Single-site chemical modification at C10 of the baccatin III core of paclitaxel and Taxol C reduces P-glycoprotein interactions in bovine brain microvessel endothelial cells

A single-site modification of paclitaxel analogs at the C10 position on the baccatin III core that reduces interaction with P-glycoprotein in bovine brain microvessel endothelial cells is described. Modification and derivatization of the C10 position were carried out using a substrate controlled hydride addition to a key C9 and C10 diketone intermediate. The analogs were tested for tubulin assembly and cytotoxicity, and were shown to retain potency similar to paclitaxel. P-glycoprotein interaction was examined using a rhodamine assay and it was found that simple hydrolysis or epimerization of the C10 acetate of paclitaxel and Taxol C can reduce interaction with the P-glycoprotein transporter that may allow for increased permeation of taxanes into the brain.     

Effect of Glycosylation and Additional Domains on the Thermostability of a Family 10 Xylanase Produced by Thermopolyspora flexuosa

The effects of different structural features on the thermostability of Thermopolyspora flexuosa xylanase XYN10A were investigated. A C-terminal carbohydrate binding module had only a slight effect, whereas a polyhistidine tag increased the thermostability of XYN10A xylanase. In contrast, glycosylation at Asn26, located in an exposed loop, decreased the thermostability of the xylanase. The presence of a substrate increased stability mainly at low pH.The thermophilic actinomycete Thermopolyspora flexuosa, previously named Nonomuraea flexuosa and before that Actinomadura flexuosa or Microtetraspora flexuosa (15), produces family 11 and family 10 xylanases, which show high thermostability (16, 17, 22). T. flexuosa xylanase XYN10A has a C-terminal family 13 carbohydrate binding module (CBM) (22). Many xylanases have an additional CBM, which can be a cellulose binding domain (CBD) or a xylan binding domain (XBD) (1, 5, 7, 22, 25, 28). XBD typically increases activity against insoluble xylan (1, 5, 24), although some XBDs also bind soluble xylans (21, 25).We studied the thermostability of T. flexuosa xylanase XYN10A and how CBM and other additional groups affect its thermostability. In addition to confirming the previously described importance of terminal regions, our study identified a loop that is important for the thermostability of T. flexuosa XYN10A. In general, identification of sites important for protein stability is necessary for targeted mutagenesis attempts to increase thermostability.The T. flexuosa xyn10A gene (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AJ508953","term_id":"57897980","term_text":"AJ508953"}}AJ508953) (22), which encodes the full-length XYN10A xylanase (1-AAST… SYNA-448) containing the catalytic domain and CBM, and a truncated gene, which encodes the catalytic domain only (1-AAST… DALN-301) were expressed in Trichoderma reesei as 3′ fusions to a sequence that encodes the Cel6A CBD (A+B) carrier polypeptide and a Kex2 cleavage site (RDKR) (27). In this article, the catalytic domain and the full-length enzyme are referred to as XYN10A and XYN10A-CBM, respectively. The catalytic domain was also produced in Escherichia coli. For production in E. coli, the sequence encoding the catalytic domain was cloned into a pKKtac vector (33) with and without an additional 3′ sequence encoding a 6×His tag at the protein C terminus (… DALNHHHHHH).The proteins were purified by hydrophobic interaction chromatography using a Phenyl Sepharose column and by ion-exchange chromatography using a DEAE Sepharose FF column (Amersham Pharmacia Biotech). The 6×His-tagged XYN10A xylanase produced in E. coli was purified by affinity chromatography using Ni-nitrilotriacetic acid (Ni-NTA) agarose beads (Qiagen).Mass spectrometric (MS) analyses were performed on a high-resolution 4.7-T hybrid quadrupole-Fourier transform ion cyclotron resonance (FT-ICR) instrument (APEX-Qe; Bruker Daltonics), which employs electrospray ionization (ESI) (see supplemental material