A cost-effective approach to microporate mammalian cells with the Neon Transfection System

Electroporation is one of the most efficient non-viral methods for transferring exogenous DNA into mammalian cells. However, the relatively high costs of electroporation kits and reagents temper the routine use of this fast and easy to perform technique in many laboratories. Several years ago, a new flexible and easy to operate electroporation device was launched under the name "Neon(TM) Transfection System". This device uses specialized pipette tips containing gold-plated electrodes as electroporation chamber. Here we report a protocol to regenerate these expensive tips as well as some other Neon(TM) kit accessories, thereby reducing the cost of electroporation at least ten-fold.publisher: Elsevier articletitle: A cost-effective approach to microporate mammalian cells with the Neon Transfection System journaltitle: Analytical Biochemistry articlelink: http://dx.doi.org/10.1016/j.ab.2014.08.017 content_type: article copyright: Copyright © 2014 Elsevier Inc. All rights reserved.status: publishe

KillerRed as a tool to study the cellular responses to peroxisome-derived oxidative stress

Many biological processes and cell fate decisions are modulated by changes in redox environment. To gain insight into how subcellular compartmentalization of reactive oxygen species (ROS) formation contributes to (site-specific) redox signaling and oxidative stress responses, it is critical to have access to tools that allow tight spatial and temporal control of ROS production. Over the past decade, the use of genetically-encoded photosensitizers has attracted growing interest of researchers because these proteins can be easily targeted to various subcellular compartments and allow controlled release of ROS when excited by light. This chapter provides guidance and practical advice on the use of po-KR, a peroxisomal variant of the phototoxic red fluorescent protein KillerRed, to address fundamental questions about how mammalian cells cope with peroxisome-derived oxidative stress.status: publishe

Trypanosoma brucei glycosomal ABC transporters: identification and membrane targeting.

Trypanosomes contain unique peroxisome-like organelles designated glycosomes which sequester enzymes involved in a variety of metabolic processes including glycolysis. We identified three ABC transporters associated with the glycosomal membrane of Trypanosoma brucei. They were designated GAT1-3 for Glycosomal ABC Transporters. These polypeptides are so-called half-ABC transporters containing only one transmembrane domain and a single nucleotide-binding domain, like their homologues of mammalian and yeast peroxisomes. The glycosomal localization was shown by immunofluorescence microscopy of trypanosomes expressing fusion constructs of the transporters with Green Fluorescent Protein. By expression of fluorescent deletion constructs, the glycosome-targeting determinant of two transporters was mapped to different fragments of their respective primary structures. Interestingly, these fragments share a short sequence motif and contain adjacent to it one--but not the same--of the predicted six transmembrane segments of the transmembrane domain. We also identified the T. brucei homologue of peroxin PEX19, which is considered to act as a chaperonin and/or receptor for cytosolically synthesized proteins destined for insertion into the peroxisomal membrane. By using a bacterial two-hybrid system, it was shown that glycosomal ABC transporter fragments containing an organelle-targeting determinant can interact with both the trypanosomatid and human PEX19, despite their low overall sequence identity. Mutated forms of human PEX19 that lost interaction with human peroxisomal membrane proteins also did not bind anymore to the T. brucei glycosomal transporter. Moreover, fragments of the glycosomal transporter were targeted to the peroxisomal membrane when expressed in mammalian cells. Together these results indicate evolutionary conservation of the glycosomal/peroxisomal membrane protein import mechanism

The peroxisomal import receptor PEX5 functions as a stress sensor, retaining catalase in the cytosol in times of oxidative stress

Accumulating evidence indicates that peroxisome functioning, catalase localization, and cellular oxidative balance are intimately interconnected. Nevertheless, it remains largely unclear why modest increases in the cellular redox state especially interfere with the subcellular localization of catalase, the most abundant peroxisomal antioxidant enzyme. This study aimed at gaining more insight into this phenomenon. Therefore, we first established a simple and powerful approach to study peroxisomal protein import and protein-protein interactions in living cells in response to changes in redox state. By employing this approach, we confirm and extend previous observations that Cys-11 of human PEX5, the shuttling import receptor for peroxisomal matrix proteins containing a C-terminal peroxisomal targeting signal (PTS1), functions as a redox switch that modulates the protein's activity in response to intracellular oxidative stress. In addition, we show that oxidative stress affects the import of catalase, a non-canonical PTS1-containing protein, more than the import of a reporter protein containing a canonical PTS1. Furthermore, we demonstrate that changes in the local redox state do not affect PEX5-substrate binding and that human PEX5 does not oligomerize in cellulo, not even when the cells are exposed to oxidative stress. Finally, we present evidence that catalase retained in the cytosol can protect against H2O2-mediated redox changes in a manner that peroxisomally targeted catalase does not. Together, these findings lend credit to the idea that inefficient catalase import, when coupled with the role of PEX5 as a redox-regulated import receptor, constitutes a cellular defense mechanism to combat oxidative insults of extra-peroxisomal origin.status: publishe

