Gerald E. Wuenschell

Metal-ion coordination by U6 small nuclear RNA contributes to catalysis in the spliceosome.

Introns are removed from nuclear messenger RNA precursors through two sequential phospho-transesterification reactions in a dynamic RNA–protein complex called the spliceosome. But whether splicing is catalysed by small nuclear RNAs in the spliceosome is unresolved. As the spliceosome is a metalloenzyme, it is important to determine whether snRNAs coordinate catalytic metals. Here we show that yeast U6 snRNA coordinates a metal ion that is required for the catalytic activity of the spliceosome. With Mg2+, U6 snRNA with a sulphur substitution for the pro-RP or pro-SP non-bridging phosphoryl oxygen of nucleotide U80 reconstitutes a fully assembled yet catalytically inactive spliceosome. Adding a thiophilic ion such as Mn2+ allows the first transesterification reaction to occur in the U6/sU80(SP)- but not the U6/sU80(RP)-reconstituted spliceosome. Mg2+ competitively inhibits the Mn2+-rescued reaction, indicating that the metal-binding site at U6/U80 exists in the wild-type spliceosome and that the site changes its metal requirement for activity in the SP spliceosome. Thus, U6 snRNA contributes to pre-messenger RNA splicing through metal-ion coordination, which is consistent with RNA catalysis by the spliceosome.

Patterns of nucleotide misincorporations during enzymatic amplification and direct large-scale sequencing of ancient DNA

Whereas evolutionary inferences derived from present-day DNA sequences are by necessity indirect, ancient DNA sequences provide a direct view of past genetic variants. However, base lesions that accumulate in DNA over time may cause nucleotide misincorporations when ancient DNA sequences are replicated. By repeated amplifications of mitochondrial DNA sequences from a large number of ancient wolf remains, we show that C/G-to-T/A transitions are the predominant type of such misincorporations. Using a massively parallel sequencing method that allows large numbers of single DNA strands to be sequenced, we show that modifications of C, as well as to a lesser extent of G, residues cause such misincorporations. Experiments where oligonucleotides containing modified bases are used as templates in amplification reactions suggest that both of these types of misincorporations can be caused by deamination of the template bases. New DNA sequencing methods in conjunction with knowledge of misincorporation processes have now, in principle, opened the way for the determination of complete genomes from organisms that became extinct during and after the last glaciation.

Nucleotide excision repair eliminates unique DNA-protein cross-links from mammalian cells

DNA-protein cross-links (DPCs) present a formidable obstacle to cellular processes because they are “superbulky” compared with the majority of chemical adducts. Elimination of DPCs is critical for cell survival because their persistence can lead to cell death or halt cell cycle progression by impeding DNA and RNA synthesis. To study DPC repair, we have used DNA methyltransferases to generate unique DPC adducts in oligodeoxyribonucleotides or plasmids to monitor both in vitro excision and in vivo repair. We show that HhaI DNA methyltransferase covalently bound to an oligodeoxyribonucleotide is not efficiently excised by using mammalian cell-free extracts, but protease digestion of the full-length HhaI DNA methyltransferase-DPC yields a substrate that is efficiently removed by a process similar to nucleotide excision repair (NER). To examine the repair of that unique DPC, we have developed two plasmid-based in vivo assays for DPC repair. One assay shows that in nontranscribed regions, DPC repair is greater than 60% in 6 h. The other assay based on host cell reactivation using a green fluorescent protein demonstrates that DPCs in transcribed genes are also repaired. Using Xpg-deficient cells (NER-defective) with the in vivo host cell reactivation assay and a unique DPC indicates that NER has a role in the repair of this adduct. We also demonstrate a role for the 26 S proteasome in DPC repair. These data are consistent with a model for repair in which the polypeptide chain of a DPC is first reduced by proteolysis prior to NER.

Glyoxalase 1 increases anxiety by reducing GABAA receptor agonist methylglyoxal

Glyoxalase 1 (Glo1) expression has previously been associated with anxiety in mice; however, its role in anxiety is controversial, and the underlying mechanism is unknown. Here, we demonstrate that GLO1 increases anxiety by reducing levels of methylglyoxal (MG), a GABAA receptor agonist. Mice overexpressing Glo1 on a Tg bacterial artificial chromosome displayed increased anxiety-like behavior and reduced brain MG concentrations. Treatment with low doses of MG reduced anxiety-like behavior, while higher doses caused locomotor depression, ataxia, and hypothermia, which are characteristic effects of GABAA receptor activation. Consistent with these data, we found that physiological concentrations of MG selectively activated GABAA receptors in primary neurons. These data indicate that GLO1 increases anxiety by reducing levels of MG, thereby decreasing GABAA receptor activation. More broadly, our findings potentially link metabolic state, neuronal inhibitory tone, and behavior. Finally, we demonstrated that pharmacological inhibition of GLO1 reduced anxiety, suggesting that GLO1 is a possible target for the treatment of anxiety disorders.

Glyoxalase 1 and its substrate methylglyoxal are novel regulators of seizure susceptibility.

