This Virtual Special Issue of Mutation Research is dedicated to Professor Bruce N. Ames in recognition of his 90th birthday in December 2018. His pioneering work in the field of chemical mutagenesis ...resulted in the well-known Ames Salmonella/mammalian-microsome mutagenicity assay that has played a pivotal role since the 1970s in the field of genetic toxicology. The assay is usually referred to as the Ames test and was gradually developed by improving the sensitivity of the test based on available scientific discoveries. When a chemical is determined to be a mutagen in the Ames test it has the potential of also being a carcinogen based on the somatic mutation theory of carcinogenesis. For nearly 20 years, I was responsible for running the Ames mutagenicity testing laboratory at SRI International on a contractual basis with commercial and government funding. Now I feel privileged having been given the opportunity to provide a historical overview of how the Ames test was developed.
The 1960s witnessed detailed studies on the genetic properties of a large number of histidine-requiring mutants of Salmonella typhimurium. The early 1970s saw development of selected strains, the ...Ames strains, for use in rapid, cheap, sensitive, and manipulable tests of chemicals and chemical mixtures for genotoxic activities. Our contribution during this latter period was an investigation into the mutagenicity of hycanthone and some of its analogues. Some lessons that this study provided are enumerated. Hycanthone is definitely a liver carcinogen in rodents predisposed by hepatic hyperplasia. Between 1969 and 1975, an estimated total of 100 kg of hycanthone was injected into some 1,000,000 humans with liver hyperplasia caused by infections with parasites. It may now be possible to assess directly the long-term impacts of hycanthone in man.
The pH dependence of the Fe(III) reduction potential, E
0′, for yeast cytochrome
c peroxidase (yCcP) and three distal pocket mutants, CcP(H52L), CcP(H52Q), and CcP(R48L/W51L/H52L), has been ...determined between pH 4 and 8. E
0′ values at pH 7.0 for the yCcP, CcP(H52L), CcP(H52Q), and CcP(R48L/W51L/H52L) are −
189, −
170, −
224, and −
146
mV, respectively. A heme-linked ionization in the reduced enzyme affects the reduction potential for yCcP and all three mutants. Apparent pK
A values for the heme-linked ionization are 7.5
±
0.2, 6.5
±
0.3, 6.4
±
0.2, and 7.0
±
0.3 for yCcP and the H52L, H52Q, and R48L/W51L/H52L mutants, respectively. A cooperative, two-proton ionization causing a spectroscopically-detectable transition was observed in the ferrous states of yCcP, CcP(H52L) and CcP(H52Q), with apparent pK
A values of 7.7
±
0.2, 7.4
±
0.1 and 7.8
±
0.1, respectively. These data indicate that: (1) the distal histidine in CcP is not the site of proton binding upon reduction of the ferric CcP, (2) the distal histidine is not one of the two groups involved in the cooperative, two-proton ionization observed in ferrous CcP, and (3) the proton-binding site is not involved in the cooperative, two-proton ionization observed in the reduced enzyme.
The pH dependence of E
0′ for three distal histidine mutants of cytochrome
c peroxidase is similar to that of wild-type enzyme.
Display omitted
Our previous study on cyclomaltodextrin glucanotransferase (CGTase) by chemical modification implied the importance of one or two histidine residues in the cyclization reaction of the enzyme. Based ...on a computer modelled three-dimensional structure of the CGTase, five histidine residues were chosen as targets for the site-directed mutagenesis. The histidine residues 98, 140, 233 and 327 were replaced by aspartate and His-177 by proline using polymerase chain reaction-mediated techniques. The CGTase variants H98D, H140D, H233D and H327D resulted in a profound decrease in the cyclizing and amylolytic activities, while mutation H177P had little influence on the activities but affected the thermal stability and the width of the pH optimum. It is suggested that His-98 functions as (or as a significant part of) the subsite 2 for the binding of the substrate in CGTase and therefore H98D destabilizes the intermediate for cyclization, but does not markedly affect the hydrolytic reactions. Mutants H140D and H233D produced only minor amounts of α-cyclodextrin, did not exhibit substrate inhibition with maltotriose and showed non-Michaelis-Menten kinetics. It is proposed that the variants H140D, H233D and H327D cause steric hindrances near the active center, while mutation H177D has similar consequences on the same site spatially.
Although lipid peroxidation associated with oxidative stress can result in cellular death, sub-lethal lipid peroxidation can gradually resolve with return to the pre-exposure state. We have shown ...that resolution of lipid peroxidation is greatly delayed in lungs or cells that are null for peroxiredoxin 6 (Prdx6) and that both the phospholipase A2 and the GSH peroxidase activities of Prdx6 are required for a maximal rate of recovery. Like other peroxiredoxins, Prdx6 can reduce H2O2 and short chain hydroperoxides, but in addition can directly reduce phospholipid hydroperoxides. This study evaluated the relative role of these two different peroxidase activities of Prdx6 in the repair of peroxidized cell membranes. The His26 residue in Prdx6 is an important component of the binding site for phospholipids. Thus, we evaluated the lungs from H26A-Prdx6 expressing mice and generated H26A-Prdx6 expressing pulmonary microvascular endothelial cells (PMVEC) by lentiviral infection of Prdx6 null cells to compare with wild type in the repair of lipid peroxidation. Isolated lungs and PMVEC were exposed to tert-butyl hydroperoxide and mice were exposed to hyperoxia (> 95% O2). Assays for lipid peroxidation in wild type control and mutant lungs and cells showed ~4-fold increase at end-exposure. Control lungs and cells showed gradual resolution during a post-exposure recovery period. However, there was no recovery from lipid peroxidation by H26A-Prdx6 lungs or PMVEC. These studies confirm an important role for Prdx6 in recovery from membrane lipid peroxidation and indicate that reduction of H2O2 or short chain hydroperoxides does not play a role in the recovery process.
