Correlation between HSP90 induction kinetics in murine leukemia cells and the amount of cisplatin over a wide range of cytostatic concentrations

The induction of HSP90 in murine erythroleukemia cells, clone F4 N, by cisplatin (DDP) was examined using indirect immunofluorescence and avidin-biotin technique, and compared with cisplatin cytotoxicity. A reverse dependence of HSP90 induction time was found on a wide range of cisplatin concentrations (0.5-10 microM), which proved to be cytostatic up to 48 h of continuous treatment. Thus, the observed induction pattern of HSP90 in F4 N cells strictly correlated with their high tolerance toward DDP. This indicates that HSP90 might be responsible, at least in part, for cisplatin resistance of F4 N cells.     

Calcium Is Essential for the Major Pseudopilin in the Type 2 Secretion System

The pseudopilus is a key feature of the type 2 secretion system (T2SS) and is made up of multiple pseudopilins that are similar in fold to the type 4 pilins. However, pilins have disulfide bridges, whereas the major pseudopilins of T2SS do not. A key question is therefore how the pseudopilins, and in particular, the most abundant major pseudopilin, GspG, obtain sufficient stability to perform their function. Crystal structures of Vibrio cholerae, Vibrio vulnificus, and enterohemorrhagic Escherichia coli (EHEC) GspG were elucidated, and all show a calcium ion bound at the same site. Conservation of the calcium ligands fully supports the suggestion that calcium ion binding by the major pseudopilin is essential for the T2SS. Functional studies of GspG with mutated calcium ion-coordinating ligands were performed to investigate this hypothesis and show that in vivo protease secretion by the T2SS is severely impaired. Taking all evidence together, this allows the conclusion that, in complete contrast to the situation in the type 4 pili system homologs, in the T2SS, the major protein component of the central pseudopilus is dependent on calcium ions for activity.In Gram-negative bacteria, the type 2 secretion system (T2SS)² is used for the secretion of several important proteins across the outer membrane (1). The T2SS is also called the terminal branch of the general secretory pathway (Gsp) (2) and, in Vibrio species, the extracellular protein secretion (Eps) apparatus (3). This sophisticated multiprotein machinery spans both the inner and the outer membrane of Gram-negative bacteria and contains 11–15 different proteins. The T2SS consists of three major subassemblies (4–9): (i) the outer membrane complex comprising mainly the crucial multisubunit secretin GspD; (ii) the pseudopilus, which consists of one major and several minor pseudopilins; and (iii) an inner membrane platform, containing the cytoplasmic secretion ATPase GspE and the membrane proteins GspL, GspM, GspC, and GspF.The pseudopilus is a key element of the T2SS that forms a helical fiber spanning the periplasm. The fiber is assembled from multiple subunits of the major pseudopilin GspG (4, 5, 10–14). The pseudopilus is thought to form a plug of the secretin pore in the outer membrane and/or to function as a piston during protein secretion. In recent years, studies of the T2SS pseudopilins led to structure determinations of all individual pseudopilins (13, 15–17). The recent structure of the helical ternary complex of GspK-GspI-GspJ suggested that these three minor pseudopilins form the tip of the pseudopilus (17). A crystal structure of GspG from Klebsiella oxytoca was in a previous study combined with electron microscopy data to arrive at a helical arrangement, with no evidence for special features, such as disulfide bridges, other covalent links, or metal-binding sites, for stabilizing this major pseudopilin or the pseudopilus (13).The pseudopilins of the T2SS share a common fold with the type 4 pilins (15–21). Pilins are proteins incorporated into pili, long appendages on the surface of bacteria forming thin, strong fibers with multiple functions (19, 21). Type 4 pilins and pseudopilins contain a prepilin leader sequence that is cleaved off by a prepilin peptidase, yielding mature protein (10, 11, 22). A distinct feature of the type 4 pilins is the occurrence of a disulfide bridge connecting β4 to a Cys in the so-called “D-region” near the C terminus (21). In a recent study (23) on the thin fibers of Gram-positive bacteria, isopeptide units appeared to be essential for providing these filaments sufficient cohesion and stability. A key question was therefore whether the major pseudopilin GspG also requires a special feature to obtain sufficient stability to perform its function.     

