Cynthia J. Brame

Mitotic Phosphorylation of Histone H3 Is Governed by Ipl1/aurora Kinase and Glc7/PP1 Phosphatase in Budding Yeast and Nematodes

Phosphorylation of histone H3 at serine 10 occurs during mitosis and meiosis in a wide range of eukaryotes and has been shown to be required for proper chromosome transmission in Tetrahymena. Here we report that Ipl1/aurora kinase and its genetically interacting phosphatase, Glc7/PP1, are responsible for the balance of H3 phosphorylation during mitosis in Saccharomyces cerevisiae and Caenorhabditis elegans. In these models, both enzymes are required for H3 phosphorylation and chromosome segregation, although a causal link between the two processes has not been demonstrated. Deregulation of human aurora kinases has been implicated in oncogenesis as a consequence of chromosome missegregation. Our findings reveal an enzyme system that regulates chromosome dynamics and controls histone phosphorylation that is conserved among diverse eukaryotes.

Methylation of histone H4 at arginine 3 occurs in vivo and is mediated by the nuclear receptor coactivator PRMT1

Posttranslational modifications of histone amino termini play an important role in modulating chromatin structure and function. Lysine methylation of histones has been well documented, and recently this modification has been linked to cellular processes involving gene transcription and heterochromatin assembly. However, the existence of arginine methylation on histones has remained unclear. Recent discoveries of protein arginine methyltransferases, CARM1 and PRMT1, as transcriptional coactivators for nuclear receptors suggest that histones may be physiological targets of these enzymes as part of a poorly defined transcriptional activation pathway. Here we show by using mass spectrometry that histone H4, isolated from asynchronously growing human 293T cells, is methylated at arginine 3 (Arg-3) in vivo. In support, a novel antibody directed against histone H4 methylated at Arg-3 independently demonstrates the in vivo occurrence of this modification and reveals that H4 Arg-3 methylation is highly conserved throughout eukaryotes. Finally, we show that PRMT1 is the major, if not exclusive, H4 Arg-3 methyltransfase in human 293T cells. These findings suggest a role for arginine methylation of histones in the transcription process.

Composition and Functional Characterization of Yeast 66S Ribosome Assembly Intermediates

The pathway and complete collection of factors that orchestrate ribosome assembly are not clear. To address these problems, we affinity purified yeast preribosomal particles containing the nucleolar protein Nop7p and developed means to separate their components. Nop7p is associated primarily with 66S preribosomes containing either 27SB or 25.5S plus 7S pre-rRNAs. Copurifying proteins identified by mass spectrometry include ribosomal proteins, nonribosomal proteins previously implicated in 60S ribosome biogenesis, and proteins not known to be involved in ribosome production. Analysis of strains mutant for eight of these proteins not previously implicated in ribosome biogenesis showed that they do participate in this pathway. These results demonstrate that proteomic approaches in concert with genetic tools provide powerful means to purify and characterize ribosome assembly intermediates.

THE ISOPROSTANES: UNIQUE PROSTAGLANDIN-LIKE PRODUCTS OF FREE-RADICAL-INITIATED LIPID PEROXIDATION*

The discovery of IsoPs as products of nonenzymatic lipid peroxidation has opened up new areas of investigation regarding the role of free radicals in human physiology and pathophysiology. The quantification of IsoPs as markers of oxidative stress status appears to be an important advance in our ability to explore the role of free radicals in the pathogenesis of human disease. An important need in the field of free-radical medicine is information regarding the clinical pharmacology of antioxidant agents. Because of the evidence implicating free radicals in the pathogenesis of a number of human diseases, large clinical trials are planned or underway to assess whether antioxidants can either prevent the development or ameliorate the pathology of certain human disorders. However, data regarding the most effective doses and combination of antioxidant agents to use in these clinical trials is lacking. As mentioned previously, administration of antioxidants suppresses the formation of IsoPs, even in normal individuals. Thus, measurement of IsoPs may provide a valuable approach to define the clinical pharmacology of antioxidants. In addition to being markers of oxidative stress, several IsoPs possess potent biological activity. The availability of additional IsoPs in synthetic form should broaden our knowledge concerning the role of these molecules as mediators of oxidant stress. Despite the fact that considerable information has been obtained since the initial report of the discovery of IsoPs [6], much remains to be understood about these molecules. With continued research in this area, we believe that much new information will emerge that will open up additional important new areas for future investigation.