Conclusion: all 3 enzymatic activities of Prdx6 contribute to the reversal of cell membrane phospholipid peroxidation. Display omitted
•Repair of peroxidized lipids did not occur with H26A-Prdx6 Delete semicolons;mutation.•Repair reflects the phospholipid hydroperoxidase and PLA2 activities of Prdx6;Move to next with "bullet mark" "P"eroxidase activity with small hydroperoxides and H2O2 does not play a role in repair.
Engineered antibodies with pH responsive cell surface target antigen-binding affinities that decrease at the acidic pH (5.5-5.8) within the endosomes have been found to have reduced susceptibility to ...degradation within the lysosomes and increased serum half-life. Such pH responsive recombinant antibodies have been developed for the treatment of cancer and cardiovascular disease. Engineered tenth type III human fibronectin (Fn3) domains are emerging as a class of target antigen-binding biopharmaceuticals that could complement or be superior to recombinant antibodies in a number of biomedical contexts. As such, there is strong motivation for demonstrating the feasibility of engineering Fn3s with pH responsive antigen binding behavior that could lead to improved Fn3 pharmacokinetics.
A yeast surface-displayed Fn3 histidine (His) mutant library screening approach yielded epidermal growth factor receptor (EGFR)-binding Fn3 domains with EGFR binding affinities that markedly decrease at endosomal pH; the first reported case of engineering Fn3s with pH responsive antigen binding. Yeast surface-displayed His mutant Fn3s, which contain either one or two His mutations, have equilibrium binding dissociation constants (KDs) that increase up to four-fold relative to wild type when pH is decreased from 7.4 to 5.5. Assays in which Fn3-displaying yeast were incubated with soluble EGFR after ligand-free incubation in respective neutral and acidic buffers showed that His mutant Fn3 pH responsiveness is due to reversible changes in Fn3 conformation and/or EGFR binding interface properties rather than irreversible unfolding.
We have established a generalizable method for efficiently constructing and screening Fn3 His mutant libraries that could enable both our laboratory and others to develop pH responsive Fn3s for use in a wide range of biomedical applications.
DNase II is an acid endonuclease that is involved in the degradation of exogenous DNA and is important for DNA fragmentation and degradation during cell death. In an effort to understand its ...catalytic mechanism, we constructed plasmids encoding nine different histidine (H)-to-leucine (L) mutants for porcine DNase II and examined the enzyme properties of the expressed mutant proteins. Of the mutants, all but H132L were secreted into the medium of expressing cells. Six of the mutated DNase II proteins (H41L, H109L, H206L, H207L, H274L and H322L) showed enzyme activity, whereas the H115L, H132L and H297L mutants exhibited very little activity. The H115L and H297L mutants were found to undergo correct protein folding, but were inactive. To further examine these mutants, we expressed H115A and H297A DNase II mutants; these mutants were inactive, but their DNase activities could be rescued with imidazole, indicating that His115 and His297 are likely to function as a general acid and a general base respectively in the catalytic centre of the enzyme. In contrast with the secreted mutants, the H132L mutant protein was found in cell lysates within 16 h after transfection. This protein was inactive, improperly folded and was drastically degraded via the proteosomal pathway after 24 h. The polypeptide of another substitution for His132 with lysine resulted in the misfolded form being retained in endoplasmic reticulum.
Two cDNA clones of rat hepatic hydroxysteroid sulfotransferase (ST) (ST-40 and ST-20) were isolated and expressed in
Escherichia coli cells. Several histidine residues in their coding regions are ...highly conserved in the ST superfamily, and histidine mutants were constructed by site-directed mutagenesis. The substitution of alanine or lysine for the histidine at position 98 in the ST-40 enzyme resulted in a loss of ST activities toward dehydroepiandrosterone (DHEA), androsterone (AD) and cortisol (CS). The mutation of histidine 98 into alanine abolished the specific binding to 3′-phosphoadenosine 5′-phosphate agarose, suggesting that the residue is located at a critical position in the 3′-phosphoadenosine 5′-phosphosulfate (PAPS) binding site. In the ST-20 enzyme, the replacement of histidine 98 with alanine also resulted in the loss of ST activity toward its preferential substrate, CS. In the ST-40 enzyme, the mutation at histidine 256 into alanine markedly reduced CS-ST activity, but DHEA-ST activity was not changed. Furthermore, selective decrease in CS-ST activity was also observed in the alanine mutant at lysine 254 or at asparagine 255 of the ST-40 enzyme. Kinetic analysis on the ST-40 and its mutant at asparagine 255 indicated that the
K
m value for CS was significantly increased in the mutant without any change in the
K
m values for 3′-phosphoadenosine 5′-phosphosulfate and DHEA. Inhibition studies demonstrated that DHEA-ST activity was competitively inhibited by AD, but not by CS in the ST-40 enzyme, whereas triethylamine, a noncompetitive inhibitor of hydroxysteroid ST, inhibited DHEA-ST activity in the ST-40 enzyme but did not inhibit CS-ST activity in either ST-40 or ST-20 enzymes. These data provide evidence that DHEA and CS bind to different sites, which probably function in a different manner in the ST-40 enzyme.
PDF