Template‐free protein structure prediction and quality assessment with an all‐atom free‐energy model

Biophysical forcefields have contributed less than originally anticipated to recent progress in protein structure prediction. Here, we have investigated the selectivity of a recently developed all‐atom free‐energy forcefield for protein structure prediction and quality assessment (QA). Using a heuristic method, but excluding homology, we generated decoy‐sets for all targets of the CASP7 protein structure prediction assessment with <150 amino acids. The decoys in each set were then ranked by energy in short relaxation simulations and the best low‐energy cluster was submitted as a prediction. For four of nine template‐free targets, this approach generated high‐ranking predictions within the top 10 models submitted in CASP7 for the respective targets. For these targets, our de‐novo predictions had an average GDT_S score of 42.81, significantly above the average of all groups. The refinement protocol has difficulty for oligomeric targets and when no near‐native decoys are generated in the decoy library. For targets with high‐quality decoy sets the refinement approach was highly selective. Motivated by this observation, we rescored all server submissions up to 200 amino acids using a similar refinement protocol, but using no clustering, in a QA exercise. We found an excellent correlation between the best server models and those with the lowest energy in the forcefield. The free‐energy refinement protocol may thus be an efficient tool for relative QA and protein structure prediction. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Peptidase inhibitors from the salivary glands of the cockroach Nauphoeta cinerea

Inhibitory activity against subtilisin, proteinase K, chymotrypsin and trypsin was detected in the salivary glands and saliva of the cockroach Nauphoeta cinerea (Blattoptera: Blaberidae). Fractionation of the salivary glands extract by affinity chromatography followed by reverse-phase HPLC yielded five subtilisin-inhibiting peptides with molecular masses ranging from 5 to 14 kDa. N-terminal sequences and subsequently full-length cDNAs of inhibitors designated NcPIa and NcPIb were obtained. The NcPIa cDNA contains 216 nucleotides and encodes a pre-peptide of 72 amino-acid residues of which 19 make up the signal peptide. The cDNA of NcPIb consists of 240 nucleotides and yields a putative secretory peptide of 80 amino-acid residues. Mature NcPIa (5906.6 Da, 53 residues) and NcPIb (6713.3 Da, 60 residues) are structurally similar (65.4% amino acid overlap) single-domain Kazal-type peptidase inhibitors. NcPIa with Arg in P1 position and typical Kazal motif VCGSD interacted stoichiometrically (1:1) with subtilisin and was slightly less active against proteinase K. NcPIb with Leu in P1 and modified Kazal motif ICGSD had similar activity on subtilisin and no on proteinase K but was active on chymotrypsin.     

Estimation of population structure in coastal Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco var. menziesii] using allozyme and microsatellite markers

Characterizing population structure using neutral markers is an important first step in association genetic studies in order to avoid false associations between phenotypes and genotypes that may arise from non-selective demographic factors. Population structure was studied in a wide sample of ∼1,300 coastal Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco var. menziesii] trees from Washington and Oregon. This sample is being used for association mapping between cold hardiness and phenology phenotypes and single-nucleotide polymorphisms in adaptive-trait candidate genes. All trees were genotyped for 25 allozyme and six simple sequence repeat (SSR) markers using individual megagametophytes. Population structure analysis was done separately for allozyme and SSR markers, as well as for both datasets combined. The parameter of genetic differentiation (θ or F _ST) was standardized to take into account high within-population variation in the SSR loci and to allow comparison with allozyme loci. Genetic distance between populations was positively and significantly correlated with geographic distance, and weak but significant clinal variation was found for a few alleles. Although the STRUCTURE simulation analysis inferred the same number of populations as used in this study and as based on previous analysis of quantitative adaptive trait variation, clustering among populations was not significant. In general, results indicated weak differentiation among populations for both allozyme and SSR loci (θ _s = 0.006–0.059). The lack of pronounced population subdivision in the studied area should facilitate association mapping in this experimental population, but we recommend taking the STRUCTURE analysis and population assignments for individual trees (Q-matrix) into account in association mapping.     

