Functional modelling of an equine bronchoalveolar lavage fluid proteome provides experimental confirmation and functional annotation of equine genome sequences.

The equine genome sequence enables the use of high-throughput genomic technologies in equine research, but accurate identification of expressed gene products and interpreting their biological relevance require additional structural and functional genome annotation. Here, we employ the equine genome sequence to identify predicted and known proteins using proteomics and model these proteins into biological pathways, identifying 582 proteins in normal cell-free equine bronchoalveolar lavage fluid (BALF). We improved structural and functional annotation by directly confirming the in vivo expression of 558 (96%) proteins, which were computationally predicted previously, and adding Gene Ontology (GO) annotations for 174 proteins, 108 of which lacked functional annotation. Bronchoalveolar lavage is commonly used to investigate equine respiratory disease, leading us to model the associated proteome and its biological functions. Modelling of protein functions using Ingenuity Pathway Analysis identified carbohydrate metabolism, cell-to-cell signalling, cellular function, inflammatory response, organ morphology, lipid metabolism and cellular movement as key biological processes in normal equine BALF. Comparative modelling of protein functions in normal cell-free bronchoalveolar lavage proteomes from horse, human, and mouse, performed by grouping GO terms sharing common ancestor terms, confirms conservation of functions across species. Ninety-one of 92 human GO categories and 105 of 109 mouse GO categories were conserved in the horse. Our approach confirms the utility of the equine genome sequence to characterize protein networks without antibodies or mRNA quantification, highlights the need for continued structural and functional annotation of the equine genome and provides a framework for equine researchers to aid in the annotation effort.

Measurement of steady-state kinetic parameters for DNA unwinding by the bacteriophage T4 Dda helicase: use of peptide nucleic acids to trap single-stranded DNA products of helicase reactions.

Measurement of steady-state rates of unwinding of double-stranded oligonucleotides by helicases is hampered due to rapid reannealing of the single-stranded DNA products. Including an oligonucleotide in the reaction mixture which can hybridize with one of the single strands can prevent reannealing. However, helicases bind to single-stranded DNA, therefore the additional oligonucleotide can sequester the enzyme, leading to slower observed rates for unwinding. To circumvent this problem, the oligonucleotide that serves as a trap was replaced with a strand of peptide nucleic acid (PNA). Fluorescence polarization was used to determine that a 15mer PNA strand does not bind to the bacteriophage T4 Dda helicase. Steady-state kinetic parameters of unwinding catalyzed by Dda were determined by using PNA as a trapping strand. The substrate consisted of a partial duplex with 15 nt of single-stranded DNA and 15 bp. In the presence of 250 nM substrate and 1 nM Dda, the rate of unwinding in the presence of the DNA trapping strand was 0.30 nM s(-1) whereas the rate was 1.34 nM s(-1) in the presence of the PNA trapping strand. PNA prevents reannealing of single-stranded DNA products, but does not sequester the helicase. This assay will prove useful in defining the complete kinetic mechanism for unwinding of oligonucleotide substrates by this helicase.

Bovine germinal vesicle oocyte and cumulus cell proteomics.

Germinal vesicle (GV) breakdown is fundamental for maturation of fully grown, developmentally competent, mammalian oocytes. Bidirectional communication between oocytes and surrounding cumulus cells (CC) is essential for maturation of a competent oocyte. However, neither the factors involved in this communication nor the mechanisms of their actions are well defined. Here, we define the proteomes of GV oocytes and their surrounding CC, including membrane proteins, using proteomics in a bovine model. We found that 4395 proteins were expressed in the CC and 1092 proteins were expressed in oocytes. Further, 858 proteins were common to both the CC and the oocytes. This first comprehensive proteome analysis of bovine oocytes and CC not only provides a foundation for signaling and cell physiology at the GV stage of oocyte development, but are also valuable for comparative studies of other stages of oocyte development at the molecular level. Furthermore, some of these proteins may represent molecular biomarkers for developmental potential of oocytes.

