Detecting peroxiredoxin hyperoxidation by one-dimensional isoelectric focusing

The activity of typical 2-cys peroxiredoxin (Prxs) can be regulated by hyperoxidation with a consequent loss of redox activity. Here we developed a simple assay to monitor the level of hyperoxidation of different typical 2-cys prxs simultaneously. This assay only requires standard equipment and can compare different samples on the same gel. It requires much less time than conventional 2D gels and gives more information than Western blotting with an antibody specific for hyperoxidized peroxiredoxin. This method could also be used to monitor protein modification with a charge difference such as phosphorylation

How are proteins reduced in the endoplasmic reticulum?

The reversal of thiol oxidation in proteins within the endoplasmic reticulum (ER) is crucial for protein folding, degradation, chaperone function, and the ER stress response. Our understanding of this process is generally poor but progress has been made. Enzymes performing the initial reduction of client proteins, as well as the ultimate electron donor in the pathway, have been identified. Most recently, a role for the cytosol in ER protein reduction has been revealed. Nevertheless, how reducing equivalents are transferred from the cytosol to the ER lumen remains an open question. We review here why proteins are reduced in the ER, discuss recent data on catalysis of steps in the pathway, and consider the implications for redox homeostasis within the early secretory pathway

Lack of an efficient endoplasmic reticulum-localized recycling system protects peroxiredoxin IV from hyperoxidation

Typical 2-cys peroxiredoxins are required to remove hydrogen peroxide from several different cellular compartments. Their activity can be regulated by hyperoxidation and consequent inactivation of the active site peroxidatic cysteine. Here we have developed a simple assay to quantify the hyperoxidation of peroxiredoxins. Hyperoxidation of peroxiredoxins can only occur efficiently in the presence of a recycling system usually based on thioredoxin and thioredoxin reductase. We demonstrate that there is a marked difference in the sensitivity of the endoplasmic reticulum-localized peroxiredoxin to hyperoxidation compared to either the cytosolic or mitochondrial enzymes. Each enzyme is equally sensitive to hyperoxidation in the presence of a robust recycling system. Our results demonstrate that the peroxiredoxin IV recycling in the ER is much less efficient than in the cytosol or mitochondria leading to the protection of peroxiredoxin IV from hyperoxidation

Folding of a single domain protein entering the endoplasmic reticulum precedes disulfide formation

The relationship between protein synthesis, folding and disulfide formation within the endoplasmic reticulum (ER) is poorly understood. Previous studies have suggested pre-existing disulfide links are absolutely required to allow protein folding and, conversely, that protein folding occurs prior to disulfide formation. To address the question of what happens first within the ER; that is, protein folding or disulfide formation, we studied folding events at the early stages of polypeptide chain translocation into the mammalian ER using stalled translation intermediates. Our results demonstrate that polypeptide folding can occur without complete domain translocation. Protein disulfide isomerase (PDI) interacts with these early intermediates, but disulfide formation does not occur unless the entire sequence of the protein domain is translocated. This is the first evidence that folding of the polypeptide chain precedes disulfide formation within a cellular context and highlights key differences between protein folding in the ER and refolding of purified proteins

Inhibition of IRE1α-mediated XBP1 mRNA cleavage by XBP1 reveals a novel regulatory process during the unfolded protein response

Background: The mammalian endoplasmic reticulum (ER) continuously adapts to the cellular secretory load by the activation of an unfolded protein response (UPR).  This stress response results in expansion of the ER, upregulation of proteins involved in protein folding and degradation, and attenuation of protein synthesis.  The response is orchestrated by three signalling pathways each activated by a specific signal transducer, either inositol requiring enzyme α (IRE1α), double-stranded RNA-activated protein kinase-like ER kinase (PERK) or activating transcription factor 6 (ATF6).  Activation of IRE1α results in its oligomerisation, autophosphorylation and stimulation of its ribonuclease activity.  The ribonuclease initiates the splicing of an intron from mRNA encoding the transcription factor, X-box binding protein 1 (XBP1), as well as degradation of specific mRNAs and microRNAs. Methods: To investigate the consequence of expression of exogenous XBP1, we generated a stable cell-line expressing spliced XBP1 mRNA under the control of an inducible promotor. Results: Following induction of expression, high levels of XBP1 protein were detected, which allowed upregulation of target genes in the absence of induction of the UPR.  Remarkably under stress conditions, the expression of exogenous XBP1 repressed splicing of endogenous XBP1 mRNA without repressing the activation of PERK. Conclusions: These results illustrate that a feedback mechanism exists to attenuate Ire1α ribonuclease activity in the presence of XBP1

