N-Methyl-N′-nitro-N-nitrosoguanidine induced DNA sequence alteration; non-random components in alkylation mutagenesis

Our approach to the study of how the molecular nature of DNA modulates the behavior of mutational sites involves the characterisation of distributions of mutations. The Escherichia coli lacI genetic/M13 cloning system allows the comparison of base substitution frequencies at a large number of sites. The observed distribution of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)-induced G:C → A:T transition (the predominant event), and A:T → G:C transition (a relatively rare event), is strikingly non-random. Some sites of G:C → A:T mutation are almost 100 times more often mutated by MNNG than the least susceptible sites. Sites of mutation, however, do not display a continuum of mutability, but rather can be strictly demarcated by their 5′ flanking base. Sites with a high frequency of occurrence share a common sequence motif, namely 5′-R-G-N-3′, which is the sole apparent feature that distinguishes them from sites less commonly mutated (i.e. 5′-Y-G-N-3′). A corollary of this defined site specificity is the absence of a strand bias in MNNG-induced lacI^−d mutation. The availability of specific or non-specific alkylation-repair systems does not appear to alter the distribution of mutation, which suggests that the observed mutational distribution is a direct reflection of the initial damage distribution. MNNG does not belong to that class of compounds typified by ultraviolet light or 4-nitroquinoline-N-oxide which exhibit both random and non-random components of mutagenesis.     

Mechanisms of spontaneous mutation in DNA repair-proficient Escherichia coli

This paper describes the DNA sequence analysis of 729 independent spontaneous lacI⁻ mutation This total is comprised of 478 novel mutations and 251 previously described events, and therefore should allow a more comprehensive view of spontaneous mutation in Escherichia coli. The spectrum is dominated by a hotspot (71% of all events). Mutations at this site consist of related addition and deletion events involving a number of repetitive sequences. Here we discuss how the frequency and proportion of these events vary in different DNA repair-deficient genetic backgrounds. The distribution of non-hotspot events includes base substitutions (38%), deletions (35%), frameshifts (14%), duplications (4%) and insertion elements (4%). G:C → A:T events dominate among base substitutions, while G:C → C:G events are the least common; the remaining types of base substitution are equally represented. Among deletions, a significant number do not display repeated sequences at their endpoints (26/72). However, almost all multiply recovered events (15/17) possess repeated sequences capable of accounting for the deletion endpoints. Similarily, over of all duplications recovered (5/7) display repeated endpoints. Single-base frameshifts are equally divided between A:T and G:C sites, in each case (−) 1 events occur 3-fold more frequently that (+)1 events. A comparative analysis of each mutational class recovered to lacI⁻ spectra available in a variety of DNA repair/metabolism-deficient strains is presented here in an attempt to assess possible contributions from chemical, physical and enzymic sources of damage.     

Spontaneous and 9-aminoacridine-induced frameshift mutagenesis: second-site frameshift mutation within the N-terminal region of the lacI gene of Escherichia coli

Summary A novel forward mutational system, based on the acquisition of an I^q-d dominant phenotype from an initial I^q− recessive state, was used to identify second-site frameshift mutation [±1(±3 _n ) events] within the N-terminal region of thelacI gene ofEscherichia coli. The DNA sequences are described of forty-six spontaneous and twenty 9-aminoacridine(9-AA)-induced second site mutations. Although −1 frameshift events dominate both spectra, the nature and site specificity of these events clearly distinguish two mutational distributions. The spontaneous distribution contains two −(A: T) frameshift hotspots; one within a monotonic A₅ run (9 occurrences), the other at a 5′-CACAACAAC-3′ sequence (12 occurrences). In contrast 17 of the 20 mutations recovered after 9-AA treatment involve the loss of a G: C pair, 14 of which occur at a single site (5′-CGGGC-3′). The striking specificity of the observed mutational hotspots is of interest since this open genetic target contains similar sequences which were infrequently recovered.     

