Glucocorticoid receptor-like Zn(Cys)4 motifs in BslI restriction endonuclease

BslI restriction endonuclease cleaves the symmetric sequence CCN(7)GG (where N=A, C, G or T). The enzyme is composed of two subunits, alpha and beta, that form a heterotetramer (alpha(2)beta(2)) in solution. The alpha subunit is believed to be responsible for DNA recognition, while the beta subunit is thought to mediate cleavage. Here, for the first time, we provide experimental evidence that BslI binds Zn(II). Specifically, using X-ray absorption spectroscopic analysis we show that the alpha subunit of BslI contains two Zn(Cys)(4)-type zinc motifs similar to those in the DNA-binding domain of the glucocorticoid receptor. This conclusion is supported by genetic analysis of the zinc-binding motifs, whereby amino acid substitutions in the zinc finger motifs are demonstrated to abolish or impair cleavage activity. An additional putative zinc-binding motif was identified in the beta subunit, consistent with the X-ray absorption data. These data were corroborated by proton induced X-ray emission measurements showing that full BslI contains at least three fully occupied Zn sites per alpha/beta heterodimer. On the basis of these data, we propose a role for the BslI Zn motifs in protein-DNA as well as protein-protein interactions.     

Mixed-model reanalysis of primate data suggests tissue and species biases in oligonucleotide-based gene expression profiles

An emerging issue in evolutionary genetics is whether it is possible to use gene expression profiling to identify genes that are associated with morphological, physiological, or behavioral divergence between species and whether these genes have undergone positive selection. Some of these questions were addressed in a recent study (Enard et al. 2002) of the difference in gene expression among human, chimp, and orangutan, which suggested an accelerated rate of divergence in gene expression in the human brain relative to liver. Reanalysis of the Affymetrix data set using analysis of variance methods to quantify the contributions of individuals and species to variation in expression of 12,600 genes indicates that as much as one-quarter of the genome shows divergent expression between primate species at the 5% level. The magnitude of fold change ranges from 1.2-fold up to 8-fold. Similar conclusions apply to reanalysis of Enard et al. 2002 parallel murine data set. However, biases inherent to short oligonucleotide microarray technology may account for some of the tissue and species effects. At high significance levels, more differences were observed in the liver than in the brain in each of the pairwise species comparisons, so it is not clear that expression divergence is accelerated in the human brain. Further, there is an apparent bias toward upregulation of gene expression in the brain in both primates and mice, whereas genes are equally likely to be up- or downregulated in the liver when these species diverge. A small subset of genes that are candidates for adaptive divergence may be identified on the basis of a high ratio of interspecific to intraspecific divergence.     

Crystal structure and biochemical analysis of the MutS.ADP.beryllium fluoride complex suggests a conserved mechanism for ATP interactions in mismatch repair

During mismatch repair ATP binding and hydrolysis activities by the MutS family proteins are important for both mismatch recognition and for transducing mismatch recognition signals to downstream repair factors. Despite intensive efforts, a MutS.ATP.DNA complex has eluded crystallographic analysis. Searching for ATP analogs that strongly bound to Thermus aquaticus (Taq) MutS, we found that ADP.beryllium fluoride (ABF), acted as a strong inhibitor of several MutS family ATPases. Furthermore, ABF promoted the formation of a ternary complex containing the Saccharomyces cerevisiae MSH2.MSH6 and MLH1.PMS1 proteins bound to mismatch DNA but did not promote dissociation of MSH2.MSH6 from mismatch DNA. Crystallographic analysis of the Taq MutS.DNA.ABF complex indicated that although this complex was very similar to that of MutS.DNA.ADP, both ADP.Mg(2+) moieties in the MutS. DNA.ADP structure were replaced by ABF. Furthermore, a disordered region near the ATP-binding pocket in the MutS B subunit became traceable, whereas the equivalent region in the A subunit that interacts with the mismatched nucleotide remained disordered. Finally, the DNA binding domains of MutS together with the mismatched DNA were shifted upon binding of ABF. We hypothesize that the presence of ABF is communicated between the two MutS subunits through the contact between the ordered loop and Domain III in addition to the intra-subunit helical lever arm that links the ATPase and DNA binding domains.     

