Charles R. Hauer

Identification and Characterization of Mycobacterial Proteins Differentially Expressed under Standing and Shaking Culture Conditions, Including Rv2623 from a Novel Class of Putative ATP-Binding Proteins

ABSTRACT The environmental signals that affect gene regulation inMycobacterium tuberculosis remain largely unknown despite their importance to tuberculosis pathogenesis. Other work has shown that several promoters, including acr (also known as hspX) (α-crystallin homolog), are upregulated in shallow standing cultures compared with constantly shaking cultures. Each of these promoters is also induced to a similar extent within macrophages. The present study used two-dimensional gel electrophoresis and mass spectrometry to further characterize differences in mycobacterial protein expression during growth under standing and shaking culture conditions. Metabolic labeling ofM. bovis BCG showed that at least 45 proteins were differentially expressed under standing and shaking culture conditions. Rv2623, CysA2-CysA3, Gap, and Acr were identified from each of four spots or gel bands that were specifically increased in bacteria from standing cultures. An additional standing-induced spot contained two comigrating proteins, GlcB and KatG. The greatest induction was observed with Rv2623, a 32-kDa protein of unknown function that was strongly expressed under standing conditions and absent in shaking cultures. Analysis using PROBE, a multiple sequence alignment and database mining tool, classified M. tuberculosis Rv2623 as a member of a novel class of ATP-binding proteins that may be involved in M. tuberculosiss response to environmental signals. These studies demonstrate the power of combined proteomic and computational approaches and demonstrate that subtle differences in bacterial culture conditions may have important implications for the study of gene expression in mycobacteria.

Autoantigens in ovarian autoimmunity associated with unexplained infertility and premature ovarian failure

OBJECTIVE To identify ovarian autoantigens associated with ovarian autoantibodies. DESIGN Hypothesis-generating prospective study. SETTING Urban infertility referral centers and academic research institution. PATIENT(S) Seventy-four patients with infertility, 19 patients with premature ovarian failure (POF), and 16 healthy control women. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Identification of autoantigens. RESULT(S) To identify major antigens for ovarian autoimmunity, sera from 74 women with unexplained infertility were screened for ovarian autoantibodies (AOAs) by immunoassay and one-dimensional Western blot. The majority of sera had immunoreactions at 50-56 kDa. Six representative positive infertility sera were used to identify antigens between 40 and 60 kD by two-dimensional Western blot and mass spectrometry. Antigens included aldehyde (retinal) dehydrogenases (ALDH1A1, ALDH1A2, and ALDH7A1), protein disulfide isomerase A3, vimentin, α-enolase, phosphoglycerate dehydrogenase, and selenium-binding protein 1 (SBP1). Sixty percent (24 out of 40) of infertility and POF sera were positive for recombinant ALDH1A1, SBP1, or enolase; 80.7% (21 out of 26) of AOA-positive sera had antibodies to one or more of the three antigens, and only 7% (1 out of 14) of AOA-negative sera had antibodies to recombinant proteins. CONCLUSION(S) ALDH1A1 and SBP1 are unique to ovarian autoimmunity associated with infertility and POF, and may provide the basis for specific tests to identify patients with ovarian autoimmunity.

Interrelationships among biological activity, disulfide bonds, secondary structure, and metal ion binding for a chemically synthesized 34-amino-acid peptide derived from α-fetoprotein

A 34-amino-acid peptide has been chemically synthesized based on a sequence from human alpha-fetoprotein. The purified peptide is active in anti-growth assays when freshly prepared in pH 7.4 buffer at 0.20 g/l, but this peptide slowly becomes inactive. This functional change is proven by mass spectrometry to be triggered by the formation of an intrapeptide disulfide bond between the two cysteine residues on the peptide. Interpeptide cross-linking does not occur. The active and inactive forms of the peptide have almost identical secondary structures as shown by circular dichroism (CD). Zinc ions bind to the active peptide and completely prevents formation of the inactive form. Cobalt(II) ions also bind to the peptide, and the UV-Vis absorption spectrum of the cobalt-peptide complex shows that: (1) a near-UV sulfur-to-metal-ion charge-transfer band had a molar extinction coefficient consistent with two thiolate bonds to Co(II); (2) the lowest-energy visible d-d transition maximum at 659 nm, also, demonstrated that the two cysteine residues are ligands for the metal ion; (3) the d-d molar extinction coefficient showed that the metal ion-ligand complex was in a distorted tetrahedral symmetry. The peptide has two cysteines, and it is speculated that the other two metal ion ligands might be the two histidines. The Zn(II)- and Co(II)-peptide complexes had similar peptide conformations as indicated by their ultraviolet CD spectra, which differed very slightly from that of the free peptide. Surprisingly, the cobalt ions acted in the reverse of the zinc ions in that, instead of stabilizing anti-growth form of the peptide, they catalyzed its loss. Metal ion control of peptide function is a saliently interesting concept. Calcium ions, in the conditions studied, apparently do not bind to the peptide. Trifluoroethanol and temperature (60 degrees C) affected the secondary structure of the peptide, and the peptide was found capable of assuming various conformations in solution. This conformational flexibility may possibly be related to the biological activity of the peptide.

