Alexander Kurosky

Haptoglobin: The Evolutionary Product of Duplication, Unequal Crossing Over, and Point Mutation

The question of how genetic material controls phenotypic expression was posed 60 years ago by Hermann Muller (1922), but the ability to answer the question awaited the development of biochemical techniques capable of dissecting and comparing purified proteins (Moore et al., 1958; Edman and Sjoquist, 1956; Ingram, 1957). Human haptoglobin has served as an unexpected source of information relating the mechanisms involved in genetic rearrangements to the linear sequence of amino acids within a protein. After developing a potent technique, starch gel electrophoresis, which proved capable of resolving and identifying previously unknown inherited variations in many human plasma proteins, Oliver Smithies and co-workers (Smithies, 1959; Smithies et al., 1962a, 1962b) demonstrated that serum haptoglobin molecules from different individuals varied one from another in a heritable way. Haptoglobin has served as an impressive paradigm of how genetic rearrangements can construct a polymorphic system and how, in the evolutionary process, old proteins can obtain new functions by utilizing amino acid sequences which have been ever so slightly altered. Excellent reviews have been written about haptoglobin, which can be consulted for further detail (Javid, 1978; Putnam, 1975; Pintera, 1971; Sutton, 1970; Giblett, 1969; Kirk, 1968; Schultze and Heremans, 1966). This review will emphasize recent developments in analyzing the composition of the haptoglobin molecules and the evolutionary implications of these findings.

Purification and Characterization of Human NTH1, a Homolog of Escherichia coli Endonuclease III

The human endonuclease III (hNTH1), a homolog of the Escherichia coli enzyme (Nth), is a DNA glycosylase with abasic (apurinic/apyrimidinic (AP)) lyase activity and specifically cleaves oxidatively damaged pyrimidines in DNA. Its cDNA was cloned, and the full-length enzyme (304 amino acid residues) was expressed as a glutathione S-transferase fusion polypeptide in E. coli. Purified wild-type protein with two additional amino acid residues and a truncated protein with deletion of 22 residues at the NH2 terminus were equally active and had absorbance maxima at 280 and 410 nm, the latter due to the presence of a [4Fe-4S]cluster, as in E. coli Nth. The enzyme cleaved thymine glycol-containing form I plasmid DNA and a dihydrouracil (DHU)-containing oligonucleotide duplex. The protein had a molar extinction coefficient of 5.0 × 104 and a pI of 10. With the DHU-containing oligonucleotide duplex as substrate, theK m was 47 nm, andk cat was ∼0.6/min, independent of whether DHU paired with G or A. The enzyme carries out β-elimination and forms a Schiff base between the active site residue and the deoxyribose generated after base removal. The prediction of Lys-212 being the active site was confirmed by sequence analysis of the peptide-oligonucleotide adduct. Furthermore, replacing Lys-212 with Gln inactivated the enzyme. However, replacement with Arg-212 yielded an active enzyme with about 85-fold lower catalytic specificity than the wild-type protein. DNase I footprinting with hNTH1 showed protection of 10 nucleotides centered around the base lesion in the damaged strand and a stretch of 15 nucleotides (with the G opposite the lesion at the 5′-boundary) in the complementary strand. Immunological studies showed that HeLa cells contain a single hNTH species of the predicted size, localized in both the nucleus and the cytoplasm.

Mitochondrial dysfunction increases allergic airway inflammation

The prevalence of allergies and asthma among the world’s population has been steadily increasing due to environmental factors. It has been described that exposure to ozone, diesel exhaust particles, or tobacco smoke exacerbates allergic inflammation in the lungs. These environmental oxidants increase the levels of cellular reactive oxygen species (ROS) and induce mitochondrial dysfunction in the airway epithelium. In this study, we investigated the involvement of preexisting mitochondrial dysfunction in the exacerbation of allergic airway inflammation. After cellular oxidative insult induced by ragweed pollen extract (RWE) exposure, we have identified nine oxidatively damaged mitochondrial respiratory chain-complex and associated proteins. Out of these, the ubiquinol-cytochrome c reductase core II protein (UQCRC2) was found to be implicated in mitochondrial ROS generation from respiratory complex III. Mitochondrial dysfunction induced by deficiency of UQCRC2 in airway epithelium of sensitized BALB/c mice prior the RWE challenge increased the Ag-induced accumulation of eosinophils, mucin levels in the airways, and bronchial hyperresponsiveness. Deficiency of UQCRC1, another oxidative damage-sensitive complex III protein, did not significantly alter cellular ROS levels or the intensity of RWE-induced airway inflammation. These observations suggest that preexisting mitochondrial dysfunction induced by oxidant environmental pollutants is responsible for the severe symptoms in allergic airway inflammation. These data also imply that mitochondrial defects could be risk factors and may be responsible for severe allergic disorders in atopic individuals.