Peroxisomes as modulators of cellular protein thiol oxidation: a new model system.

Aims: Peroxisomes are ubiquitous, single-membrane-bounded organelles that contain considerable amounts of enzymes involved in the production or breakdown of hydrogen peroxide (H2O2), a key signaling molecule in multiple biological processes and disease states. Despite this, the role of this organelle in cross-compartmental H2O2 signaling remains largely unclear, mainly because of the difficulty to modulate peroxisomal H2O2 production in a selective manner. This study aimed at establishing and validating a cellular model suitable to decipher the complex signaling processes associated with peroxisomal H2O2 release. Results: Here, we report the development of a human cell line that can be used to selectively generate H2O2 inside peroxisomes in a time- and dose-controlled manner. In addition, we provide evidence that peroxisomederived H2O2 can oxidize redox-sensitive cysteine residues in multiple proteins within (e.g., peroxiredoxin-5 [PRDX5]) and outside (e.g., nuclear factor kappa B subunit 1 [NFKB1] and subunit RELA proto-oncogene [RELA], phosphatase and tensin homolog [PTEN], forkhead box O3 [FOXO3], and peroxin 5 [PEX5]) the peroxisomal compartment. Furthermore, we show that the extent of protein oxidation depends on the subcellular location of the target protein and is inversely correlated to catalase activity and cellular glutathione content. Finally, we demonstrate that excessive H2O2 production inside peroxisomes does not induce their selective degradation, at least not under the conditions examined. Innovation: This study describes for the first time a powerful model system that can be used to examine the role of peroxisome-derived H2O2 in redox-regulated (patho)physiological processes, a research area in need of further investigation and innovative approaches. Conclusion: Our results provide unambiguous evidence that peroxisomes can serve as regulatory hubs in thiolbased signaling networks

Peroxisomes as Modulators of Cellular Protein Thiol Oxidation: A New Model System

AIMS: Peroxisomes are ubiquitous, single-membrane-bounded organelles that contain considerable amounts of enzymes involved in the production or breakdown of hydrogen peroxide (H2O2), a key signaling molecule in multiple biological processes and disease states. Despite this, the role of this organelle in cross-compartmental H2O2 signaling remains largely unclear, mainly because of the difficulty to modulate peroxisomal H2O2 production in a selective manner. This study aimed at establishing and validating a cellular model suitable to decipher the complex signaling processes associated with peroxisomal H2O2 release. RESULTS: Here, we report the development of a human cell line that can be used to selectively generate H2O2 inside peroxisomes in a time- and dose-controlled manner. In addition, we provide evidence that peroxisome-derived H2O2 can oxidize redox-sensitive cysteine residues in multiple proteins within (e.g., peroxiredoxin-5 [PRDX5]) and outside (e.g., nuclear factor kappa B subunit 1 [NFKB1] and subunit RELA proto-oncogene [RELA], phosphatase and tensin homolog [PTEN], forkhead box O3 [FOXO3], and peroxin 5 [PEX5]) the peroxisomal compartment. Furthermore, we show that the extent of protein oxidation depends on the subcellular location of the target protein and is inversely correlated to catalase activity and cellular glutathione content. Finally, we demonstrate that excessive H2O2 production inside peroxisomes does not induce their selective degradation, at least not under the conditions examined. INNOVATION: This study describes for the first time a powerful model system that can be used to examine the role of peroxisome-derived H2O2 in redox-regulated (patho)physiological processes, a research area in need of further investigation and innovative approaches. CONCLUSION: Our results provide unambiguous evidence that peroxisomes can serve as regulatory hubs in thiol-based signaling networks.status: publishe