Role of active-site residues 107 and 108 of glutathione S-transferase mGSTA4-4 in determining the catalytic properties of the enzyme for 4-hydroxynonenal.

The murine alpha-class glutathione S-transferase mGSTA4-4 displays a high catalytic activity with 4-hydroxynonenal (4-HNE), a cytotoxic product of lipid peroxidation. The X-ray crystal structure of mGSTA4-4 was used to design mutations targeting the 4-HNE binding site, with the goal of defining the structural elements of the mGSTA4-4 protein necessary for the high conjugative activity with 4-HNE. Two candidate positions, 107 and 108, were investigated. Of these, residue 108 appears to be significant in codetermining the catalytic properties of mGSTA4-4 toward 4-HNE. Systematic mutagenesis of amino acid 108 indicated that high activity toward 4-HNE is contingent on the presence of an aliphatic, hydrophobic side chain in this position. In particular, replacement of the wild-type V108 with leucine led to a more than fivefold increase in both absolute activity of the enzyme for 4-HNE and its selectivity for 4-HNE over the model substrate 1-chloro-2,4-dinitrobenzene, due to a selective increase of the turnover number for 4-HNE with no change in the affinity of the protein for this substrate and no changes in the kinetic parameters for 1-chloro-2,4-dinitrobenzene. In contrast, the A107L mutation decreased activity of the enzyme for both 4-HNE and CDNB and partially reversed the positive effect of the V108L mutation in a double mutant.

Amino acid residue 104 in an alpha-class glutathione S-transferase is essential for the high selectivity and specificity of the enzyme for 4-hydroxynonenal.

Murine mGSTA4-4 is a glutathione S-transferase with high activity and specificity for products of lipid peroxidation, including the cytotoxic 4-hydroxynonenal (4-HNE). Physiological relevance of this enzyme in the defense against effects of oxidative stress can be inferred from the above biochemical properties, and has been also directly demonstrated by us in vivo. The identification of residues responsible for the high activity toward 4-HNE is facilitated by the availability of X-ray crystal structures of mGSTA4-4 and of hGSTA1-1, a structurally related enzyme which lacks activity for 4-HNE. Residues likely to be involved in 4-HNE recognition were identified by molecular modeling. One such residue, M104, was mutated to E104, as present in hGSTA1-1. The resulting M104E mutant had unchanged catalytic properties toward the model substrate 1-chloro-2,4-dinitrobenzene. However, the Km of mGSTA4-4(M104E) for 4-HNE was increased more than sevenfold, while the Vmax for that substrate remained essentially unchanged. We conclude that M104 codetermines the recognition and binding of 4-HNE to the active center of mGSTA4-4.

Crystal structure of a murine glutathione S-transferase in complex with a glutathione conjugate of 4-hydroxynon-2-enal in one subunit and glutathione in the other: evidence of signaling across the dimer interface.

mGSTA4-4, a murine glutathione S-transferase (GST) exhibiting high activity in conjugating the lipid peroxidation product 4-hydroxynon-2-enal (4-HNE) with glutathione (GSH), was crystallized in complex with the GSH conjugate of 4-HNE (GS-Hna). The structure has been solved at 2.6 A resolution, which reveals that the active site of one subunit of the dimeric enzyme binds GS-Hna, whereas the other binds GSH. A marked asymmetry between the two subunits is evident. Most noticeable are the differences in the conformation of arginine residues 69 and 15. In all GST structures published previously, the guanidino groups of R69 residues from both subunits stack at the dimer interface and are related by a (pseudo-) 2-fold axis. In the present structure of mGSTA4-4, however, the two R69 side chains point in opposite directions, although their guanidino groups remain in contact. In the subunit with bound GSH, R69 also interacts with R15, and the guanidino group of R15 points away from the active site, whereas in the subunit that binds GS-Hna, R15 pivots into the active site, which breaks its interaction with R69. According to our previous results [Nanduri et al. (1997) Arch. Biochem. Biophys. 335, 305-310], the availability of R15 in the active site assists the conjugation of 4-HNE with GSH. We propose a model for the catalytic mechanism of mGSTA4-4 in conjugating 4-HNE with GSH-i.e., the guanidino group of R15 is available in the active site of only one subunit at any given time and the stacked pair of R69 residues act as a switch that couples the concerted movement of the two R15 side chains. The alternate occupancy of 4-HNE in the two subunits has been confirmed by our kinetic analysis that shows the negative cooperativity of mGSTA4-4 for 4-HNE. Disruption of the signaling between the subunits by mutating the R69 residues released the negative cooperativity with 4-HNE.