Effect of a neutralizing monoclonal antibody to cholesteryl ester transfer protein on the redistribution of apolipoproteins A-IV and E among human lipoproteins

The effect of inhibiting cholesteryl ester transfer protein (CETP) on the in vitro redistribution of apolipoproteins(apo) A-IV and apoE among lipoproteins in whole plasma was studied in seven normal male subjects. Plasmas were incubated in the presence of a purified monoclonal antibody TP2 (Mab TP2) that neutralizes the activity of CETP. Mab TP2 had no effect on lecithin:cholesterol acyltransferase (LCAT) activity. Prior to and following a 6-h incubation at 37 degrees C in the presence of Mab TP2 or a control mouse myeloma immunoglobulin (IgG), plasmas were gel-filtered on Sephacryl S-300 and the distribution of apoA-IV and apoE among lipoproteins was determined by radioimmunoassay. Incubation (i.e., with active LCAT and CETP) increased the amount of apoA-IV associated with lipoproteins by 240%. When CETP activity was inhibited during incubation, the amount of apoA-IV that became lipoprotein-associated was significantly increased (315% of basal). Plasma incubation also caused a redistribution of apoE from high density lipoproteins (HDL) to larger lipoproteins (131% of basal); however, when CETP was inhibited, significantly greater amounts of apoE became associated with the larger particles (155% of basal). These effects were observed in all seven subjects. Increased movement of apoE from HDL to triglyceride-rich particles was not due to displacement by apoA-IV since loss of apoE from HDL was still observed when no movement of apoA-IV onto HDL occurred, such as during LCAT or combined LCAT and CETP inhibition. We speculate that low CETP activity (e.g., in species such as rats) may lead to an increased content of HDL apoA-IV and also to apoE enrichment of triglyceride-rich lipoproteins, augmenting their clearance.     

The base of the proteasome regulatory particle exhibits chaperone-like activity

Protein substrates of the proteasome must apparently be unfolded and translocated through a narrow channel to gain access to the proteolytic active sites of the enzyme. Protein folding in vivo is mediated by molecular chaperones. Here, to test for chaperone activity of the proteasome, we assay the reactivation of denatured citrate synthase. Both human and yeast proteasomes stimulate the recovery of the native structure of citrate synthase. We map this chaperone-like activity to the base of the regulatory particle of the proteasome, that is, to the ATPase-containing assembly located at the substrate-entry ports of the channel. Denatured but not native citrate synthase is bound by the base complex. Ubiquitination of citrate synthase is not required for its binding or refolding by the base complex of the proteasome. These data suggest a model in which ubiquitin-protein conjugates are initially tethered to the proteasome by specific recognition of their ubiquitin chains; this step is followed by a nonspecific interaction between the base and the target protein, which promotes substrate unfolding and translocation.     

Ubiquitin-proteasome-dependent degradation of a mitofusin, a critical regulator of mitochondrial fusion

The mitochondrion is a dynamic membranous network whose morphology is conditioned by the equilibrium between ongoing fusion and fission of mitochondrial membranes. In the budding yeast, Saccharomyces cerevisiae, the transmembrane GTPase Fzo1p controls fusion of mitochondrial outer membranes. Deletion or overexpression of Fzo1p have both been shown to alter the mitochondrial fusion process indicating that maintenance of steady-state levels of Fzo1p are required for efficient mitochondrial fusion. Cellular levels of Fzo1p are regulated through degradation of Fzo1p by the F-box protein Mdm30p. How Mdm30p promotes degradation of Fzo1p is currently unknown. We have now determined that during vegetative growth Mdm30p mediates ubiquitylation of Fzo1p and that degradation of Fzo1p is an ubiquitin-proteasome-dependent process. In vivo, Mdm30p associates through its F-box motif with other core components of Skp1-Cullin-F-box (SCF) ubiquitin ligases. We show that the resulting SCF(Mdm30p) ligase promotes ubiquitylation of Fzo1p at mitochondria and its subsequent degradation by the 26S proteasome. These results provide the first demonstration that a cytosolic ubiquitin ligase targets a critical regulatory molecule at the mitochondrial outer membrane. This study provides a framework for developing an understanding of the function of Mdm30p-mediated Fzo1p degradation in the multistep process of mitochondrial fusion.     

Cold habituation does not improve manual dexterity during rest and exercise in 5 °C

When exposed to a cold environment, a barehanded person experiences pain, cold sensation, and reduced manual dexterity. Both acute (e.g. exercise) and chronic (e.g. cold acclimatization or habituation) processes might lessen these negative effects. The purpose of this experiment was to determine the effect of cold habituation on physiology, perception, and manual dexterity during rest, exercise, and recovery in 5 °C. Six cold weather athletes (CWA) and eight non habituated men (NON) volunteered to participate in a repeated measures cross-over design. The protocol was conducted in 5 °C and was 90 min of resting cold exposure, 30 min of cycle ergometry exercise (50 % VO_{2 peak}), and 60 min of seated recovery. Core and finger skin temperature, metabolic rate, Purdue Pegboard dexterity performance, hand pain, thermal sensation, and mood were quantified. Exercise-induced finger rewarming (EIFRW) was calculated for each hand. During 90 min of resting exposure to 5 °C, the CWA had a smaller reduction in finger temperature, a lower metabolic rate, less hand pain, and less negative mood. Despite this cold habituation, dexterity performance was not different between groups. In response to cycle ergometry, EIFRW was greater in CWA (~12 versus 7 °C) and occurred at lower core temperatures (37.02 versus 37.31 °C) relative to NON but dexterity was not greater during post-exercise recovery. The current data indicate that cold habituated men (i.e., CWA) do not perform better on the Purdue Pegboard during acute cold exposure. Furthermore, despite augmented EIFRW in CWA, dexterity during post-exercise recovery was similar between groups.     

