The Response of Haloferax volcanii to Salt and Temperature Stress: A Proteome Study by Label‐Free Mass Spectrometry

In‐depth proteome analysis of the haloarchaeal model organism Haloferax volcanii has been performed under standard, low/high salt, and low/high temperature conditions using label‐free mass spectrometry. Qualitative analysis of protein identification data from high‐pH/reversed‐phase fractionated samples indicates 61.1% proteome coverage (2509 proteins), which is close to the maximum recorded values in archaea. Identified proteins match to the predicted proteome in their physicochemical properties, with only a small bias against low‐molecular‐weight and membrane‐associated proteins. Cells grown under low and high salt stress as well as low and high temperature stress are quantitatively compared to standard cultures by sequential window acquisition of all theoretical mass spectra (SWATH‐MS). A total of 2244 proteins, or 54.7% of the predicted proteome, are quantified across all conditions at high reproducibility, which allowed for global analysis of protein expression changes under these stresses. Of these, 2034 are significantly regulated under at least one stress condition. KEGG pathway enrichment analysis shows that several major cellular pathways are part of H. volcanii’s universal stress response. In addition, specific pathways (purine, cobalamin, and tryptophan) are affected by temperature stress. The most strongly downregulated proteins under all stress conditions, zinc finger protein HVO_2753 and ribosomal protein S14, are found oppositely regulated to their immediate genetic neighbors from the same operon.     

Explicit-solvent molecular dynamics simulations of the polysaccharide schizophyllan in water

Schizophyllan is a beta(1-->3)-D-glucan polysaccharide with beta(1-->6)-branched lateral glucose residues that presents a very stiff triple-helical structure under most experimental conditions. Despite the remarkable stability of this structure (which persists up to 120 degrees C in aqueous solution), schizophyllan undergoes a major change of state around 7 degrees C in water that has been hypothesized to result from an order-disorder transition in the lateral residues. This hypothesis is only supported by indirect experimental evidence and detailed knowledge (at the atomic level) concerning hydrogen-bonding networks, interactions with the solvent molecules, orientational freedom of the lateral residues, and orientational correlations among them is still lacking. In this study explicit-solvent molecular dynamics simulations of a schizophyllan fragment (complemented by simulations of its tetrasaccharide monomer) are performed at three different temperatures (273 K, 350 K, and 450 K) and with two different types of boundary conditions (finite nonperiodic or infinite periodic fragment) as an attempt to provide detailed structural and dynamical information about the triple-helical conformation in solution and the mechanism of the low-temperature transition. These simulations suggest that three important driving forces for the high stability of the triple helix are i), the limited conformational work involved in its formation; ii), the formation of a dense hydrogen-bonding network at its center; and iii), the formation of interchain hydrogen bonds between main-chain and lateral glucose residues. However, these simulations evidence a moderate and continuous variation of the simulated observables upon increasing the temperature, rather than a sharp transition between the two lowest temperatures (that could be associated with the state transition). Although water-mediated hydrogen-bonded association of neighboring lateral residues is observed, this interaction is not strong enough to promote the formation of an ordered state (correlated motions of the lateral residues), even at the lowest temperature considered.     

Kinetic mechanism and nucleotide specificity of NADH peroxidase

NADH peroxidase is a flavoprotein isolated from Streptococcus faecalis which catalyzes the pyridine nucleotide-dependent reduction of hydrogen peroxide to water. Initial velocity, product, and dead-end inhibition studies have been performed at pH 7.5 and support a ping-pong kinetic mechanism. In the absence of hydrogen peroxide, both transhydrogenation between NADH and thioNAD, and isotope exchange between [14C]NADH and NAD, have been demonstrated, although in both these experiments, the maximal velocity of nucleotide exchange was less than 1.5% the maximal velocity of the peroxidatic reaction. We propose that NADH binds tightly to both oxidized and two-electron reduced enzyme. NADH oxidation proceeds stereospecifically with the transfer of the 4S hydrogen to enzyme, and then, via exchange, to water. No primary tritium kinetic isotope effect was observed, and no statistically significant primary deuterium kinetic isotope effects on V/K were determined, although primary deuterium kinetic isotope effects on V were observed in the presence and absence of sodium acetate. NADH peroxidase thus shares with other flavoprotein reductases striking kinetic, spectroscopic, and stereochemical similarities. On this basis, we propose a chemical mechanism for the peroxide cleaving reaction catalyzed by NADH peroxidase which involves the obligate formation of a flavinperoxide, and peroxo bond cleavage by nucleophilic attack by enzymatic dithiols.     