Tapasin Is a Facilitator, Not an Editor, of Class I MHC Peptide Binding

Tapasin has been proposed to function as a peptide editor to displace lower affinity peptides and/or to favor the binding of high affinity peptides. Consistent with this, cell surface HLA-B8 molecules in tapasin-deficient cells were less stable and the peptide repertoire was substantially altered. However, the binding affinities of peptides expressed in the absence of tapasin were unexpectedly higher, not lower. The peptide repertoire from cells expressing soluble tapasin was similar in both appearance and affinity to that presented in the presence of full-length tapasin, but the HLA-B8 molecules showed altered cell surface stability characteristics. Similarly, the binding affinities of HLA-A*0201-associated peptides from tapasin+ and tapasin− cells were equivalent, although steady state HLA-A*0201 cell surface expression was decreased and the molecules demonstrated reduced cell surface stability on tapasin− cells. These data are inconsistent with a role for tapasin as a peptide editor. Instead, we propose that tapasin acts as a peptide facilitator. In this role, it stabilizes the peptide-free conformation of class I MHC molecules in the endoplasmic reticulum and thus increases the number and variety of peptides bound to class I MHC. Full-length tapasin then confers additional stability on class I MHC molecules that are already associated with peptides.

Characterization of the Lysyl Adducts Formed from Prostaglandin H2 via the Levuglandin Pathway

Prostaglandin H(2) has been demonstrated to rearrange to gamma-ketoaldehyde prostanoids termed levuglandins E(2) and D(2). As gamma-dicarbonyl molecules, the levuglandins react readily with amines. We sought to characterize the adducts formed by synthetic levuglandin E(2) and prostaglandin H(2)-derived levuglandins with lysine. Using liquid chromatography/electrospray mass spectrometry, we found that the reaction predominantly produces lysyl-levuglandin Schiff base adducts that readily dehydrate to form lysyl-anhydrolevuglandin Schiff base adducts. These adducts were characterized by examination of their mass spectra, by analysis of the products of their reaction with sodium cyanide, sodium borohydride, and methoxylamine and by the mass spectra derived from collision-induced dissociation in tandem mass spectrometry. The Schiff base adducts also are formed on peptide-bound lysyl residues. In addition, synthetic levuglandin E(2) and prostaglandin H(2)-derived levuglandins produced pyrrole-derived lactam and hydroxylactam adducts upon reaction with lysine as determined by tandem mass spectrometry. A marked time dependence in the formation of these adducts was observed: Schiff base adducts formed very rapidly and robustly, whereas the lactam and hydroxylactam adducts formed more slowly but accumulated throughout the time of the experiment. These findings provide a basis for investigating protein modification induced by oxygenation of arachidonic acid by the cyclooxygenases.

New developments in the isoprostane pathway: identification of novel highly reactive γ-ketoaldehydes (isolevuglandins) and characterization of their protein adducts

