pH dependence studies provide insight into the structure and mechanism of thimet oligopeptidase (EC 3.4.24.15)

Thimet oligopeptidase (EC 3.4.24.15; TOP) is a Zn(II) endopeptidase implicated in physiological regulation of processes involving neuropeptides. The present study clarifies the active site structure and mechanism of catalysis of TOP. The enzyme exhibited a bell-shaped pH dependence of activity having an acidic limb due to a protonation event with a pK(a) of 5.7 and a basic limb with pK(a) of 8.8. The acidic limb can be attributed to protonation of a residue affecting k(cat) while the alkaline limb may be due to conformational change. Mutation of Tyr612 to Phe resulted in more than 400-fold decrease in activity. This result, supported by modeling studies, implicates Tyr612 in transition state stabilization analogous to the role of His231 of thermolysin.     

Human sterol 27-hydroxylase (CYP27) overexpressor transgenic mouse model. Evidence against 27-hydroxycholesterol as a critical regulator of cholesterol homeostasis

CYP27-overexpressed transgenic mice were generated with the use of a human full-length CYP27 coding region cloned into a ubiquitous expression vector. Positive transgenic mice were identified by tail DNA genotyping and high fecal 27-hydroxycholesterol content. The levels of 27-hydroxycholesterol were found to be 3-5 times higher in the circulation and the tissues of the overexpressed mice when compared with littermate controls. There were no gross morphological differences between the overexpressed mice and their controls. Total cholesterol and triglyceride levels were not affected by overexpression of CYP27. Serum lathosterol was also normal, suggesting a normal rate of cholesterol synthesis. Serum levels of 7alpha-hydroxycholesterol were unaffected, suggesting a normal rate of bile acid formation in the pathway involving cholesterol 7alpha-hydroxylase. Biliary bile acid composition was slightly affected by CYP27 overexpression in female but not in male mice. Fecal levels of neutral steroids were slightly but significantly increased in overexpressor female mice but not in male mice. Levels of 24-hydroxycholesterol in the circulation were significantly reduced in the overexpressed mice, probably as a consequence of a recently described catabolic pathway involving CYP27. Combined with the results of our previous work on mice with a disruption of the CYP27 gene, the present results suggest that the levels of 27-hydroxycholesterol are not of critical importance for cholesterol homeostasis in mice.     

Manipulation of a nuclear NAD+ salvage pathway delays aging without altering steady-state NAD+ levels

Yeast deprived of nutrients exhibit a marked life span extension that requires the activity of the NAD(+)-dependent histone deacetylase, Sir2p. Here we show that increased dosage of NPT1, encoding a nicotinate phosphoribosyltransferase critical for the NAD(+) salvage pathway, increases Sir2-dependent silencing, stabilizes the rDNA locus, and extends yeast replicative life span by up to 60%. Both NPT1 and SIR2 provide resistance against heat shock, demonstrating that these genes act in a more general manner to promote cell survival. We show that Npt1 and a previously uncharacterized salvage pathway enzyme, Nma2, are both concentrated in the nucleus, indicating that a significant amount of NAD(+) is regenerated in this organelle. Additional copies of the salvage pathway genes, PNC1, NMA1, and NMA2, increase telomeric and rDNA silencing, implying that multiple steps affect the rate of the pathway. Although SIR2-dependent processes are enhanced by additional NPT1, steady-state NAD(+) levels and NAD(+)/NADH ratios remain unaltered. This finding suggests that yeast life span extension may be facilitated by an increase in the availability of NAD(+) to Sir2, although not through a simple increase in steady-state levels. We propose a model in which increased flux through the NAD(+) salvage pathway is responsible for the Sir2-dependent extension of life span.     

Comparative non-shivering thermogenesis in adjacent populations of the common spiny mouse (Acomys cahirinus) from opposite slopes: the effects of increasing salinity

We compared non-shivering thermogenesis between two adjacent populations of the common spiny mouse Acomys cahirinus from different habitats, in relation to increasing salinity. Individuals were captured from the north- and south-facing slopes of the same valley, that represent "Mediterranean" and "desert" habitats, respectively. We hypothesized that the two populations of mice would differ in their thermoregulatory capacities, reflecting their need to cope with the environmental stress in each habitat. We measured resting metabolic rate by recording oxygen consumption, body temperature and response to an injection of exogenous noradrenaline. Mice were maintained on diets with increasing levels of salt intake to examine their abilities to cope with increasing osmotic stress. Mice from north-facing slopes generally had a higher resting metabolic rate and a higher increase in oxygen consumption in response to noradrenaline than mice from south-facing slopes. Increasing salinity decreased resting metabolic rate values, body temperature, and oxygen consumption in response to noradrenaline in both populations, and diminished slope-dependant differences. We suggest that these differences could be a result of an ongoing adaptive process to different climatic conditions, typical of the Mediterranean region, that are a demonstrable example of evolution in action.     