Primary and secondary structural analyses of glutathione S-transferase π from human placenta☆

Abstract The primary structure of glutathione S-transferase (GST) π from a single human placenta was determined. The structure was established by chemical characterization of tryptic and cyanogen bromide peptides as well as automated sequence analysis of the intact enzyme. The structural analysis indicated that the protein is comprised of 209 amino acid residues and gave no evidence of post-translational modifications. The amino acid sequence differed from that of the deduced amino acid sequence determined by nucleotide sequence analysis of a cDNA clone (Kano, T., Sakai, M., and Muramatsu, M., 1987, Cancer Res. 47 , 5626–5630) at position 104 which contained both valine and isoleucine whereas the deduced sequence from nucleotide sequence analysis identified only isoleucine at this position. These results demonstrated that in the one individual placenta studied at least two GST π genes are coexpressed, probably as a result of allelomorphism. Computer assisted consensus sequence evaluation identified a hydrophobic region in GST π (residues 155–181) that was predicted to be either a buried transmembrane helical region or a signal sequence region. The significance of this hydrophobic region was interpreted in relation to the mode of action of the enzyme especially in regard to the potential involvement of a histidine in the active site mechanism. A comparison of the chemical similarity of five known human GST complete enzyme structures, one of π, one of μ, two of α, and one microsomal, gave evidence that all five enzymes have evolved by a divergent evolutionary process after gene duplication, with the microsomal enzyme representing the most divergent form.

Nuclear Heat Shock Response and Novel Nuclear Domain 10 Reorganization in Respiratory Syncytial Virus-Infected A549 Cells Identified by High-Resolution Two-Dimensional Gel Electrophoresis

ABSTRACT The pneumovirus respiratory syncytial virus (RSV) is a leading cause of epidemic respiratory tract infection. Upon entry, RSV replicates in the epithelial cytoplasm, initiating compensatory changes in cellular gene expression. In this study, we have investigated RSV-induced changes in the nuclear proteome of A549 alveolar type II-like epithelial cells by high-resolution two-dimensional gel electrophoresis (2DE). Replicate 2D gels from uninfected and RSV-infected nuclei were compared for changes in protein expression. We identified 24 different proteins by peptide mass fingerprinting after matrix-assisted laser desorption ionization-time of flight mass spectrometry (MS), whose average normalized spot intensity was statistically significant and differed by ±2-fold. Notable among the proteins identified were the cytoskeletal cytokeratins, RNA helicases, oxidant-antioxidant enzymes, the TAR DNA binding protein (a protein that associates with nuclear domain 10 [ND10] structures), and heat shock protein 70- and 60-kDa isoforms (Hsp70 and Hsp60, respectively). The identification of Hsp70 was also validated by liquid chromatography quadropole-TOF tandem MS (LC-MS/MS). Separate experiments using immunofluorescence microscopy revealed that RSV induced cytoplasmic Hsp70 aggregation and nuclear accumulation. Data mining of a genomic database showed that RSV replication induced coordinate changes in Hsp family proteins, including the 70, 70-2, 90, 40, and 40-3 isoforms. Because the TAR DNA binding protein associates with ND10s, we examined the effect of RSV infection on ND10 organization. RSV induced a striking dissolution of ND10 structures with redistribution of the component promyelocytic leukemia (PML) and speckled 100-kDa (Sp100) proteins into the cytoplasm, as well as inducing their synthesis. Our findings suggest that cytoplasmic RSV replication induces a nuclear heat shock response, causes ND10 disruption, and redistributes PML and Sp100 to the cytoplasm. Thus, a high-resolution proteomics approach, combined with immunofluorescence localization and coupled with genomic response data, yielded unexpected novel insights into compensatory nuclear responses to RSV infection.