Epilepsy is a complex disease characterized by a predisposition toward seizures. There are numerous barriers to the successful treatment of epilepsy. For instance, current antiepileptic drugs have adverse side effects and variable efficacies. Furthermore, the pathophysiologic basis of epilepsy remains largely elusive. Therefore, investigating novel genes and biologic processes underlying epilepsy may provide valuable insight and enable the development of new therapeutic agents. We previously identified methylglyoxal (MG) as an endogenous γ‐aminobutyric acid (GABAA) receptor agonist. Here, we investigated the role of MG and its catabolic enzyme, glyoxalase 1 (GLO1), in seizures.

Inhibition of GLO1 in Glioblastoma Multiforme Increases DNA-AGEs, Stimulates RAGE Expression, and Inhibits Brain Tumor Growth in Orthotopic Mouse Models

Cancers that exhibit the Warburg effect may elevate expression of glyoxylase 1 (GLO1) to detoxify the toxic glycolytic byproduct methylglyoxal (MG) and inhibit the formation of pro-apoptotic advanced glycation endproducts (AGEs). Inhibition of GLO1 in cancers that up-regulate glycolysis has been proposed as a therapeutic targeting strategy, but this approach has not been evaluated for glioblastoma multiforme (GBM), the most aggressive and difficult to treat malignancy of the brain. Elevated GLO1 expression in GBM was established in patient tumors and cell lines using bioinformatics tools and biochemical approaches. GLO1 inhibition in GBM cell lines and in an orthotopic xenograft GBM mouse model was examined using both small molecule and short hairpin RNA (shRNA) approaches. Inhibition of GLO1 with S-(p-bromobenzyl) glutathione dicyclopentyl ester (p-BrBzGSH(Cp)2) increased levels of the DNA-AGE N2-1-(carboxyethyl)-2′-deoxyguanosine (CEdG), a surrogate biomarker for nuclear MG exposure; substantially elevated expression of the immunoglobulin-like receptor for AGEs (RAGE); and induced apoptosis in GBM cell lines. Targeting GLO1 with shRNA similarly increased CEdG levels and RAGE expression, and was cytotoxic to glioma cells. Mice bearing orthotopic GBM xenografts treated systemically with p-BrBzGSH(Cp)2 exhibited tumor regression without significant off-target effects suggesting that GLO1 inhibition may have value in the therapeutic management of these drug-resistant tumors.

Abstract 2039: Potential therapeutic peptides for neuroblastoma.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Neuroblastoma (NB) is a form of cancer that starts in certain types of very primitive developing nerve cells found in an embryo or fetus. This type of cancer occurs in infants and young children. It is rarely found in children older than 10 years. NB is by far the most common cancer in infants (less than 1 year old). It accounts for about 7% of all cancers in children. There are approximately 650 new cases of NB each year in the United States. The types of treatment used may include surgery, chemotherapy, retinoid therapy, and radiation therapy. In many cases, especially when the cancer has spread too far to be completely removed by surgery, chemotherapy is the main treatment. Most children with NB will need to have chemotherapy. In most cases, treatment involves a combination of anticancer drugs. These therapies by-in-large always have significant side-effects. The goal of all therapies aimed at eradicating cancer, including NB, is to selectively destroy cancer cells while sparing normal tissue. Most chemotherapeutic or radiotherapeutic agents function by damaging DNA or interfering with DNA replication. Rapid advances in recent years have contributed significantly to identifying and understanding the roles of key proteins in DNA damage response pathways. (As a result, the nuclear protein PCNA has been shown to be a component of all DNA replication/repair processes in the human cell). Logically then, it is reasonable to assume that by altering the cellular response to DNA damage, perhaps through manipulating genes or proteins related to DNA repair, cell cycle checkpoint control, damage tolerance or apoptosis, the effectiveness of conventional cancer therapy can potentially be enhanced or used as novel targets for drug discovery. Of course, also selectively sensitizing cancer cells relative to normal tissues should improve the therapeutic ratio for anti-cancer treatment. Our laboratorys recent data indicate that the use of PCNA derived synthetic peptides promote NB cytotoxicity. The hypothesis driving our work is that specific peptides derived from the PCNA protein sequence (caPeptides) have the potential ability to block the binding of several cellular proteins that participate in DNA replication, repair, cell cycle control, transcription, and chromosomal recombination in cancer cells to full length PCNA. By disrupting the naturally occurring interaction between PCNA and the proteins that bind to, or interact, with PCNA, cellular functions that recruit PCNA would be disrupted in the NB cell. These peptides, either alone or in combination with other cancer therapy agents are potentially useful cancer chemotherapeutics or augmentors of the pharmacodynamic effect of specific anti-cancer chemotherapeutics. Citation Format: Linda H. Malkas, Long Gu, Mingway Li, Shanna Smith, Robert Lingeman, Gerald Wuenschell, Lei Zhang, Robert Hickey. Potential therapeutic peptides for neuroblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2039. doi:10.1158/1538-7445.AM2013-2039