The bicyclic endoperoxide prostaglandin (PG) H2 undergoes nonenzymatic rearrangement not only to PGE2 and PGD2, but also to levuglandins (LG) E2 and D2, which are highly reactive γ‐ketoaldehydes. Isoprostanes (IsoPs) are PG‐like compounds that are produced by nonenzymatic peroxidation of arachidonic acid. PGH2‐like endoperoxides are intermediates in this pathway. Therefore, we explored whether the IsoP endoperoxides also undergo rearrangement to form IsoLGs. Oxidation of arachidonic acid in vitro resulted in the formation of abundant quantities of compounds that were established to be IsoLGs by using mass spectrometric analyses. However, the formation of IsoLGs could not be detected in biological systems subjected to an oxidant stress. We hypothesized that this was due to extremely rapid adduction of IsoLGs to proteins. This notion was supported by the finding that LGE2 adducted to albumin at a rate that exceeded that of 4‐hydroxynonenal by several orders of magnitude: >50% of LGE2 had adducted within 20 s. We therefore undertook to characterize the nature of LG adducts. Using liquid chromatography electrospray tandem mass spectrometry, we established that LGs form oxidized pyrrole adducts (lactams and hydroxylactams) with the ε‐amino group of lysine. Oxidation of low density lipoprotein resulted in readily detectable IsoLG adducts on apolipoprotein B after enzymatic digestion of the protein to individual amino acids. These studies identify a novel class of ketoaldehydes produced by the IsoP pathway that form covalent protein adducts at a rate that greatly exceeds that of other known aldehyde products of lipid peroxidation. Elucidation of the nature of the adducts formed by IsoLGs provides the basis to explore the formation of IsoLGs in vivo and investigate the potential biological ramifications of their formation in settings of oxidant injury.—Roberts, L. J., II, Salomon, R. G., Morrow, J. D., Brame, C. J. New developments in the isoprostane pathway: identification of novel highly reactive γ‐ketoaldehydes (isolevuglandins) and characterization of their protein adducts. FASEB J. 13, 1157–1168 (1999)

Measurement of chronic oxidative and inflammatory stress by quantification of isoketal/levuglandin gamma-ketoaldehyde protein adducts using liquid chromatography tandem mass spectrometry.

Measurement of F2-isoprostanes (F2-IsoPs) has been independently verified as one of the most reliable approaches to assess oxidative stress in vivo. However, the rapid clearance of F2-IsoPs makes the timing of sample collection critical for short-lived oxidative insults. Isoketals (IsoKs) are γ-ketoaldehydes formed via the IsoP pathway of lipid peroxidation that rapidly react with lysyl residues of proteins to form stable protein adducts. Oxidative stress can also activate cyclooxygenases to produce prostaglandin H2, which can form two specific isomers of IsoK—levuglandin (LG) D2 and E2. Because adducted proteins are not rapidly cleared, IsoK/LG protein adduct levels can serve as a dosimeter of oxidative and inflammatory damage over prolonged periods of time as well as brief episodes of injury. Quantification of IsoK/LG protein adducts begins with liquid-phase extraction to separate proteins from lipid membranes, allowing measurement of both IsoK/LG protein adducts and F2-IsoP from the same sample if desired. IsoK/LG-lysyl-lactam adducts are measured by liquid chromatography tandem mass spectrometry after proteolytic digestion of extracted proteins, solid-phase extraction and preparative HPLC.

A Molecular Genetics Laboratory Course Applying Bioinformatics and Cell Biology in the Context of Original Research

Research based laboratory courses have been shown to stimulate student interest in science and to improve scientific skills. We describe here a project developed for a semester-long research-based laboratory course that accompanies a genetics lecture course. The project was designed to allow students to become familiar with the use of bioinformatics tools and molecular biology and genetic approaches while carrying out original research. Students were required to present their hypotheses, experiments, and results in a comprehensive lab report. The lab project concerned the yeast casein kinase 1 (CK1) protein kinase Yck2. CK1 protein kinases are present in all organisms and are well conserved in primary structure. These enzymes display sequence features that differ from other protein kinase subfamilies. Students identified such sequences within the CK1 subfamily, chose a sequence to analyze, used available structural data to determine possible functions for their sequences, and designed mutations within the sequences. After generating the mutant alleles, these were expressed in yeast and tested for function by using two growth assays. The student response to the project was positive, both in terms of knowledge and skills increases and interest in research, and several students are continuing the analysis of mutant alleles as summer projects.

Test-Enhanced Learning: The Potential for Testing to Promote Greater Learning in Undergraduate Science Courses

Testing can be a learning event. Research from cognitive science suggests that repeated retrieval using various formats can enhance long-term learning, that feedback enhances this benefit, that testing can potentiate further study, and that benefits of testing go beyond rote memory. These observations open opportunities for teaching and research.