Evolution of the redox function in mammalian Apurinic/ apyrimidinic endonuclease

Human apurinic/apyrimidinic endonuclease (hApe1) encodes two important functional activities: an essential base excision repair (BER) activity and a redox activity that regulates expression of a number of genes through reduction of their transcription factors, AP-1, NFκB, HIF-1α, CREB, p53 and others. The BER function is highly conserved from prokaryotes (E. coli exonuclease III) to humans (hApe1). Here, we provide evidence supporting a redox function unique to mammalian Apes. An evolutionary analysis of Ape sequences reveals that, of the 7 Cys residues, Cys 93, 99, 208, 296, and 310 are conserved in both mammalian and non-mammalian vertebrate Apes, while Cys 65 is unique to mammalian Apes. In the zebrafish Ape (zApe), selected as the vertebrate sequence most distant from human, the residue equivalent to Cys 65 is Thr 58. The wild-type zApe enzyme was tested for redox activity in both in vitro EMSA and transactivation assays and found to be inactive, similar to C65A hApe1. Substitution of Thr 58 with Cys in zApe, however, resulted in a redox active enzyme, suggesting that a Cys residue in this position is indeed critical for redox function. In order to further probe differences between redox active and inactive enzymes, we have determined the crystal structures of vertebrate redox inactive enzymes, the C65A human Ape1 enzyme and the zApe enzyme at 1.9 and 2.3 Å, respectively. Our results provide new insights on the redox function and highlight a dramatic gain-of-function activity for Ape1 in mammals not found in non-mammalian vertebrates or lower organisms

Interactions of APE1 with a redox inhibitor: Evidence for an alternate conformation of the enzyme

Apurinic/apyrimidinic endonuclease (APE1) is an essential base excision repair protein that also functions as a reduction and oxidation (redox) factor in mammals. Through a thiol-based mechanism, APE1 reduces a number of important transcription factors, including AP-1, p53, NF-κB, and HIF-1α. What is known about the mechanism to date is that the buried residues Cys 65 and Cys 93 are critical for APE1’s redox activity. To further detail the redox mechanism, we developed a chemical footprinting−mass spectrometric assay using N-ethylmaleimide (NEM), an irreversible Cys modifier, to characterize the interaction of the redox inhibitor, E3330, with APE1. When APE1 was incubated with E3330, two NEM-modified products were observed, one with two and a second with seven added NEMs; this latter product corresponds to a fully modified APE1. In a similar control reaction without E3330, only the +2NEM product was observed in which the two solvent-accessible Cys residues, C99 and C138, were modified by NEM. Through hydrogen−deuterium amide exchange with analysis by mass spectrometry, we found that the +7NEM-modified species incorporates approximately 40 more deuterium atoms than the native protein, which exchanges nearly identically as the +2NEM product, suggesting that APE1 can be trapped in a partially unfolded state. E3330 was also found to increase the extent of disulfide bond formation involving redox critical Cys residues in APE1 as assessed by liquid chromatography and tandem mass spectrometry, suggesting a basis for its inhibitory effects on APE1’s redox activity. Collectively, our results suggest that APE1 adopts a partially unfolded state, which we propose is the redox active form of the enzyme

Base excision repair apurinic/apyrimidinic endonucleases in apicomplexan parasite Toxoplasma gondii

DNA repair is essential for cell viability and proliferation. In addition to reactive oxygen produced as a byproduct of their own metabolism, intracellular parasites also have to manage oxidative stress generated as a defense mechanism by the host. The spontaneous loss of DNA bases due to hydrolysis and oxidative DNA damage in intracellular parasites is great, but little is known about the type of DNA repair machineries that exist in these early-branching eukaryotes. However, it is clear, processes similar to DNA base excision repair (BER) must exist to rectify spontaneous and host-mediated damage in Toxoplasma gondii. Here we report that T. gondii, an opportunistic protozoan pathogen, possesses two apurinic/apyrimidinic (AP) endonucleases that function in DNA BER. We characterize the enzymatic activities of Toxoplasma exonuclease III (ExoIII, or Ape1) and endonuclease IV (EndoIV, or Apn1), designated TgAPE and TgAPN, respectively. Over-expression of TgAPN in Toxoplasma conferred protection from DNA damage, and viable knockouts of TgAPN were not obtainable. We generated an inducible TgAPN knockdown mutant using a ligand-controlled destabilization domain to establish that TgAPN is critical for Toxoplasma to recover from DNA damage. The importance of TgAPN and the fact that humans lack any observable APN family activity highlights TgAPN as a promising candidate for drug development to treat toxoplasmosis

Honeybee Colony Vibrational Measurements to Highlight the Brood Cycle

Insect pollination is of great importance to crop production worldwide and honey bees are amongst its chief facilitators. Because of the decline of managed colonies, the use of sensor technology is growing in popularity and it is of interest to develop new methods which can more accurately and less invasively assess honey bee colony status. Our approach is to use accelerometers to measure vibrations in order to provide information on colony activity and development. The accelerometers provide amplitude and frequency information which is recorded every three minutes and analysed for night time only. Vibrational data were validated by comparison to visual inspection data, particularly the brood development. We show a strong correlation between vibrational amplitude data and the brood cycle in the vicinity of the sensor. We have further explored the minimum data that is required, when frequency information is also included, to accurately predict the current point in the brood cycle. Such a technique should enable beekeepers to reduce the frequency with which visual inspections are required, reducing the stress this places on the colony and saving the beekeeper time

Collating and validating indigenous and local knowledge to apply multiple knowledge systems to an environmental challenge: A case-study of pollinators in India