Distribution of 1000 sequenced T-DNA tags in the Arabidopsis genome

Induction of knockout mutations by T-DNA insertion mutagenesis is widely used in studies of plant gene functions. To assess the efficiency of this genetic approach, we have sequenced PCR amplified junctions of 1000 T-DNA insertions and analysed their distribution in the Arabidopsis genome. Map positions of 973 tags could be determined unequivocally, indicating that the majority of T-DNA insertions landed in chromosomal domains of high gene density. Only 4.7% of insertions were found in interspersed, centromeric, telomeric and rDNA repeats, whereas 0.6% of sequenced tags identified chromosomally integrated segments of organellar DNAs. 35.4% of T-DNAs were localized in intervals flanked by ATG and stop codons of predicted genes, showing a distribution of 62.2% in exons and 37.8% in introns. The frequency of T-DNA tags in coding and intergenic regions showed a good correlation with the predicted size distribution of these sequences in the genome. However, the frequency of T-DNA insertions in 3'- and 5'-regulatory regions of genes, corresponding to 300 bp intervals 3' downstream of stop and 5' upstream of ATG codons, was 1.7-2.3-fold higher than in any similar interval elsewhere in the genome. The additive frequency of insertions in 5'-regulatory regions and coding domains provided an estimate for the mutation rate, suggesting that 47.8% of mapped T-DNA tags induced knockout mutations in Arabidopsis.     

Protein structure prediction via combinatorial assembly of sub-structural units

Following the hierarchical nature of protein folding, we propose a three-stage scheme for the prediction of a protein structure from its sequence. First, the sequence is cut to fragments that are each assigned a structure. Second, the assigned structures are combinatorially assembled to form the overall 3D organization. Third, highly ranked predicted arrangements are completed and refined. This work focuses on the second stage of this scheme: the combinatorial assembly. We present CombDock, a combinatorial docking algorithm. CombDock gets an ordered set of protein sub-structures and predicts the inter-contacts that define their overall organization. We reduce the combinatorial assembly to a graph-theory problem, and give a heuristic polynomial solution to this computationally hard problem. We applied CombDock to various examples of structural units of two types: protein domains and building blocks, which are relatively stable sub-structures of domains. Moreover, we tested CombDock using increasingly distorted input, where the native structural units were replaced by similarly folded units extracted from homologous proteins and, in the more difficult cases, from globally unrelated proteins. The algorithm is robust, showing low sensitivity to input distortion. This suggests that CombDock is a useful tool in protein structure prediction that may be applied to large target proteins.     

MASS: multiple structural alignment by secondary structures

We present a novel method for multiple alignment of protein structures and detection of structural motifs. To date, only a few methods are available for addressing this task. Most of them are based on a series of pairwise comparisons. In contrast, MASS (Multiple Alignment by Secondary Structures) considers all the given structures at the same time. Exploiting the secondary structure representation aids in filtering out noisy results and in making the method highly efficient and robust. MASS disregards the sequence order of the secondary structure elements. Thus, it can find non-sequential and even non-topological structural motifs. An important novel feature of MASS is subset alignment detection: It does not require that all the input molecules be aligned. Rather, MASS is capable of detecting structural motifs shared only by a subset of the molecules. Given its high efficiency and capability of detecting subset alignments, MASS is suitable for a broad range of challenging applications: It can handle large-scale protein ensembles (on the order of tens) that may be heterogeneous, noisy, topologically unrelated and contain structures of low resolution.     

Multiple structural alignment by secondary structures: algorithm and applications

We present MASS (Multiple Alignment by Secondary Structures), a novel highly efficient method for structural alignment of multiple protein molecules and detection of common structural motifs. MASS is based on a two-level alignment, using both secondary structure and atomic representation. Utilizing secondary structure information aids in filtering out noisy solutions and achieves efficiency and robustness. Currently, only a few methods are available for addressing the multiple structural alignment task. In addition to using secondary structure information, the advantage of MASS as compared to these methods is that it is a combination of several important characteristics: (1) While most existing methods are based on series of pairwise comparisons, and thus might miss optimal global solutions, MASS is truly multiple, considering all the molecules simultaneously; (2) MASS is sequence order-independent and thus capable of detecting nontopological structural motifs; (3) MASS is able to detect not only structural motifs, shared by all input molecules, but also motifs shared only by subsets of the molecules. Here, we show the application of MASS to various protein ensembles. We demonstrate its ability to handle a large number (order of tens) of molecules, to detect nontopological motifs and to find biologically meaningful alignments within nonpredefined subsets of the input. In particular, we show how by using conserved structural motifs, one can guide protein-protein docking, which is a notoriously difficult problem. MASS is freely available at http://bioinfo3d.cs.tau.ac.il/MASS/.     

Distinct patterns of apoptosis in the lung during liquid ventilation compared with gas ventilation

To determine whether liquid ventilation (LV) causes less cell injury and improves lung function compared with conventional gas ventilation (GV), we analyzed pulmonary physiological profiles, lung histology, and cell death in 110- and 120-day preterm lambs, which were randomized to receive either ventilation modality on FI(O(2)) = 1. LV lungs were well expanded with adequate pulmonary function, whereas GV animals exhibited marked atelectasis, poor pulmonary function, and increased mortality. Both ventilatory strategies induced marked lung cell apoptosis, but with distinct patterns of distribution. Although GV induced apoptosis of epithelium primarily in the lining and within the lumina of bronchioles, LV induced significant apoptosis much more homogeneously throughout lung parenchyma including alveoli and interstitial spaces. These studies suggest that although both forms of ventilation cause regional apoptosis, LV more effectively delivers oxygen and recruits the lung more homogeneously than GV.