for details).Xylanase activity was measured with a 3,5-dinitrosalicylic acid assay by using 1% solubilized birchwood xylan as a substrate (33). The optimum temperature, residual activity, and half-life assays were performed as described earlier (36). SWISS-MODEL (4) was used to automatically model T. flexuosa XYN10A and XYN10A-CBM (PDB codes for the modeling templates are 1v6w and 1e0w, respectively [12, 14]).The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that the masses of XYN10A xylanase and XYN10A-CBM produced in Trichoderma reesei were ∼37 kDa and ∼50 kDa, respectively (Fig. ​(Fig.1A).1A). MS analysis of the 6×His-tagged XYN10A produced in E. coli (SDS-PAGE not shown) indicated the presence of a single protein form (Fig. ​(Fig.1B),1B), with a measured mass of 34,943.25 Da. This is consistent with the theoretical mass of 6×His-tagged XYN10A (34,942.93 Da). In contrast, XYN10A produced in T. reesei was heterogeneously modified, and six protein forms (numbered 1 to 6) were detected (Fig. ​(Fig.1B).1B). The mass of form 1 (34,120.76 Da) is in excellent agreement with the calculated mass of XYN10A (34120.73 Da). The masses of forms 2 and 3, with mass increments of ∼203 and ∼162 Da, respectively, suggested protein glycosylation (+203 Da = GlcNAc; +162 Da = Man). There are two potential sites for N-glycosylation in XYN10A, Asn26 and Asn95. These six protein forms were resolved only by the high-resolution FT-ICR MS technique, not by SDS-PAGE (eluted as a single band [Fig. ​[Fig.1A1A]).Open in a separate windowFIG. 1.(A) SDS-PAGE of purified XYN10A and XYN10A-CBM produced in Trichoderma reesei. Lane 1, molecular weight markers; lane 2, catalytic domain (XYN10A); lane 3, full-length enzyme (XYN10A-CBM). (B) ESI FT-ICR mass spectra of XYN10A with a 6×His tag produced in E. coli (bottom) and XYN10A produced in T. reesei (top). Only the expanded view at m/z 1260 to 1300, with the signals representing the most abundant protein ion charge state z = 27+, is presented. For the measured and calculated masses of the protein forms identified, see the supplemental material.In order to locate the glycosylation site or sites, XYN10A proteins produced in E. coli and T. reesei were subjected to on-line pepsin digestion (see supplemental material for details). The sequence coverage for XYN10A xylanase produced in E. coli was 62%. For XYN10A produced in T. reesei, a lower sequence coverage was obtained, but three glycopeptides (residues 20 to 44, 20 to 46, and 20 to 59), carrying one GlcNAc residue, were detected (glycopeptides A to C in Fig. S1B in the supplemental material). A triply charged glycopeptide A was further analyzed by collision-induced dissociation (CID) measurement (see inset in Fig. S1B in the supplemental material). A ladder of b-type fragment ions further identified this peptide and verified Asn26 as the N-glycosylation site in XYN10A, carrying GlcNAc(Man) as a glycan core structure.The additional sequences attached to the catalytic domain affected the thermostability of XYN10A xylanase. The deletion of the native C-terminal CBM domain (XYN10A produced in T. reesei) slightly decreased (∼2°C) the apparent temperature optimum in the region of 70 to 75°C (Table ​(Table11 and Fig. ​Fig.2A).2A). However, at 80°C, the deletion of the CBM domain increased the activity (Fig. ​(Fig.2A).2A). Furthermore, the half-life in the presence of the substrate at 80°C was lower when the CBM was present (Table ​(Table22).Open in a separate windowFIG. 2.Enzyme activity and stability profiles. (A) Enzyme activity as a function of temperature. The enzymes were incubated for 30 min at each temperature at pH 7. (B) Enzyme inactivation as a function of temperature. The enzyme samples were incubated without the substrate for 30 min at each temperature (pH 7), and the residual activity was measured at 70°C. Values are means ± standard deviations (error bars) for three experiments. Symbols: ⧫, XYN10A xylanase produced in T. reesei; ⋄, XYN10A-CBM produced in T. reesei; ▪, XYN10A produced in E. coli; □, XYN10A-6×His produced in E. coli.

TABLE 1.