The Chemistry and Biology of Cholera Toxi

(1980). The Chemistry and Biology of Cholera Toxi. Critical Reviews in Biochemistry: Vol. 9, No. 3, pp. 171-206.

Variable expression of pathogenesis-related protein allergen in mountain cedar (Juniperus ashei) pollen

Allergic diseases have been increasing in industrialized countries. The environment is thought to have both direct and indirect modulatory effects on disease pathogenesis, including alterating on the allergenicity of pollens. Certain plant proteins known as pathogenesis-related proteins appear to be up-regulated by certain environmental conditions, including pollutants, and some have emerged as important allergens. Thus, the prospect of environmentally regulated expression of plant-derived allergens becomes yet another potential environmental influence on allergic disease. We have identified a novel pathogenesis-related protein allergen, Jun a 3, from mountain cedar (Juniperus ashei) pollen. The serum IgE from patients with hypersensitivity to either mountain cedar or Japanese cedar were shown to bind to native and recombinant Jun a 3 in Western blot analysis and ELISA. Jun a 3 is homologous to members of the thaumatin-like pathogenesis-related (PR-5) plant protein family. The amounts of Jun a 3 extracted from mountain cedar pollen varied up to 5-fold in lots of pollen collected from the same region in different years and between different regions during the same year. Thus, Jun a 3 may contribute not only to the overall allergenicity of mountain cedar pollen, but variable levels of Jun a 3 may alter the allergenic potency of pollens produced under different environmental conditions.

Differential expression of α, μ and π classes of isozymes of glutathione S-transferase in bovine lens, cornea, and retina

Abstract Isozyme characterization of glutathione S-transferase (GST) isolated from bovine ocular tissue was undertaken. Two isozymes of lens, GST 7.4 and GST 5.6, were isolated and found to be homodimers of a Mr 23,500 subunit. Amino acid sequence analysis of a 20-residue region of the amino terminus was identical for both isozymes and was identical to GST ψ and GST μ of human liver. Antibodies raised against GST ψ cross-reacted with both lens isozymes. Although lens GST 5.6 and GST 7.4 demonstrated chemical and immunological relatedness, they were distinctly different as evidenced by their pI and comparative peptide fingerprint. A corneal isozyme, GST 7.2, was also isolated and established to be a homodimer of Mr 24,500 subunits. Sequence analysis of the amino-terminal region indicated it to be about 67% identical with the GST π isozyme of human placenta. Antibodies raised against GST π cross-reacted with cornea GST 7.2. Another corneal isozyme, GST 8.7, was found to be a homodimer of Mr 27,000 subunits. Sequence analysis revealed it to have a blocked amino-terminus. GST 8.7 immunologically cross-reacted with the antibodies raised against cationic isozymes of human liver indicating it to be of the α class. Two isozymes of retina, GST 6.8 and GST 6.3, were isolated and identified to be heterodimers of subunits of Mr 23,500 and 24,500. Amino-terminal sequence analysis gave identical results for both retina GST 6.8 and GST 6.3. The sequence analysis of the Mr 23,500 subunit was identical to that obtained for lens GSTs. Similarly, sequence analysis of the Mr 24,500 subunit was identical to that obtained for the cornea GST 7.2 isozyme. Both the retina isozymes cross-reacted with antibodies raised against human GST ψ as well as GST π. The results of these studies indicated that all three major classes of GST isozymes were expressed in bovine eye but the GST genes were differentially expressed in lens, cornea, and retina. In lens only the μ class of GST was expressed, whereas cornea expressed α and π classes and retina expressed μ and π classes of GST isozymes.

