Danielle Hagstrom

Redox-regulated cargo binding and release by the peroxisomal targeting signal receptor, Pex5.

Background: Pex5 transports PTS1 proteins to peroxisomes, releases them there, and returns to the cytosol. Results: Several steps of the import cycle are controlled by redox-sensitive oligomeric states of Pex5. Conclusion: Cargo release from Pex5 is achieved by a redox-regulated oligomer to dimer transition of Pex5 and aided by Pex8. Significance: This redox regulation of Pex5 function provides the first mechanistic view of cargo release. In its role as a mobile receptor for peroxisomal matrix cargo containing a peroxisomal targeting signal called PTS1, the protein Pex5 shuttles between the cytosol and the peroxisome lumen. Pex5 binds PTS1 proteins in the cytosol via its C-terminal tetratricopeptide domains and delivers them to the peroxisome lumen, where the receptor·cargo complex dissociates. The cargo-free receptor is exported to the cytosol for another round of import. How cargo release and receptor recycling are regulated is poorly understood. We found that Pex5 functions as a dimer/oligomer and that its protein interactions with itself (homo-oligomeric) and with Pex8 (hetero-oligomeric) control the binding and release of cargo proteins. These interactions are controlled by a redox-sensitive amino acid, cysteine 10 of Pex5, which is essential for the formation of disulfide bond-linked Pex5 forms, for high affinity cargo binding, and for receptor recycling. Disulfide bond-linked Pex5 showed the highest affinity for PTS1 cargo. Upon reduction of the disulfide bond by dithiothreitol, Pex5 transitioned to a noncovalent dimer, concomitant with the partial release of PTS1 cargo. Additionally, dissipation of the redox balance between the cytosol and the peroxisome lumen caused an import defect. A hetero-oligomeric interaction between the N-terminal domain (amino acids 1–110) of Pex5 and a conserved motif at the C terminus of Pex8 further facilitates cargo release, but only under reducing conditions. This interaction is also important for the release of PTS1 proteins. We suggest a redox-regulated model for Pex5 function during the peroxisomal matrix protein import cycle.

Planarian brain regeneration as a model system for developmental neurotoxicology

Abstract Freshwater planarians, famous for their regenerative prowess, have long been recognized as a valuable in vivo animal model to study the effects of chemical exposure. In this review, we summarize the current techniques and tools used in the literature to assess toxicity in the planarian system. We focus on the planarians particular amenability for neurotoxicology and neuroregeneration studies, owing to the planarians unique ability to regenerate a centralized nervous system. Zooming in from the organismal to the molecular level, we show that planarians offer a repertoire of morphological and behavioral readouts while also being amenable to mechanistic studies of compound toxicity. Finally, we discuss the open challenges and opportunities for planarian brain regeneration to become an important model system for modern toxicology.

The unique degradation pathway of the PTS2 receptor, Pex7, is dependent on the PTS receptor/coreceptor, Pex5 and Pex20.

In Pichia pastoris, the PTS2 receptor, Pex7, is selectively degraded in a regulated manner. The shuttling of Pex7, and consequently its degradation, depends on the receptor recycling pathways used by Pex5 and Pex20 and relies on an interaction between Pex7 and Pex20. The shuttling and stability of Pex7 are divergent from those of Pex5 and Pex20.

Planarian cholinesterase: in vitro characterization of an evolutionarily ancient enzyme to study organophosphorus pesticide toxicity and reactivation

The freshwater planarian Dugesia japonica has recently emerged as an animal model for developmental neurotoxicology and found to be sensitive to organophosphorus (OP) pesticides. While previous activity staining of D. japonica, which possess a discrete cholinergic nervous system, has shown acylthiocholine catalysis, it is unknown whether this is accomplished through an acetylcholinesterase (AChE), butyrylcholinesterase (BChE), or a hybrid esterase and how OP exposure affects esterase activity. Here, we show that the majority of D. japonica cholinesterase (DjChE) activity departs from conventional AChE and BChE classifications. Inhibition by classic protonable amine and quaternary reversible inhibitors (ethopropazine, donepezil, tacrine, edrophonium, BW284c51, propidium) shows that DjChE is far less sensitive to these inhibitors than human AChE, suggesting discrete differences in active center and peripheral site recognition and structures. Additionally, we find that different OPs (chlorpyrifos oxon, paraoxon, dichlorvos, diazinon oxon, malaoxon) and carbamylating agents (carbaryl, neostigmine, physostigmine, pyridostigmine) differentially inhibit DjChE activity in vitro. DjChE was most sensitive to diazinon oxon and neostigmine and least sensitive to malaoxon and carbaryl. Diazinon oxon-inhibited DjChE could be reactivated by the quaternary oxime, pralidoxime (2-PAM), and the zwitterionic oxime, RS194B, with RS194B being significantly more potent. Sodium fluoride (NaF) reactivates OP-DjChE faster than 2-PAM. As one of the most ancient true cholinesterases, DjChE provides insight into the evolution of a hybrid enzyme before the separation into distinct AChE and BChE enzymes found in higher vertebrates. The sensitivity of DjChE to OPs and capacity for reactivation validate the use of planarians for OP toxicology studies.

Multi-Behavioral Endpoint Testing Of An 87-Chemical Compound Library In Freshwater Planarians

There is an increased recognition in the field of toxicology of the value of medium-to-high-throughput screening methods using in vitro and alternative animal models. We have previously introduced the asexual freshwater planarian Dugesia japonica as a new alternative animal model and proposed that it is particularly well-suited for the study of developmental neurotoxicology. In this article, we discuss how we have expanded and automated our screening methodology to allow for fast screening of multiple behavioral endpoints, developmental toxicity, and mortality. Using an 87-compound library provided by the National Toxicology Program, consisting of known and suspected neurotoxicants, including drugs, flame retardants, industrial chemicals, polycyclic aromatic hydrocarbons (PAHs), pesticides, and presumptive negative controls, we further evaluate the benefits and limitations of the system for medium-throughput screening, focusing on the technical aspects of the system. We show that, in the context of this library, planarians are the most sensitive to pesticides with 16/16 compounds causing toxicity and the least sensitive to PAHs, with only 5/17 causing toxicity. Furthermore, while none of the presumptive negative controls were bioactive in adult planarians, 2/5, acetaminophen and acetylsalicylic acid, were bioactive in regenerating worms. Notably, these compounds were previously reported as developmentally toxic in mammalian studies. Through parallel screening of adults and developing animals, planarians are thus a useful model to detect such developmental-specific effects, which was observed for 13 chemicals in this library. We use the data and experience gained from this screen to propose guidelines for best practices when using planarians for toxicology screens.

Biochemically characterizing the subcellular localization of peroxisomal proteins by fractionation, protease protection, and carbonate extraction.

Traditional biochemical approaches, as well as the complementary methods of living cell fluorescence microscopy and immunofluorescence microscopy, can serve to characterize the subcellular localization of proteins and organelles. This chapter describes methods for isolation of crude organelle fractions from methanol- or oleate-grown Pichia pastoris, followed by protease protection and carbonate extraction assays to dissect the subcellular localization of peroxisomal matrix and membrane proteins. These biochemical tools can be used to analyze the targeting efficiency of proteins to the peroxisome membrane and matrix, as well as the topology of membrane proteins.