Molecular cloning and characterization of a thioredoxin from Taiwanofungus camphorata

Abstract Background Thioredoxin (Trx) is reduced by thioredoxin reductase. Trx is used in ribonucleoide reduction, assimilatory sulfate reduction, in modulation of protein sulfhydryl groups, and refolding proteins. Results A TcTrx (Tc: Taiwanofungus camphorata) cDNA (640 bp, GenBank AY838902.1) encoding a putative thioredoxin (Trx) of 135 amino acid residues with calculated molecular mass of 16.17 kDa was cloned from Taiwanofungus c amphorata. The deduced amino acid sequence containing a motif (Cys-Gly-Pro-Cys) that is highly conserved among the reported Trxs. A three dimensional structural model of the TcTrx has been created based on the known structure of Malassezia sympodialis Trx (MsTrx, PDB ID: 2j23). To characterize the TcTrx, the codon optimized coding region was subcloned into an expression vector and transformed into Saccharomyces cerevisiae. The recombinant His8-tagged TcTrx was expressed and purified by Ni affinity chromatography. The purified enzyme showed a band of approximately 32 kDa (expected dimeric form) on a 12% SDS-PAGE. The molecular mass determined by MALDI-TOF is 33.16 kDa which suggests that the purified enzyme is a dimeric enzyme. Furthermore, the enzyme exhibited TcTrx activity via insulin assay. The Michaelis constant (K M ) value for insulin was 3.78 × 10−2 mM. The enzyme’s half-life of deactivation was 13 min at 45°C. The enzyme was most active at pH 7. Conclusions A three dimensional structural model of T. camphorata Trx based on its TcTrx cDNA sequence. The active form of the TcTrx has been successfully expressed in yeast. The enzyme possesses Trx activity and is capable of reduction of disulfide bonds during the formation of newly synthesized proteins. </jats:sec

Proteomic analysis of rhein-induced cyt: ER stress mediates cell death in breast cancer cells

Rhein is a natural product purified from herbal plants such as Rheum palmatum, which has been shown to have anti-angiogenesis and anti-tumor metastasis properties. However, the biological effects of rhein on the behavior of breast cancers are not completely elucidated. To evaluate whether rhein might be useful in the treatment of breast cancer and its cytotoxic mechanism, we analyzed the impact of rhein treatment on differential protein expression as well as redox regulation in a non-invasive breast cancer cell line, MCF-7, and an invasive breast cancer cell line, MDA-MB-231, using lysine- and cysteine-labeling two-dimensional difference gel electrophoresis (2D-DIGE) combined with MALDI-TOF/TOF mass spectrometry. This proteomic study revealed that 73 proteins were significantly changed in protein expression; while 9 proteins were significantly altered in thiol reactivity in both MCF-7 and MDA-MB-231 cells. The results also demonstrated that rhein-induced cytotoxicity in breast cancer cells mostly involves dysregulation of cytoskeleton regulation, protein folding, the glycolysis pathway and transcription control. A further study also indicated that rhein promotes misfolding of cellular proteins as well as unbalancing of the cellular redox status leading to ER-stress. Our work shows that the current proteomic strategy offers a high-through-put platform to study the molecular mechanisms of rhein-induced cytotoxicity in breast cancer cells. The identified differentially expressed proteins might be further evaluated as potential targets in breast cancer therapy

A Lattice Study of (Dˉ1D∗)±(\bar{D}_1 D^{*})^\pm Near-threshold Scattering

In this exploratory lattice study, low-energy near threshold scattering of the $(\bar{D}_1 D^{*})^\pm$ meson system is analyzed using lattice QCD with $N_f=2$ twisted mass fermion configurations. Both s-wave ($J^P=0^-$) and p-wave ($J^P=1^+$) channels are investigated. It is found that the interaction between the two charmed mesons is attractive near the threshold in both channels. This calculation provides some hints in the searching of resonances or bound states around the threshold of $(\bar{D}_1 D^{*})^\pm$ system.Comment: 20 pages, 15 figures, matches the version on PR

Characterisation of the MacA/MacB/TolC tripartite pump that confers resistance to macrolides in E. coli

Gram-negative bacteria possess tripartite pumps, composed of a membrane fusion protein (MFP), an inner membrane protein (IMP) and an outer membrane protein (OMP), to transport drugs across the inner and outer membranes. The plasmid encoding MacA, MacB and TolC confers resistance to the macrolide erythromycin in the host E. coli cell Kam3, indicating the three proteins are assembled and actively functional. MFPs are believed to have an important role in the stabilizing the pump complex; intriguingly, we found that the MFP MacA not only interacts directly with the IMP MacB and the OMP TolC, but regulates the function of MacB, apparently increasing its affinity for both ATP and erythromycin. As MacB hydrolyzes ATP there is a burst in phosphate production that is symptomatic of the reaction being rate- limited by product release; but the burst disappeared in the presence of MacA. Since MacA caused only a marginal increase in the k(_cat), but a significant decrease in the Km, for the steady-state ATPase activity, this suggests that the disappearance of the phosphate burst is due to a decrease in the rate of hydrolysis, rather than an increase in the rate of product release. This kinetic behaviour indicates that MacA promotes and stabilizes the ATP-binding form of the transporter. MacA regulates the activity of MacB via its ẞ-strand domain since S. aureus MacA, which lacks the coiled coil structure that is present in E. coli MacA and believed to be involved in the interaction with TolC, was able to abolish the Pi burst catalysed by MacB, in direct analogy with the effect of E. coli MacA on MacB. Analytical ultracentrifugation, mass spectrometry and atomic force microscopy indicated that MacB forms dimers, in analogy to ABC- transporters that require a pair of NBDs to bind ATP. Our data suggests a direct role for MacA in facilitating the delivery of drugs by MacB to TolC: by enhancing the binding of drugs to MacB and stabilizing the reorientation of MacB to the outward- facing conformation

ATP-Binding Cassette Transporter VcaM from Vibrio cholerae is Dependent on the Outer Membrane Factor Family for Its Function.

Vibrio cholerae ATP-binding cassette transporter VcaM (V. cholerae ABC multidrug resistance pump) has previously been shown to confer resistance to a variety of medically important drugs. In this study, we set to analyse its properties both in vitro in detergent-solubilised state and in vivo to differentiate its dependency on auxiliary proteins for its function. We report the first detailed kinetic parameters of purified VcaM and the rate of phosphate (Pi) production. To determine the possible functional dependencies of VcaM on the tripartite efflux pumps we then utilized different E. coli strains lacking the principal secondary transporter AcrB (Acriflavine resistance protein), as well as cells lacking the outer membrane factor (OMF) TolC (Tolerance to colicins). Consistent with the ATPase function of VcaM we found it to be susceptible to sodium orthovanadate (NaOV), however, we also found a clear dependency of VcaM function on TolC. Inhibitors targeting secondary active transporters had no effects on either VcaM-conferred resistance or Hoechst 33342 accumulation, suggesting that VcaM might be capable of engaging with the TolC-channel without periplasmic mediation by additional transporters. Our findings are indicative of VcaM being capable of a one-step substrate translocation from cytosol to extracellular space utilising the TolC-channel, making it the only multidrug ABC-transporter outside of the MacB-family with demonstrable TolC-dependency