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

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

ERp18 regulates the activation of ATF6α during the unfolded protein response

Activation of the ATF6α signaling pathway is initiated by trafficking of ATF6α from the ER to the Golgi apparatus. Its subsequent proteolysis releases a transcription factor that translocates to the nucleus causing downstream gene activation. How ER retention, Golgi trafficking, and proteolysis of ATF6α are regulated and whether additional protein partners are required for its localization and processing remain unresolved. Here, we show that ER‐resident oxidoreductase ERp18 associates with ATF6α following ER stress and plays a key role in both trafficking and activation of ATF6α. We find that ERp18 depletion attenuates the ATF6α stress response. Paradoxically, ER stress accelerates trafficking of ATF6α to the Golgi in ERp18‐depleted cells. However, the translocated ATF6α becomes aberrantly processed preventing release of the soluble transcription factor. Hence, we demonstrate that ERp18 monitors ATF6α ER quality control to ensure optimal processing following trafficking to the Golgi

ERp18 regulates the activation of ATF6α during the unfolded protein response

Activation of the ATF6α signaling pathway is initiated by trafficking of ATF6α from the ER to the Golgi apparatus. Its subsequent proteolysis releases a transcription factor that translocates to the nucleus causing downstream gene activation. How ER retention, Golgi trafficking, and proteolysis of ATF6α are regulated and whether additional protein partners are required for its localization and processing remain unresolved. Here, we show that ER‐resident oxidoreductase ERp18 associates with ATF6α following ER stress and plays a key role in both trafficking and activation of ATF6α. We find that ERp18 depletion attenuates the ATF6α stress response. Paradoxically, ER stress accelerates trafficking of ATF6α to the Golgi in ERp18‐depleted cells. However, the translocated ATF6α becomes aberrantly processed preventing release of the soluble transcription factor. Hence, we demonstrate that ERp18 monitors ATF6α ER quality control to ensure optimal processing following trafficking to the Golgi

Phenological mismatch affects individual fitness and population growth in the winter moth

Climate change can severely impact species that depend on temporary resources by inducing phenological mismatches between consumer and resource seasonal timing. In the winter moth, warmer winters caused eggs to hatch before their food source, young oak leaves, became available. This phenological mismatch changed the selection on the temperature sensitivity of egg development rate. However, we know little about the fine-scale fitness consequences of phenological mismatch at the individual level and how this mismatch affects population dynamics in the winter moth. To determine the fitness consequences of mistimed egg hatching relative to timing of oak budburst, we quantified survival and pupation weight in a feeding experiment. We found that mismatch greatly increased mortality rates of freshly hatched caterpillars, as well as affecting caterpillar growth and development time. We then investigated whether these individual fitness consequences have population-level impacts by estimating the effect of phenological mismatch on population dynamics, using our long-term data (1994-2021) on relative winter moth population densities at four locations in The Netherlands. We found a significant effect of mismatch on population density with higher population growth rates in years with a smaller phenological mismatch. Our results indicate that climate change-induced phenological mismatch can incur severe individual fitness consequences that can impact population density in the wild.</p

Timing manipulations reveal the lack of a causal link across timing of annual-cycle stages in a long-distance migrant

Organisms need to time their annual-cycle stages, like breeding and migration, to occur at the right time of the year. Climate change has shifted the timing of annual-cycle stages at different rates, thereby tightening or lifting time constraints of these annual-cycle stages, a rarely studied consequence of climate change. The degree to which these constraints are affected by climate change depends on whether consecutive stages are causally linked (scenario I) or whether the timing of each stage is independent of other stages (scenario II). Under scenario I, a change in timing in one stage has knock-on timing effects on subsequent stages, whereas under scenario II, a shift in the timing of one stage affects the degree of overlap with previous and subsequent stages. To test this, we combined field manipulations, captivity measurements and geolocation data. We advanced and delayed hatching dates in pied flycatchers (Ficedula hypoleuca) and measured how the timing of subsequent stages (male moult and migration) were affected. There was no causal effect of manipulated hatching dates on the onset of moult and departure to Africa. Thus, advancing hatching dates reduced the male moult–breeding overlap with no effect on the moult–migration interval. Interestingly, the wintering location of delayed males was more westwards, suggesting that delaying the termination of breeding carries over to winter location. Because we found no causal linkage of the timing of annual-cycle stages, climate change could shift these stages at different rates, with the risk that the time available for some becomes so short that this will have major fitness consequences

The mammalian cytosolic thioredoxin reductase pathway acts via a membrane 1 protein to reduce ER-localised proteins

Folding of proteins entering the mammalian secretory pathway requires the insertion of the correct disulfides. Disulfide formation involves both an oxidative pathway for their insertion and a reductive pathway to remove incorrectly formed disulfides. Reduction of these disulfides is critical for correct folding and degradation of misfolded proteins. Previously, we showed that the reductive pathway is driven by NADPH generated in the cytosol. Here, by reconstituting the pathway using purified proteins and ER microsomal membranes, we demonstrate that the thioredoxin reductase system provides the minimal cytosolic components required for reducing proteins within the ER lumen. In particular, saturation of the pathway and its protease sensitivity demonstrates the requirement for a membrane protein to shuttle electrons from the cytosol to the ER. These results provide compelling evidence for the critical role of the cytosol in regulating ER redox homeostasis ensuring correct protein folding and facilitating the degradation of misfolded ER proteins

