, were observed mainly in radiation enteritis samples. Stress, inflammatory responses, and antioxidant metabolism were altered in radiation enteritis as were genes coding for recruitment of lymphocytes and macrophages. The Rho/HSP27 (HSPB1)/zyxin pathway, involved in tissue contraction and myofibroblast transdifferentiation, was also altered in radiation enteritis, suggesting that this pathway could be related to the fibrogenic process. Our results provide a global and integrated view of the alteration of gene expression associated with radiation enteritis. They suggest that radiation enteritis is a dynamic process involving constant remodeling of each structural component of the intestinal tissue, i.e. the mucosa, the mesenchyme, and blood vessels. Functional studies will be necessary to validate the present results.     

Putative membrane assembly of EtpM-colicin V chimeras

EtpM of the enterohemorrhagic E. coli O157:H7 is a bitopic membrane protein of the type II protein secretion apparatus. There is a twin-arginine (RR) motif in front of its signal anchor, suggesting a Tat-dependent membrane targeting of EtpM. By exploiting the periplasmic bactericidal activity of colicin V (ColV), we constructed EtpM-ColV fusions and studied the EtpM-mediated translocation of ColV. The wild type strain and the DeltatatC mutant were killed by the expressed fusions and were fully protected from the killing effect by the ColV-specific immunity protein. In contrast, cold-inactivation of YidC, which is generally required for integral membrane protein assembly, significantly attenuated the killing effect in the cold-sensitive yidC mutant. These results confirmed the predicted N(in)-C(out) EtpM topology, and suggests an EtpM-mediated, Tat-independent and YidC-dependent translocation of ColV.     

Rational design of lipid for membrane protein crystallization

The lipidic cubic phase has been used to grow crystals of membrane proteins for high-resolution structure determination. However, the original, so-called, in meso method does not work reliably at low temperatures, where proteins are generally more stable, because the hosting lipid turns solid. The need existed therefore for a lipid that forms the cubic phase and that supports crystal growth at low temperatures. We created a database of phase diagrams and used it to design such a lipid. X-ray diffraction showed that the new lipid exhibits designed phase behavior. Further, it produces diffraction quality membrane protein crystals by the in meso method at 6 degrees C. This demonstrates that lipidic materials, like their protein counterparts are amenable to rational design. The same approach as used in this study should find application in extending the range of membrane proteins crystallizable by the in meso method and in tailoring transport of cubic phases for controlled delivery and uptake.     

RNase HI overproduction is required for efficient full-length RNA synthesis in the absence of topoisomerase I in Escherichia coli

It has long been known that Escherichia coli cells deprived of topoisomerase I (topA null mutants) do not grow. Because mutations reducing DNA gyrase activity and, as a consequence, negative supercoiling, occur to compensate for the loss of topA function, it has been assumed that excessive negative supercoiling is somehow involved in the growth inhibition of topA null mutants. However, how excess negative supercoiling inhibits growth is still unknown. We have previously shown that the overproduction of RNase HI, an enzyme that degrades the RNA portion of an R-loop, can partially compensate for the growth defects because of the absence of topoisomerase I. In this article, we have studied the effects of gyrase reactivation on the physiology of actively growing topA null cells. We found that growth immediately and almost completely ceases upon gyrase reactivation, unless RNase HI is overproduced. Northern blot analysis shows that the cells have a significantly reduced ability to accumulate full-length mRNAs when RNase HI is not overproduced. Interestingly, similar phenotypes, although less severe, are also seen when bacterial cells lacking RNase HI activity are grown and treated in the same way. All together, our results suggest that excess negative supercoiling promotes the formation of R-loops, which, in turn, inhibit RNA synthesis.     

Thermodynamic and spectroscopic studies of copper (II) complexes with three bis(amide) ligands derived from L-tartaric acid

The interactions of three bis(amide) ligands derived from tartaric acid with copper (II) were investigated in aqueous solution by a combination of potentiometry, UV-vis spectrophotometry, electron paramagnetic resonance (EPR), and mass spectrometry. The formation constants of the complexes were measured and their relative structures were reported. The sites of complexation of these ligands are investigated based mostly on their electronic and EPR spectra and on the comparison with the behaviour of some analog compounds.     

Phylogenomic Analysis of the PEBP Gene Family in Cereals

The TFL1 and FT genes, which are key genes in the control of flowering time in Arabidopsis thaliana, belong to a small multigene family characterized by a specific phosphatidylethanolamine-binding protein domain, termed the PEBP gene family. Several PEBP genes are found in dicots and monocots, and act on the control of flowering time. We investigated the evolution of the PEBP gene family in cereals. First, taking advantage of the complete rice genome sequence and EST databases, we found 19 PEBP genes in this species, 6 of which were not previously described. Ten genes correspond to five pairs of paralogs mapped on known duplicated regions of the rice genome. Phylogenetic analysis of Arabidopsis and rice genes indicates that the PEBP gene family consists of three main homology classes (the so-called TFL1-LIKE, MFT-LIKE, and FT-LIKE subfamilies), in which gene duplication and/or loss occurred independently in Arabidopsis and rice. Second, phylogenetic analyses of genomic and EST sequences from five cereal species indicate that the three subfamilies of PEBP genes have been conserved in cereals. The tree structure suggests that the ancestral grass genome had at least two MFT-like genes, two TFL1-like genes, and eight FT-like genes. A phylogenomic approach leads to some hypotheses about conservation of gene function within the subfamilies. [Reviewing Editor: Dr. Yves Van de Peer]