Construction of acetate auxotrophs of Neisseria meningitidis to study host-meningococcal endotoxin interactions

To facilitate studies of the molecular determinants of host-meningococcal lipooligosaccharide (endotoxin) interactions at patho-physiologically relevant endotoxin concentrations (i.e. < or =10 ng/ml), we have generated acetate auxotrophs NMBACE1 from encapsulated Neisseria meningitidis (serogroup B, strain NMB) and NMBACE2 from an isogenic bacterial mutant lacking the polysialic acid capsule. Growth of the auxotrophs in medium containing [(14)C]acetate yielded (14)C-lipooligosaccharides containing approximately 600 cpm/ng. Gel sieving resolved 14C-lipooligosaccharide-containing aggregates with an estimated molecular mass of > or =20 x 10(6) Da (peak A) and approximately 1 x 10(6) Da (peak B) from both strains. Lipooligosaccharides in peaks A and B had the same fatty acid composition and SDS-polyacrylamide gel electrophoresis profile. 14C-Labeled capsule copurified with (14)C-lipooligosaccharides in peak B from NMBACE1, whereas the other aggregates contained only 14C-lipooligosaccharide. For all aggregates, lipopolysaccharide-binding protein and soluble CD14-induced delivery of lipooligosaccharides to endothelial cells and cell activation correlated with disaggregation of lipooligosaccharides. These processes were inhibited by the presence of capsule but unaffected by the size of the aggregates. In contrast, endotoxin activation of cells containing membrane CD14 was unaffected by capsule but diminished when endotoxin was presented in larger aggregates. These findings demonstrate that the physical presentation of lipooligosaccharide, including possible interactions with capsule, affect the ability of meningococcal endotoxin to interact with and activate specific host targets.     

Synthesis and antiviral activity of P1' arylsulfonamide azacyclic urea HIV protease inhibitors

A series of novel azacyclic urea HIV protease inhibitors bearing a benzenesulfonamide group at P1' were synthesized utilizing a parallel synthesis method. Structural studies of early analogs bound in the enzyme active site were used to design more potent inhibitors. The effects of substituting the P1' benzenesulfonyl group on antiviral activity and protein binding are described.     

Synthesis and activity of 1-aryl-1'-imidazolyl methyl ethers as non-thiol farnesyltransferase inhibitors

A series of imidazole-containing methyl ethers (4-5) have been designed and synthesized as potent and selective farnesyltransferase inhibitors (FTIs) by transposition of the D-ring to the methyl group on the imidazole of the previously reported FTIs 3. Several compounds such as 4h and 5b demonstrate superior enzymatic activity to the current benchmark compound tipifarnib (1) with IC(50) values in the lower subnanomolar range, while maintaining excellent cellular activity comparable to tipifarnib. The compounds are characterized as being simple, easier to make, and possess no chiral center involved.     

Design, synthesis, and activity of 4-quinolone and pyridone compounds as nonthiol-containing farnesyltransferase inhibitors

As a part of our efforts to identify potent inhibitors of farnesyltransferase (FTase), modification of the structure of tipifarnib through structure-based design was undertaken by replacing the 2-quinolones with 4-quinolones and pyridones, and subsequent relocation of the D-ring to the N-methyl group on the imidazole ring. This study has yielded a novel series of potent and selective FTase inhibitors. The X-ray structure of tipifarnib (1) in complex with FTase was described.     

Aryl tetrahydropyridine inhibitors of farnesyltransferase: bioavailable analogues with improved cellular potency

Inhibitors of farnesyltransferase are effective against a variety of tumors in mouse models of cancer. Clinical trials to evaluate these agents in humans are ongoing. In our effort to develop new farnesyltransferase inhibitors, we have discovered bioavailable aryl tetrahydropyridines that are potent in cell culture. The design, synthesis, SAR and biological properties of these compounds will be discussed.     

Backbone dynamics of the human MIA protein studied by (15)N NMR relaxation: implications for extended interactions of SH3 domains

The melanoma inhibitory activity (MIA) protein is a clinically valuable marker in patients with malignant melanoma as enhanced values diagnose metastatic melanoma stages III and IV. Here, we report the backbone dynamics of human MIA studied by (15)N NMR relaxation experiments. The folded core of human MIA is found to be rigid, but several loops connecting beta-sheets, such as the RT-loop for example, display increased mobility on picosecond to nanosecond time scales. One of the most important dynamic features is the pronounced flexibility of the distal loop, comprising residues Asp 68 to Ala 75, where motions on time scales up to milliseconds occur. Further, significant exchange contributions are observed for residues of the canonical binding site of SH3 domains including the RT-loop, the n-Src loop, for the loop comprising residues 13 to 19, which we refer to as the"disulfide loop", in part for the distal loop, and the carboxyl terminus of human MIA. The functional importance of this dynamic behavior is discussed with respect to the biological activity of several point mutations of human MIA. The results of this study suggest that the MIA protein and the recently identified highly homologous fibrocyte-derived protein (FDP)/MIA-like (MIAL) constitute a new family of secreted proteins that adopt an SH3 domain-like fold in solution with expanded ligand interactions.