There is an important role for indigenous and local knowledge in a Multiple Evidence Base to make decisions about the use of biodiversity and its management. This is important both to ensure that the knowledge base is complete (comprising both scientific and local knowledge) and to facilitate participation in the decision making process. We present a novel method to gather evidence in which we used a peer-to-peer validation process among farmers that we suggest is analogous to scientific peer review. We used a case-study approach to trial the process focussing on pollinator decline in India. Pollinator decline is a critical challenge for which there is a growing evidence base, however, this is not the case world–wide. In the state of Orissa, India, there are no validated scientific studies that record historical pollinator abundance, therefore local knowledge can contribute substantially and may indeed be the principle component of the available knowledge base. Our aim was to collate and validate local knowledge in preparation for integration with scientific knowledge from other regions, for the purpose of producing a Multiple Evidence Base to develop conservation strategies for pollinators. Farmers reported that vegetable crop yields were declining in many areas of Orissa and that the abundance of important insect crop pollinators has declined sharply across the study area in the last 10–25 years, particularly Apis cerana, Amegilla sp. and Xylocopa sp. Key pollinators for commonly grown crops were identified; both Apris cerana and Xylocopa sp. were ranked highly as pollinators by farmer participants. Crop yield declines were attributed to soil quality, water management, pests, climate change, overuse of chemical inputs and lack of agronomic expertise. Pollinator declines were attributed to the quantity and number of pesticides used. Farmers suggested that fewer pesticides, more natural habitat and the introduction of hives would support pollinator populations. This process of knowledge creation was supported by participants, which led to this paper being co-authored by both scientists and farmers

Feeding and Foraging Behaviors of Subterranean Termites (Isoptera:Rhinotermitidae).

Six studies were done on feeding and foraging behaviors of three species of subterranean termites, Coptotermes formosanus Shiraki, Reticulitermes flavipes (Kollar), and R. virginicus (Banks) (Isoptera: Rhinotermitidae). When presented with four equal wood blocks, C. formosanus did not forage randomly but concentrated on a few preferred blocks. When R. flavipes and C. formosanus were each placed in foraging arenas with linear, successive wood blocks, C. formosanus consumed a greater percentage dry mass of a wood block before moving to the next block (10.3%) than did R. flavipes (4.2%). Groups of C. formosanus were offered wood blocks that differed in initial moisture content. Wood feeding rate, number of workers, and number of soldiers were highest in the high moisture treatment. Large changes in wood moisture occurred and were affected by the presence of termites. Groups of termites from five colonies of C. formosanus were presented with wood blocks that had been previously damaged: (1) by nestmates, (2) by conspecifics from another colony, (3) by R. virginicus, and (4) no damage. Coptotermes formosanus preferred wood previously damaged by conspecifics, regardless of colony origin, over wood damaged by R. virginicus or undamaged wood. Additionally, they preferred wood damaged by R. virginicus over undamaged wood. Wood surface area (mm\sp2) exposed per unit feeding was higher for C. formosanus and R. flavipes than for R. virginicus. Wood surface area was sometimes reduced, rather than increased, as a result of feeding by R. virginicus. Groups of C. formosanus were dyed with 0%, 0.5% or 1% concentrations of the dye, Sudan Red 7B. Dyed termites had lower numbers of symbiotic protozoans, lower feeding rates, and lower survivorship than did non-dyed termites. These studies suggest that toxic baits for remedial control of termites should be placed at areas of strong foraging since termites are predisposed to stay at rewarding sites. Baits should be highly moist. Areas of structures with previous termite damage should be carefully monitored for reinfestation. These species may differ in their roles as wood decomposers. the popular termite marker, Sudan Red 7B, is not totally innocuous to C. formosanus

Mass Spectrometric Approaches to Probing the Redox Function of Ape1

Indiana University-Purdue University Indianapolis (IUPUI)Human apurinic/apyrimidinic endonuclease 1 (hApe1) is a multi-functional protein having two major functions: apurinic/apyrimidinic endonuclease activity for DNA damage repair and redox activity for gene regulation. Many studies have shown the action of Ape1 in the base excision repair pathway leading to cell survival. It has also been reported that Ape1 reduces a number of important transcription factors that are involved in cancer promotion and progression. Though the repair activity is well understood, the redox mechanism is not yet clear. What is known about Ape1 is its structure and that it contains seven cysteines (C65, C93, C99, C138, C208, C296, and C310), none of which are disulfide bonded. Two of these cysteines, C99 and C138, are solvent-accessible, and C65, C93, and C99 are located in the redox domain. It is believed that one or more cysteines are involved in the redox function and is hypothesized that hApe1 reduces the down-stream transcription factors by a disulfide exchange mechanism. E3330, (2E)-3-[5-(2,3-dimethoxy-6-methyl-1,4-benzoquninoyl)]2-nonyl-2-propenoic acid, is a specific inhibitor for the redox function of hApe1. The interaction mechanism is not known. Using N-Ethylmaleimide (NEM) chemical footprinting, combined with Hydrogen/Deuterium Exchange (HDX) data, we propose that a locally unfolded form coexists with the folded form in an equilibrium that is driven by irreversible NEM labeling, and that E3330 interacts with and stabilizes this locally unfolded form. This locally unfolded form is thereby proposed to be the redox-active form. We further support this claim with LC-MS/MS analysis showing an increase of disulfide bonds induced by E3330 among the cysteines in the redox domain, which would be too far apart from each other in the folded form to form a disulfide bond. We also studied three analogs of E3330. The need for an E3330 analog is to develop a more efficient and effective compound that would allow for sub-micromolar levels of activity (E3330 requires a micromolar amount). Study of the analogs will also allow us to gain perspective of the mechanism or mechanisms of E3330’s activity in Ape1’s redox function