The zinc finger of the CSN-associated deubiquitinating enzyme USP15 is essential to rescue the E3 ligase Rbx1

The COP9 signalosome (CSN) is a conserved protein complex found in all eukaryotic cells and involved in the regulation of the ubiquitin (Ub)/26S proteasome system. It binds numerous proteins, including the Ub E3 ligases and the deubiquitinating enzyme Ubp12p, the S. pombe ortholog of human USP15. We found that USP15 copurified with the human CSN complex. Isolated CSN complex exhibited protease activity that deubiquitinated poly-Ub substrates and was completely inhibited by o-phenanthroline (OPT), a metal-chelating agent. Surprisingly, the recombinant USP15 was also not able to cleave isopeptide bonds of poly-Ub chains in presence of OPT. Detailed analysis of USP sequences led to the discovery of a novel zinc (Zn) finger in USP15 and related USPs. Mutation of a single conserved cysteine residue in the predicted Zn binding motif resulted in the loss of USP15 capability to degrade poly-Ub substrates, indicating that the Zn finger is essential for the cleavage of poly-Ub chains. Moreover, pulldown experiments demonstrated diminished binding of tetra-Ub to mutated USP15. Cotransfection of USP15 and the Ub ligase Rbx1 revealed that the wild-type deubiquitinating enzyme, but not the USP15 mutant with a defective Zn finger, stabilized Rbx1 toward the Ub system, most likely by reversing poly/autoubiquitination. In summary, a functional Zn finger of USP15 is needed to maintain a conformation essential for disassembling poly-Ub chains, a prerequisite for rescuing the E3 ligase Rbx1.     

Development of a high-throughput screening assay for inhibitors of aggrecan cleavage using luminescent oxygen channeling (AlphaScreen )

Aggrecan is one of the most important structural components of joint cartilage, and members of the metalloprotease (MMP) and ADAM (a disintegrin and metalloproteinase) protease families have been shown to degrade aggrecan in vivo. A robust assay for aggrecan-degrading activity suitable for high-throughput screening (HTS) was set up and measured using AlphaScreen. In this technology, beads brought into proximity through cross-linking and stimulated with laser light generate a signal through luminescent oxygen tunneling, the outcome of which is a time-resolved fluorescent signal. Specific antibodies to the carbohydrate side chains of aggrecan were harnessed to create a scaffold whereby aggrecan could form a cross-link between donor and acceptor AlphaScreen detector beads. Digested aggrecan, which failed to form a cross-link, generated no signal, so that inhibitors of the digestion could be detected as a restoration of signal. The development of this assay and its validation for HTS are described in this report.     

Comparison of assay technologies for a nuclear receptor assay screen reveals differences in the sets of identified functional antagonists

Many assay technologies currently exist to develop high-throughput screening assays, and the number of choices continues to increase. Results from a previous study comparing assay technologies in our laboratory do not support the common assumption that the same hits would be found regardless of which assay technology is used. To extend this investigation, a nuclear receptor antagonist assay was developed using 3 assay formats: AlphaScreen, time-resolved fluorescence (TRF), and time-resolved fluorescence resonance energy transfer (TR-FRET). Compounds ( approximately 42000) from the Novartis library were evaluated in all 3 assay formats. A total of 128 compounds were evaluated in dose-response experiments, and 109 compounds were confirmed active from all 3 formats. The AlphaScreen, TRF, and TR-FRET assay technologies identified 104, 23, and 57 active compounds, respectively, with only 18 compounds active in all 3 assay formats. A total of 128 compounds were evaluated in a cell-based functional assay, and 35 compounds demonstrated activity in this cellular assay. Furthermore, 34, 11, and 16 hits that were originally identified in the dose-response experiment by AlphaScreen, TRF, and TR-FRET assay technologies, respectively, were functionally active. The results of the study indicated that AlphaScreen identified the greatest number of functional antagonists.