Degradation of p21cip1 in Cells Productively Infected with Human Cytomegalovirus

ABSTRACT Human cytomegalovirus (HCMV) stimulates arrested cells to enter the cell cycle by activating cyclin-dependent kinases (Cdks), notably Cdk2. Several mechanisms are involved in the activation of Cdk2. HCMV causes a substantial increase in the abundance of cyclin E and stimulates translocation of Cdk2 from the cytoplasm to the nucleus. Further, the abundance of the Cdk inhibitors (CKIs) p21cip1/waf1(p21cip1) and p27kip1 is substantially reduced. The activity of cyclin E/Cdk2 increases as levels of CKIs, particularly p21cip1, fall. We have previously shown that these phenomena contribute to priming the cell for efficient replication of HCMV. In this study, the mechanisms responsible for the decrease in p21cip1 levels after HCMV infection were investigated by measuring p21cip1 RNA and protein levels in permissive human lung (LU) fibroblasts after HCMV infection. Northern blot analysis revealed that p21cip1 RNA levels increased briefly at 3 h after HCMV infection and then decreased to their nadir at 24 h; thereafter, RNA levels increased to about 60% of the preinfection level. Western blot analysis demonstrated that the relative abundance of p21cip1 protein roughly paralleled the observed changes in initial RNA levels; however, the final levels of protein were much lower than preinfection levels. After a transient increase at 3 h postinfection, p21cip1 abundance declined sharply over the next 24 h and remained at a very low level through 96 h postinfection. The disparity between p21cip1 RNA and protein levels suggested that the degradation of p21cip1 might be affected in HCMV-infected cells. Treatment of HCMV-infected cells with MG132, an inhibitor of proteasome-mediated proteolysis, provided substantial protection of p21cip1 in mock-infected cells, but MG132 was much less effective in protecting p21cip1 in HCMV-infected cells. The addition of E64d or Z-Leu-Leu-H, each an inhibitor of calpain activity, to HCMV-infected cells substantially increased the abundance of p21cip1 in a concentration-dependent manner. To verify that p21cip1 was a substrate for calpain, purified recombinant p21cip1 was incubated with either m-calpain or μ-calpain, which resulted in rapid proteolysis of p21cip1. E64d inhibited the proteolysis of p21cip1 catalyzed by either m-calpain or μ-calpain. Direct measurement of calpain activity in HCMV-infected LU cells indicated that HCMV infection induced a substantial and sustained increase in calpain activity, although there was no change in the abundance of either m- or μ-calpain or the endogenous calpain inhibitor calpastatin. The observed increase of calpain activity was consistent with the increases in intracellular free Ca2+ and phospholipid degradation in HCMV-infected LU cells reported previously from our laboratory. Considered together, these results suggest that the increase in calpain activity observed following HCMV infection contributes significantly to the reduction of p21cip1 levels and the resultant cell cycle progression.

Viral-mediated inhibition of antioxidant enzymes contributes to the pathogenesis of severe respiratory syncytial virus bronchiolitis.

RATIONALE Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infections in children, for which no specific treatment or vaccine is currently available. We have previously shown that RSV induces reactive oxygen species in cultured cells and oxidative injury in the lungs of experimentally infected mice. The mechanism(s) of RSV-induced oxidative stress in vivo is not known. OBJECTIVES To measure changes of lung antioxidant enzymes expression/activity and activation of NF-E2-related factor 2 (Nrf2), a transcription factor that regulates detoxifying and antioxidant enzyme gene expression, in mice and in infants with naturally acquired RSV infection. METHODS Superoxide dismutase 1 (SOD 1), SOD 2, SOD 3, catalase, glutathione peroxidase, and glutathione S-transferase, as well as Nrf2 expression, were measured in murine bronchoalveolar lavage, cell extracts of conductive airways, and/or in human nasopharyngeal secretions by Western blot and two-dimensional gel electrophoresis. Antioxidant enzyme activity and markers of oxidative cell injury were measured in either murine bronchoalveolar lavage or nasopharyngeal secretions by colorimetric/immunoassays. MEASUREMENTS AND MAIN RESULTS RSV infection induced a significant decrease in the expression and/or activity of SOD, catalase, glutathione S-transferase, and glutathione peroxidase in murine lungs and in the airways of children with severe bronchiolitis. Markers of oxidative damage correlated with severity of clinical illness in RSV-infected infants. Nrf2 expression was also significantly reduced in the lungs of viral-infected mice. CONCLUSIONS RSV infection induces significant down-regulation of the airway antioxidant system in vivo, likely resulting in lung oxidative damage. Modulation of oxidative stress may pave the way toward important advances in the therapeutic approach of RSV-induced acute lung disease.