Purification and in vitro folding of recombinant human thrombopoietin receptor expressed in Escherichia coli

Thrombopoietin receptor (TPOR) is a member of the cytokine receptor superfamily expressed primarily on hematopoietic cells. TPOR plays an important role in regulating platelet production. Due to its low expression level in human tissue, studies on the biochemical and biophysical properties of TPOR have been limited. In the present study, an extracellular domain of recombinant human TPOR (rh TPOR-EN) was expressed in Escherichia coli as inclusion bodies. Purification was achieved by metal chelated chromatography under denaturing condition and was refolded by gel filtration chromatography. Far UV circular dichroism spectroscopy and surface plasmon resonance experiments were performed to demonstrate that the protein was in a refolded state and could bind with its ligand. Thus, a production and purification scheme was developed by which sufficient quantities of rh TPOR-EN can be made available for biochemical and biophysical characterizations.     

Determinants of trachoma endemicity using Chlamydia trachomatis ompA DNA sequencing

A six-year prospective study of Chlamydia trachomatis infection and ocular disease in Tanzanian village children was conducted to identify the determinants of trachoma endemicity using sequencing of ompA. Overall, 749 conjunctival samples were obtained, with 176 children sampled in both 1989 and 1995. 31.1% (233/749) were positive by PCR-enzyme immunoassay, and 76% (176/233) of the positives were sequenced in variable domains (VD) 1 to 4 (22 children in both 1989 and 1995). Twenty-six ompA genotypes of serovar A, and 19 of B/Ba were identified, and only 20% of genotypes identified in 1995 matched those found in 1989. In particular, B/Ba genotypes exhibited a 15-base region in VD 2 with increased nucleotide substitution, and these types were associated with age and water availability. Homotypic infection and infection with multiple genotypes and high chlamydial load did predict subsequent severe trachoma (odds ratio (OR) = 10.14, 95% confidence interval (CI): 1.71, 60.23; OR = 6.40, 95% CI: 0.75, 54.41; OR = 6.74, 95% CI: 0.82, 55.38, respectively). And, multitypic infection was clustered with residence of village and associated with familial cattle ownership. In conclusion, high ompA polymorphism and the inability of some hosts to clear infection with the same ompA genotype suggest two distinct but converging mechanisms of endemic severe trachoma.     

Mechanism of fenretinide (4-HPR)-induced cell death

4-HPR (fenretinide) is a synthetic analog of retinoic acid (RA) whose potential as a chemopreventative agent has gained support from in vitro and animal experiments and in limited clinical trials. Comparative analyses of cellular, biochemical, and molecular properties of fenretinide with RA using various tissue culture cells reveal that a key distinction between these two retinoids lies in the ability of fenretinide to induce programmed cell death, also known as apoptosis. Here we review the composite evidence for induction of apoptosis in fenretinide-treated cells. Assays used to validate apoptosis in various cell types are also summarized. Apoptosis in response to fenretinide primarily occurs by a receptor-independent mechanism, which is accompanied by increases in signaling molecules, e.g., ceramide, and cysteine-dependent aspartate-directed proteases, termed caspases, including execution caspase-3. Both caspase-3 inhibitor DEVD-CHO and ceramide synthase inhibitor fumonisin B₁ (FB₁) block fenretinide-induced apoptosis. Increase in caspase-3 appears to result from fenretinide-elicited stabilization of procaspase-3 zymogen. We also review apoptotic regulatory proteins such as inhibitor of apoptosis (IAPs) and second mitochondria-derived activator of caspase (SMACs) that participate in the coordinate control of caspase activities. The existence of a large number of proteins capable of modulating apoptosis via activation or inhibition of caspases, coupled with the fact that both the initiation and execution phases of apoptosis utilize pre-existing zymogens, which, once set in motion, culminates in an irreversible apoptotic cascade, raise the possibility that the on/off switch of apoptosis is linked to an intricate intracellular regulatory network, capable of responding to external stimuli such as fenretinide. This network functions to provide checks/balances of the need for apoptosis as well as to minimize and prevent untimely errors in apoptosis. We suggest that dynamic and coordinated regulation of apoptosis by such a hypothetical network in vivo may involve co-localization of pro- and anti-apoptotic proteins and their respective activators/inhibitors in a macromolecular modular unit which we propose to be named caspasomes. Fenretinide also induces apoptosis by elevating reactive oxygen species (ROS), unrelated to changes in ceramide-caspases. Thus multiple, distinct pathways contribute to the induction of apoptosis by fenretinide.     

Hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase

Human angiotensin-converting enzyme-related carboxypeptidase (ACE2) is a zinc metalloprotease whose closest homolog is angiotensin I-converting enzyme. To begin to elucidate the physiological role of ACE2, ACE2 was purified, and its catalytic activity was characterized. ACE2 proteolytic activity has a pH optimum of 6.5 and is enhanced by monovalent anions, which is consistent with the activity of ACE. ACE2 activity is increased approximately 10-fold by Cl(-) and F(-) but is unaffected by Br(-). ACE2 was screened for hydrolytic activity against a panel of 126 biological peptides, using liquid chromatography-mass spectrometry detection. Eleven of the peptides were hydrolyzed by ACE2, and in each case, the proteolytic activity resulted in removal of the C-terminal residue only. ACE2 hydrolyzes three of the peptides with high catalytic efficiency: angiotensin II () (k(cat)/K(m) = 1.9 x 10(6) m(-1) s(-1)), apelin-13 (k(cat)/K(m) = 2.1 x 10(6) m(-1) s(-1)), and dynorphin A 1-13 (k(cat)/K(m) = 3.1 x 10(6) m(-1) s(-1)). The ACE2 catalytic efficiency is 400-fold higher with angiotensin II () as a substrate than with angiotensin I (). ACE2 also efficiently hydrolyzes des-Arg(9)-bradykinin (k(cat)/K(m) = 1.3 x 10(5) m(-1) s(-1)), but it does not hydrolyze bradykinin. An alignment of the ACE2 peptide substrates reveals a consensus sequence of: Pro-X((1-3 residues))-Pro-Hydrophobic, where hydrolysis occurs between proline and the hydrophobic amino acid.     

Generation of double-stranded breaks in hypernegatively supercoiled DNA by Drosophila topoisomerase IIIbeta,a type IA enzyme

Drosophila topoisomerase (topo) IIIbeta is a member of the type IA family of DNA topoisomerases, which generates a single-stranded break to form a covalent complex with the 5'-end of DNA. We show here that a purified preparation of topo IIIbeta is able to convert a hypernegatively supercoiled substrate into primarily nicked, but also linear, DNA at enzyme/DNA molar ratios of 5:1 or greater. Although the optimal temperature for the relaxation activity is between 37 and 45 degrees C, maximal cleavage occurs between 23 and 30 degrees C, a temperature range that is more physiologically relevant for fruit flies. The cleavage products require protease treatment to enter the gel, they are stable over time, they are reversible, and they are not observed with a Y332F active site mutant, which further supports the idea that topo IIIbeta possesses an endonucleolytic cleavage activity. This cleavage activity appears to be specific for highly unwound, or single strand-containing substrates. Southern blot analysis of the cleavage products demonstrates that the topo IIIbeta cleavage activity is concentrated primarily in highly A/T-rich regions. These results suggest that topo IIIbeta may function as a reversible endonuclease in vivo by recognizing and cleaving/rejoining DNA structures with single-stranded character.