Peaks of the optimum temperatures (30-min assay)^a

In silico evidence for functional specialization after genome duplication in yeast

A fairly recent whole-genome duplication (WGD) event in yeast enables the effects of gene duplication and subsequent functional divergence to be characterized. We examined 15 ohnolog pairs (i.e. paralogs from a WGD) out of c . 500 Saccharomyces cerevisiae ohnolog pairs that have persisted over an estimated 100 million years of evolution. These 15 pairs were chosen for their high levels of asymmetry, i.e. within the pair, one ohnolog had evolved much faster than the other. Sequence comparisons of the 15 pairs revealed that the faster evolving duplicated genes typically appear to have experienced partially – but not fully – relaxed negative selection as evidenced by an average nonsynonymous/synonymous substitution ratio (d N /d S _avg=0.44) that is higher than the slow-evolving genes' ratio (d N /d S _avg=0.14) but still <1. Increased number of insertions and deletions in the fast-evolving genes also indicated loosened structural constraints. Sequence and structural comparisons indicated that a subset of these pairs had significant differences in their catalytically important residues and active or cofactor-binding sites. A literature survey revealed that several of the fast-evolving genes have gained a specialized function. Our results indicate that subfunctionalization and even neofunctionalization has occurred along with degenerative evolution, in which unneeded functions were destroyed by mutations.     

Group-specific component (Gc): subtypes in the Finnish population. Description of a new allele and an apparent mother-child incompatibility

The group-specific component (Gc) subtypes were determined in 575 adult Finns by immunoblotting after isoelectric focusing in agarose gel. The gene frequencies were Gc1S = 0.661, Gc1F = 0.139 and Gc2 = 0.200. This material included one rare allele, a more acidically focusing Gc 2 (named Gc 2A18). The phenotypes of 200 mother-child pairs studied were in accordance with the three-allelic mode of inheritance. An apparent mother-child incompatibility observed during routine paternity testing is reported.     

Epigenetics and atherosclerosis

The contribution of epigenetic mechanisms to cardiovascular diseases remains poorly understood. Hypomethylation of genomic DNA is present in human atherosclerotic lesions and methylation changes also occur at the promoter level of several genes involved in the pathogenesis of atherosclerosis, such as extracellular superoxide dismutase, estrogen receptor-α, endothelial nitric oxide synthase and 15-lipoxygenase. So far, no clear data is available about histone modification marks in atherosclerotic lesions. It remains unclear whether epigenetic changes are causally related to the pathogenetic features, such as clonal proliferation of lesion smooth muscle cells, lipid accumulation and modulation of immune responses in the lesions, or whether they merely represent a consequence of the ongoing pathological process. However, epigenetic changes could at least partly explain poorly understood environmental and dietary effects on atherogenesis and the rapid increases and decreases in the incidence of coronary heart disease observed in various populations. RNAi mechanisms may also contribute to the epigenetic regulation of vascular cells. Therapies directed towards modification of the epigenetic status of vascular cells might provide new tools to control atherosclerosis-related cardiovascular diseases.     

Single-enzyme kinetics with fluorogenic substrates: lessons learnt and future directions

Single-molecule fluorescence techniques have developed into powerful tools for studying the kinetics of biological reactions at the single-molecule level. Using fluorogenic substrates, enzymatic reactions can be observed in real-time with single-turnover resolution. The turnover sequence contains all kinetic information, giving access to reaction substeps and dynamic processes such as fluctuations in the reaction rate. Despite their clearly proven potential, the accuracy of current measurements is limited by the availability of substrates with 1:1 stoichiometry and the signal-to-noise ratio of the measurement. In this review we summarize the state-of-the-art and discuss these limitations using experiments performed with α-chymotrypsin as an example. We are further providing an overview of recent efforts aimed at the improvement of fluorogenic substrates and the development of new detection schemes. These detection schemes utilize nanophotonic structures such as zero mode waveguides or nanoantennas. Nanophotonic approaches reduce the size of the effective detection volume and are a powerful strategy to increase the signal-to-noise ratio. We believe that a combination of improved substrates and novel detection schemes will pave the way for performing accurate single-enzyme experiments in biologically relevant conditions.