Transcriptional Regulation of Rat CYP2A3 by Nuclear Factor 1 IDENTIFICATION OF A NOVEL NFI-A ISOFORM, AND EVIDENCE FOR TISSUE-SELECTIVE INTERACTION OF NFI WITH THE CYP2A3 PROMOTER IN VIVO

Rat CYP2A3 and its mouse and human orthologs are expressed preferentially in the olfactory mucosa. We found previously that an element in the proximal promoter region of CYP2A3 (the nasal predominant transcriptional activating (NPTA) element), which is similar to a nuclear factor 1 (NFI)-binding site, is critical for transcriptional activation of CYP2A3 in vitro. We proposed that this element might be important for tissue-selective CYP2A3 expression. The goals of the present study were to characterize NPTA-binding proteins and to obtain more definitive evidence for the role of NFI in the transcriptional activation of CYP2A3. The NPTA-binding proteins were isolated by DNA-affinity purification from rat olfactory mucosa. Mass spectral analysis indicated that isoforms corresponding to all four NFI genes were present in the purified NPTA-binding fraction. Further analysis of NPTA-binding proteins led to the identification of a novel NFI-A isoform, NFI-A-short, which was derived from alternative splicing of the NFI-A transcript. Transient transfection assay showed that NFI-A2, an NFI isoform previously identified in the olfactory mucosa, transactivated the CYP2A3 promoter, whereas NFI-A-short, which lacks the transactivation domain, counteracted the activation. Chromatin immunoprecipitation assays indicated that NFI proteins are associated with the CYP2A3 promoter in vivo, in rat olfactory mucosa, but essentially not in the liver where the CYP2A3 promoter is hypermethylated and CYP2A3 is not expressed. These data strongly support a role for NFI transcription factors in the transcriptional activation of CYP2A3.

Mercury impairment of mouse thymocyte survival in vitro: involvement of cellular thiols.

Heavy metals are well known to be able to induce immunotoxicity, but comparative metal studies related to apoptosis have not been conducted. In the present study, the effects of arsenic, cadmium, gold, lead, manganese, and mercury on thymocytes from BALB/c mice were analyzed. Thymic cells were cultured for 3–24 h in vitro in the absence or presence of metal, and markers of apoptosis or cell death, including annexin V binding, DNA loss/oligonucleosomal fragmentation, 7-amino-actinomycin D uptake (loss of impermeance), changes of the mitochondrial membrane potential (JC-1 fluorescence), and Western analysis of cellular thiols, were assayed. Mercury (Hg) was the only metal shown to be consistently toxic with the dose and times utilized. Cadmium (Cd) was the only other metal tested that also produced some significant level of DNA loss; however, the induction of apoptosis by Cd was not as consistent as that observed with Hg. When Hg was added with 2-mercaptoethanol (2-ME), Hg produced greater toxicity. Endogenous DNA synthesis by thymocytes was immediately inhibited by Hg and Hg + 2-ME. The Hg + 2-ME-induced apoptosis appeared to be associated with altered levels of cellular thiols, in that glutathione (GSH) depletion was significant in comparison to the non-metal control and Hg alone. The increased Hg-induced toxicity in the presence of 2-ME likely was due to the ability of 2-ME to enhance (10- to 20-fold) the cellular uptake of Hg. Western analysis with biotin maleimide demonstrated that Hg + 2-ME and to a lesser extent the positive control dexamethasone eliminated many reactive thiols; the major thiol-reactive protein still reactive with the maleimide probe had an approximate Molecular Mass of 45 kD. Surprisingly, Hg alone enhanced the expression of this thiol-expressing protein, which by Mass Spectrometry (MS)/MS analysis was shown to be β-actin. Hg also produced the appearance of yet to be identified new proteins. Based on the results with Hg + 2-ME, it is suggested that numerous protein thiols participate in maintenance of cell survival and their loss is associated with apoptosis. The increased expression of new thiol-reactive proteins or thiol-reactive proteins with altered electrophoretic profiles needs to be further investigated. However, the enhanced toxicity attributed to Hg + 2-ME suggests that increased intracellular oxidative stress, observed as increased depletion of GSH, is responsible for the accelerated cell death. This work was supported by National Institutes of Health grant ES11135. The authors acknowledge use of the Immunology and Biological Mass Spectrometry Facilities of Wadsworth Center. We also especially appreciate the assistance of Kathy Lubowski, Joan Pedersen-Lane, and Renjie Song.