Inactivation of mammalian Ero 1α is catalysed by specific protein disulfide isomerases

Disulfide formation within the endoplasmic reticulum is a complex process requiring a disulfide exchange protein such as protein disulfide isomerase and a mechanism to form disulfides de novo. In mammalian cells, the major pathway for de novo disulfide formation involves the enzyme Ero1α which couples oxidation of thiols to the reduction of molecular oxygen to form hydrogen peroxide. Ero1α activity is tightly regulated by a mechanism that requires the formation of regulatory disulfides. These regulatory disulfides are reduced to activate and reform to inactive the enzyme. To investigate the mechanism of inactivation we analysed regulatory disulfide formation in the presence of various oxidants under controlled oxygen concentration. Neither molecular oxygen, nor hydrogen peroxide was able to oxidise Ero1α efficiently to form the correct regulatory disulfides. However, specific members of the PDI family such as PDI or ERp46 were able to catalyse this process. Further studies showed that both active sites of PDI contribute to the formation of regulatory disulfides in Ero1α and that the PDI substrate binding domain is crucial to allow electron transfer between the two enzymes. These results demonstrate a simple feedback mechanism of regulation of mammalian Ero1α involving its primary substrate

Characterization of a cryogenic beam source for atoms and molecules

We present a combined experimental and theoretical study of beam formation from a cryogenic buffer gas cell. Atoms and molecules are loaded into the cell by laser ablation of a target, and are cooled and swept out of the cell by a flow of cold helium. We study the thermalization and flow dynamics inside the cell and measure how the speed, temperature, divergence and extraction efficiency of the beam are influenced by the helium flow. We use a finite element model to simulate the flow dynamics and use the predictions of this model to interpret our experimental results.Comment: 10 pages, 14 figure

ERdj5 is the ER reductase that catalyzes the removal of non-native disulfides and correct folding of the LDL receptor

ERdj5 is a member of the protein disulfide isomerase family of proteins localized to the endoplasmic reticulum (ER) of mammalian cells. To date, only a limited number of substrates for ERdj5 are known. Here we identify a number of endogenous substrates that form mixed disulfides with ERdj5, greatly expanding its client repertoire. ERdj5 previously had been thought to exclusively reduce disulfides in proteins destined for dislocation to the cytosol for degradation. However, we demonstrate here that for one of the identified substrates, the low-density lipoprotein receptor (LDLR), ERdj5 is required not for degradation, but rather for efficient folding. Our results demonstrate that the crucial role of ERdj5 is to reduce non-native disulfides formed during productive folding and that this requirement is dependent on its interaction with BiP. Hence, ERdj5 acts as the ER reductase, both preparing misfolded proteins for degradation and catalyzing the folding of proteins that form obligatory non-native disulfides

Cytosolic thioredoxin reductase 1 is required for correct disulfide formation in the ER

Folding of proteins entering the secretory pathway in mammalian cells frequently requires the insertion of disulfide bonds. Disulfide insertion can result in covalent linkages found in the native structure as well as those that are not, so‐called non‐native disulfides. The pathways for disulfide formation are well characterized, but our understanding of how non‐native disulfides are reduced so that the correct or native disulfides can form is poor. Here, we use a novel assay to demonstrate that the reduction in non‐native disulfides requires NADPH as the ultimate electron donor, and a robust cytosolic thioredoxin system, driven by thioredoxin reductase 1 (TrxR1 or TXNRD1). Inhibition of this reductive pathway prevents the correct folding and secretion of proteins that are known to form non‐native disulfides during their folding. Hence, we have shown for the first time that mammalian cells have a pathway for transferring reducing equivalents from the cytosol to the ER, which is required to ensure correct disulfide formation in proteins entering the secretory pathway

Deceleration of a supersonic beam of SrF molecules to 120 m/s

We report on the deceleration of a beam of SrF molecules from 290 to 120~m/s. Following supersonic expansion, the molecules in the $X^2\Sigma$ ($v=0$, $N=1$) low-field seeking states are trapped by the moving potential wells of a traveling-wave Stark decelerator. With a deceleration strength of 9.6 km/s$^2$ we have demonstrated the removal of 85 % of the initial kinetic energy in a 4 meter long modular decelerator. The absolute amount of kinetic energy removed is a factor 1.5 higher compared to previous Stark deceleration experiments. The demonstrated decelerator provides a novel tool for the creation of highly collimated and slow beams of heavy diatomic molecules, which serve as a good starting point for high-precision tests of fundamental physics