IRE1 signaling increases PERK expression during chronic ER stress.

The Unfolded Protein Response (UPR) is an essential cellular process activated by the accumulation of unfolded proteins within the Endoplasmic Reticulum (ER), a condition referred to as ER stress. Three ER anchored receptors, IRE1, PERK and ATF6 act as ER stress sensors monitoring the health of the ER. Upon detection of ER stress, IRE1, PERK and ATF6 initiate downstream signaling pathways collectively referred to as the UPR. The overarching aim of the UPR is to restore ER homeostasis by reducing ER stress, however if that is not possible, the UPR transitions from a pro-survival to a pro-death response. While our understanding of the key signaling pathways central to the UPR is well defined, the same is not true of the subtle signaling events that help fine tune the UPR, supporting its ability to adapt to varying amplitudes or durations of ER stress. In this study, we demonstrate cross talk between the IRE1 and PERK branches of the UPR, wherein IRE1 via XBP1s signaling helps to sustain PERK expression during prolonged ER stress. Our findings suggest cross talk between UPR branches aids adaptiveness thereby helping to support the plasticity of UPR signaling responses.

Unfolding the Interactions between Endoplasmic Reticulum Stress and Oxidative Stress.

Oxidative stress is caused by an imbalance in cellular redox state due to the accumulation of reactive oxygen species (ROS). While homeostatic levels of ROS are important for cell physiology and signaling, excess ROS can induce a variety of negative effects ranging from damage to biological macromolecules to cell death. Additionally, oxidative stress can disrupt the function of redox-sensitive organelles including the mitochondria and endoplasmic reticulum (ER). In the case of the ER, the accumulation of misfolded proteins can arise due to oxidative stress, leading to the onset of ER stress. To combat ER stress, cells initiate a highly conserved stress response called the unfolded protein response (UPR). While UPR signaling, within the context of resolving ER stress, is well characterised, how UPR mediators respond to and influence oxidative stress is less defined. In this review, we evaluate the interplay between oxidative stress, ER stress and UPR signaling networks. Specifically, we assess how UPR signaling mediators can influence antioxidant responses.

Liquid chromatography-tandem mass spectrometry glycoproteomic study of porcine IgG and detection of subtypes.

RATIONALE: While high-throughput proteomic methods have been widely applied to monoclonal antibodies and human immunoglobulin gamma (IgG) samples, less information is available on porcine IgG. As pigs are considered one of the most suitable species for xenotransplantation, it is important to characterize IgG amino acid sequences and glycosylation profiles, which is the focus of this study. METHODS: Three different purified porcine IgG samples, including wild-type and knockout species, were digested with trypsin and enriched for glycopeptides. Digestion mixtures were spiked with a mixture of six standard peptides. Analysis was performed using electrospray ionization liquid chromatography-tandem mass spectrometry (MS/MS) in standard MS/MS data-dependent acquisition mode on a hybrid triple quadrupole time-of-flight mass spectrometer. RESULTS: To facilitate the classification of subtypes detected experimentally, UniprotKB database entries were organized using comparative alignment scores. Sequences were grouped based on 11 different subtypes as translated from GenBank entries. Proteomic searches were accomplished automatically using specialized software, whereas glycoprotein searches were performed manually by monitoring the extracted chromatograms of diagnostic MS/MS glycan fragments and studying their corresponding mass spectra; 40-50 non-glycosylated peptides and 4-5 glycosylated peptides were detected in each sample, with several glycoforms per sequence. CONCLUSIONS: Proteomic analysis of porcine IgG is complicated by factors such as the presence of several subtypes, redundant heavy chain (HC) sequences in protein databases, and the lack of consistent cross-referencing between databases. Aligning and comparing HC sequences were necessary to eliminate redundancy. This study highlights the complexity of pig IgG and shows the importance of MS in proteomics and glycoproteomics.