DNA splicing by directed ligation (SDL)

Splicing by directed ligation (SDL) is a method of in-phase joining of PCR-generated DNA fragments that is based on a pre-designed combination of class IIS restriction endonuclease recognition and cleavage sites. Since these enzymes cleave outside of their recognition sites, the resulting sticky end can have any desired sequence, and the site itself can be removed and does not appear in the final spliced DNA product. SDL is based on the addition of class IIS recognition sites onto primers used to amplify DNA sequences. Cleavage of the PCR products results in elimination of the recognition site-containing flanking sequences and leaves the DNA fragments crowned with protruding ends. With careful design of the sticky ends, several segments can be ligated together in a predetermined order in a single reaction. SDL requires fewer rounds of amplification than overlap extension methods, and is particularly useful for creating a series of recombinants that differ in one segment.     

Superhelical DNA studied by solution scattering and computer models

We present here recent results on the structure of superhelical DNA and its changes with salt concentration between 0.01 and 1.5 M NaCl. Scattering curves of two different superhelical DNAs were determined by static light scattering. The measured radii of gyration do not change significantly with salt concentration. Small-angle neutron scattering, together with calculations from a Monte Carlo model, allows to determine the superhelix diameter. Measured and simulated scattering curves agreed almost quantitatively. Experimentally we find that the diameter decreases from 16.0±0.9 nm at 10 mM to 9.0±0.7 nm at 100 mM NaCl. The superhelix diameter from the simulated conformations decreased from 18.0±1.5 nm at 10 mM to 9.4±1.5 nm at 100 mM NaCl. At higher salt concentrations up to 1.5 M NaCl, the diameter stays constant at 9 nm.This revised version was published online in October 2005 with corrections to the Cover Date.     

Fluorescence resonance energy transfer in the study of nucleic acids

Recent data are reviewed on the employment of fluorescence resonance energy transfer (FRET) in studying hybridization and higher structures of nucleic acids as well as their enzyme- and ribozyme-catalyzed reactions.     

Thermodynamic, kinetic and structural basis for recognition and repair of abasic sites in DNA by apurinic/apyrimidinic endonuclease from human placenta

X-ray analysis of enzyme–DNA interactions is very informative in revealing molecular contacts, but provides neither quantitative estimates of the relative importance of these contacts nor information on the relative contributions of specific and nonspecific interactions to the total affinity of enzymes for specific DNA. A stepwise increase in the ligand complexity approach is used to estimate the relative contributions of virtually every nucleotide unit of synthetic DNA containing abasic sites to its affinity for apurinic/apyrimidinic endonuclease (APE1) from human placenta. It was found that APE1 interacts with 9–10 nt units or base pairs of single-stranded and double-stranded ribooligonucleotides and deoxyribooligonucleotides of different lengths and sequences, mainly through weak additive contacts with internucleotide phosphate groups. Such nonspecific interactions of APE1 with nearly every nucleotide within its DNA-binding cleft provides up to seven orders of magnitude (ΔG° ~ −8.7 to −9.0 kcal/mol) of the enzyme affinity for any DNA substrate. In contrast, interactions with the abasic site together with other specific APE1–DNA interactions provide only one order of magnitude (ΔG° ~ −1.1 to −1.5 kcal/mol) of the total affinity of APE1 for specific DNA. We conclude that the enzyme's specificity for abasic sites in DNA is mostly due to a great increase (six to seven orders of magnitude) in the reaction rate with specific DNA, with formation of the Michaelis complex contributing to the substrate preference only marginally.