Evidence for a functional monomeric form of the bacteriophage T4 DdA helicase. Dda does not form stable oligomeric structures.

The active form of many helicases is oligomeric, possibly because oligomerization provides multiple DNA binding sites needed for unwinding of DNA. In order to understand the mechanism of the bacteriophage T4 Dda helicase, the potential requirement for oligomerization was investigated. Chemical cross-linking and high pressure gel filtration chromatography provided little evidence for the formation of an oligomeric species. The specific activity for ssDNA stimulated ATPase activity was independent of Dda concentration. Dda was mutated to produce an ATPase-deficient protein (K38A Dda) by altering a residue within a conserved, nucleotide binding loop. The helicase activity of K38A Dda was inactivated, although DNA binding properties were similar to Dda. In the presence of limiting DNA substrate, the rate of unwinding by Dda was not changed; however, the amplitude of product formation was reduced in the presence of increasing concentrations of K38A Dda. The reduction was between that expected for a monomeric or dimeric helicase based on simple competition for substrate binding. When unwinding of DNA was measured in the presence of excess DNA substrate, addition of K38A Dda caused no reduction in the observed rate for strand separation. Taken together, these results indicate that oligomerization of Dda is not required for DNA unwinding.

Naturally occurring human glutathione S-transferase GSTP1-1 isoforms with isoleucine and valine in position 104 differ in enzymic properties.

Glutathione S-transferase P1-1 isoforms, differing in a single amino acid residue (Ile104 or Val104), have been previously identified in human placenta [Ahmad, H., Wilson, D. E., Fritz, R. R., Singh, S. V., Medh, R. D., Nagle, G. T., Awasthi, Y. C. & Kurosky, A. (1990) Arch. Biochem. Biophys. 278, 398-408]. In the present report, the enzymic properties of these two proteins are compared. [I104]glutathione S-transferase P1-1 has been expressed from its cDNA in Escherichia coli and purified to homogeneity by affinity chromatography; the cDNA has been mutated to replace Ile104 by Val104, and [V104]glutathione S-transferase P1-1 was expressed and isolated as described for [I104]glutathione S-transferase P1-1. The two enzymes differed in their specific activity and affinity for electrophilic substrates (KM values for 1-chloro-2,4-dinitrobenzene were 0.8 mM and 3.0 mM for [I-104]glutathione S-transferase P1-1 and [V-104]glutathione S-transferase P1-1, respectively), but were identical in their affinity for glutathione. In addition, the two enzymes were distinguishable by their heat stability, with half-lives at 45 degrees C of 19 min and 51 min, respectively. The resistance to heat denaturation was differentially modulated by the presence of substrates. These data, in conjunction with molecular modeling, indicate that the residue in position 104 helps to define the geometry of the hydrophobic substrate-binding site, and may also influence activity by interacting with residues directly involved in substrate binding.

Mechanism of differential catalytic efficiency of two polymorphic forms of human glutathione S-transferase P1-1 in the glutathione conjugation of carcinogenic diol epoxide of chrysene.