Molecular Cloning, Primary Structure, and Properties of a New Glycoamidase from the Fungus Aspergillus tubigensis

A new glycoamidase, peptide-N 4-(N-acetyl-β-d-glucosaminyl)asparagine amidase (PNGase) At, was discovered in the eukaryote Aspergillus tubigensis. The enzyme was purified to homogeneity, and the DNA sequence was determined by cloning in Escherichia coli. Over 80% of the deduced amino acid sequence was verified independently by Edman analysis and/or electrospray ionization-mass spectrometry of protease fragments of native PNGase At. This glycoamidase contains 12 potential asparagine-linked glycosylation sites, of which at least 9 sites are occupied with typical high mannose oligosaccharides. PNGase At consists of two non-identical glycosylated subunits that are derived from a single polypeptide gene precursor. Evidence is presented suggesting that autocatalysis is involved in subunit formation. PNGase At is an important new tool for analysis of asparagine-linked glycans; it can hydrolyze a broad range of glycopeptides, including those with core-linked α1→6 or α1→3 fucose, under conditions not favorable with existing glycoamidases.

Unique cyanide adduct in human serum albumin: potential as a surrogate exposure marker.

Cyanide (CN = HCN + CN(-)) is a renowned poison and neurotoxicant that is prevalent throughout the environment. Despite a plethora of studies conducted over the last half century, relatively little is known of its potential to cause adverse health outcomes at sublethal exposures. CN exposure is normally determined from blood, but because CN is rapidly metabolized and cleared from this compartment (t(1/2) < 1 h), it is common for several half-lives to have passed before blood samples are drawn for analysis. This variable, coupled with a very narrow toxic index and metabolic diversity within the human population, has rendered accurate assessment of CN exposure, and consequently any predictions of possible adverse health outcomes, highly problematic. Prior studies by us showed the potential of Cys-SCN adducts within human serum albumin (HSA) to act as retrospective surrogates of CN exposure. Here, we report the discovery of a stable, SCN adduct at Cys(567) formed by the reaction of CN with the C-terminal Cys(558)Cys(567) disulfide bond of HSA. Treatment of HSA purified from human serum with base in guanidine hydrochloride releases a readily detectable, uniquely modified, C-terminal-19-mer peptide from Cys(567)-SCN moieties in all the samples examined thus far. Inclusion of a HSA-Cys(567)-S(13)C(15)N labeled internal standard permits quantitation of the Cys(567)-SCN adduct by LC-MS/MS in selective reaction monitoring (SRM) of the surrogate peptide with high sensitivity and good precision. Reaction of CN in vitro with the Cys(558)Cys(567) disulfide bond in HSA is specific, rapid, and concentration dependent within a putative, physiologically relevant range. Data from various human sera demonstrate the potential usefulness of this adduct as a biomarker of CN exposure.

Significance of mutations on the structural perturbation of thymidylate synthase: implications for their involvement in subunit exchange.

Wild‐type thymidylate synthase (WT‐TS) from Escherichia coli and several of its mutants showed varying degrees of susceptibility to trypsin. While WT‐TS was resistant to trypsin as were the mutants C146S, K48E, and R126K, others such as Y94A, Y94F, C146W, and R126E were digested but at different rates from one another. The peptides released from the mutants were identified by mass spectrometry and Edman sequence analysis. The known crystal structures for WT‐TS, Y94F, and R126E, surprisingly, showed no structural differences that could explain the difference in their susceptibility to trypsin. One explanation is that the mutations could perturb the dynamic equilibrium of the dimeric state of the mutants as to increase their dissociation to monomers, which being less structured than the dimer, would be hydrolyzed more readily by trypsin. Earlier studies appear to support this proposal since conditions that promote subunit dissociation in solutions of R126E with other inactive mutants, such as dilution, low concentrations of urea, and elevated pH, greatly enhance the rate of restoration of TS activity. Analytic ultracentrifuge studies with various TSs in urea, or at pH 9.0, or that have been highly diluted are, for the most part, in agreement with this thesis, since these conditions are associated with an increase in dissociation to monomers, particularly with the mutant TSs. However, these studies do not rule out the possibility that conformation differences among the various TS dimers are responsible for the differences in susceptibility to trypsin, particularly at high concentrations of protein where the WT‐TS and mutants are mainly dimers.