The kinetics of the conjugation of glutathione (GSH) with anti-1, 2-dihydroxy-3,4-oxy-1,2,3,4-tetrahydrochrysene (anti-CDE), the activated form of the widespread environmental pollutant chrysene, catalyzed by two naturally occurring polymorphic forms of the pi class human GSH S-transferase (hGSTP1-1), has been investigated. The polymorphic forms of hGSTP1-1, which differ in their primary structure by a single amino acid in position 104, exhibited preference for the GSH conjugation of (+)-anti-CDE, which is a far more potent carcinogen than (-)-anti-CDE. When concentration of anti-CDE was varied (5-200 microM and the GSH concentration was kept constant at 2 mM, both hGSTP1-1(I104) and hGSTP1-1(V104) obeyed Michaelis-Menten kinetics. However, the Vmax of GSH conjugation of anti-CDE was approximately 5.3-fold higher for the V104 variant than for the I104 form. Calculation of catalytic efficiency (kcat/Km) thus resulted in a value for hGSTP1-1(V104), 28 mM-1 s-1, that was 7.0-fold higher than that for hGSTP1-1(I104), 4 mM-1 s-1. The mechanism of the differences in the kinetic properties of hGSTP1-1 isoforms toward anti-CDE was investigated by molecular modeling of the two proteins with GSH conjugation products in their active sites. These studies revealed that the enantioselectivity of hGSTP1-1 for (+)-anti-CDE and the differential catalytic efficiencies of the V104 and I104 forms of hGSTP1-1 in the GSH conjugation of (+)-anti-CDE were due to the differences in the active-site architecture of the two proteins. The results of the present study, for the first time, provide evidence for the toxicological relevance of GSTP1-1 polymorphism in humans and suggest that the population polymorphism of hGSTP1-1 variants with disparate enzyme activities may, at least in part, account for the differential susceptibility of individuals to environmental carcinogens such as anti-CDE and possibly other similar carcinogens.

Active site architecture of polymorphic forms of human glutathione S-transferase P1-1 accounts for their enantioselectivity and disparate activity in the glutathione conjugation of 7beta,8alpha-dihydroxy-9alpha,10alpha-ox y-7,8,9,10-tetrahydrobenzo(a)pyrene.

In this study, we demonstrate that the active site architecture of the human glutathione (GSH) S-transferase Pi (GSTP1-1) accounts for its enantioselectivity in the GSH conjugation of 7beta,8alpha-dihydroxy-9alpha,10alpha-oxy-7,8,9, 10-tetrahydrobenzo(a) pyrene (anti-BPDE), the ultimate carcinogen of benzo(a)pyrene. Furthermore, we report that the two polymorphic forms of human GSTP1-1, differing in their primary structure by a single amino acid in position 104, have disparate activity toward (+)-anti-BPDE, which can also be rationalized in terms of their active site structures. When concentration of (+)-anti-BPDE, which among four BPDE isomers is the most potent carcinogen, was varied and GSH concentration was kept constant at 2 mM (saturating concentration), both forms of hGSTP1-1 [hGSTP1-1(V104) and hGSTP1-1(I104)] obeyed Michaelis-Menten kinetics. The V(max) of GSH conjugation of (+)-anti-BPDE was approximately 3.4-fold higher for hGSTP1-1(V104) than for hGSTP1-1(I104). Adherence to Michaelis-Menten kinetics was also observed for both isoforms when (-)-anti-BPDE, which is a weak carcinogen, was used as the variable substrate. However, (-)-anti-BPDE was a relatively poor substrate for both isoforms as compared with (+)-anti-BPDE. Moreover, there were no significant differences between hGSTP1-1(V104) and hGSTP1-1(I104) in either V(max) or K(m) for (-)-anti-BPDE. The mechanism of differences in kinetic properties and enantioselectivity of hGSTP1-1 variants toward anti-BPDE was investigated by modeling of the two proteins with conjugation product molecules in their active sites. Molecular modeling studies revealed that the differences in catalytic properties of hGSTP1-1 variants as well as the enantioselectivity of hGSTP1-1 in the GSH conjugation of anti-BPDE can be rationalized in terms of the architecture of their active sites. Our results suggest that the population polymorphism of hGSTP1-1 variants with disparate enzyme activities may, at least in part, account for the differential susceptibility of individuals to carcinogens such as anti-BPDE and possibly other similar carcinogens.