Spatial Separation of HLA-DM/HLA-DR Interactions within MIIC and Phagosome-Induced Immune Escape

SummaryMajor Histocompatibility Complex (MHC) class II molecules, including Human Leukocyte Antigen (HLA)-DR, present peptide fragments from proteins degraded in the endocytic pathway. HLA-DR is targeted to late-endocytic structures named MHC class II-containing Compartments (MIIC), where it interacts with HLA-DM. This chaperone stabilizes HLA-DR during peptide exchange and is critical for successful peptide loading. To follow this process in living cells, we have generated cells containing HLA-DR3/Cyan Fluorescent Protein (CFP), HLA-DM/Yellow Fluorescent Protein (YFP), and invariant chain. HLA-DR/DM interactions were observed by Fluorescence Resonance Energy Transfer (FRET). These interactions were pH insensitive, yet occurred only in internal structures and not at the limiting membrane of MIIC. In a cellular model of infection, phagosomes formed a limiting membrane surrounding internalized Salmonella. HLA-DR and HLA-DM did not interact in Salmonella-induced vacuoles, and HLA-DR was not loaded with antigens. The absence of HLA-DR/DM interactions at the limiting membrane prevents local loading of MHC class II molecules in phagosomes. This may allow these bacteria to successfully evade the immune system

Two essential Thioredoxins mediate apicoplast biogenesis, protein import, and gene expression in Toxoplasma gondii.

Apicomplexan parasites are global killers, being the causative agents of diseases like toxoplasmosis and malaria. These parasites are known to be hypersensitive to redox imbalance, yet little is understood about the cellular roles of their various redox regulators. The apicoplast, an essential plastid organelle, is a verified apicomplexan drug target. Nuclear-encoded apicoplast proteins traffic through the ER and multiple apicoplast sub-compartments to their place of function. We propose that thioredoxins contribute to the control of protein trafficking and of protein function within these apicoplast compartments. We studied the role of two Toxoplasma gondii apicoplast thioredoxins (TgATrx), both essential for parasite survival. By describing the cellular phenotypes of the conditional depletion of either of these redox regulated enzymes we show that each of them contributes to a different apicoplast biogenesis pathway. We provide evidence for TgATrx1's involvement in ER to apicoplast trafficking and TgATrx2 in the control of apicoplast gene expression components. Substrate pull-down further recognizes gene expression factors that interact with TgATrx2. We use genetic complementation to demonstrate that the function of both TgATrxs is dependent on their disulphide exchange activity. Finally, TgATrx2 is divergent from human thioredoxins. We demonstrate its activity in vitro thus providing scope for drug screening. Our study represents the first functional characterization of thioredoxins in Toxoplasma, highlights the importance of redox regulation of apicoplast functions and provides new tools to study redox biology in these parasites

Comparing the Efficacy of Bevacizumab and Ranibizumab in Patients with Diabetic Macular Edema (BRDME):The BRDME Study, a Randomized Trial

Purpose: To generate conclusive evidence regarding the noninferiority of intravitreal bevacizumab compared with ranibizumab in patients with diabetic macular edema (DME). Design: Comparative, randomized, double-masked, multicenter, noninferiority clinical trial. Participants: Eligible patients were older than 18 years, diagnosed with type 1 or type 2 diabetes mellitus, with glycosylated hemoglobin of less than 12%, central area thickness of more than 325 μm, and visual impairment from DME with a best-corrected visual acuity (BCVA) between 24 letters and 78 letters. Methods: From June 2012 through February 2018, a total of 170 participants were randomized to receive 6 monthly injections of either 1.25 mg bevacizumab (n = 86) or 0.5 mg ranibizumab (n = 84). Main Outcome Measures: Primary outcome was change in BCVA from baseline to month 6 compared between the 2 treatment arms. The noninferiority margin was 3.5 letters. Results: The difference in mean BCVA between treatment arms was 1.8 letters in favor of ranibizumab after 6 months of follow-up; BCVA improved by 4.9±6.7 letters in the bevacizumab group and 6.7±8.7 letters in the ranibizumab group. The lower bound of the 2-sided 90% confidence interval (CI) was –3.626 letters, exceeding the noninferiority margin of 3.5 letters. Central area thickness decreased more with ranibizumab (138.2±114.3 μm) compared with bevacizumab (64.2±104.2 μm). In a post hoc subgroup analysis, participants with a worse BCVA at baseline (≤69 letters) improved by 6.7±7.0 letters with bevacizumab and 10.4±10.0 letters with ranibizumab, and central area thickness decreased significantly more in the ranibizumab arm of this subgroup compared with the bevacizumab arm. Participants with an initially better BCVA at baseline (≥70 letters) did not demonstrate differences in BCVA or OCT outcomes between treatment arms. Conclusions: Based on change in BCVA from baseline to month 6, the noninferiority of 1.25 mg bevacizumab to 0.5 mg ranibizumab was not confirmed. Only the subgroup of patients with a lower BCVA at baseline showed better visual acuity and anatomic outcomes with ranibizumab. Our study confirmed the potential differential efficacy of anti–vascular endothelial growth factor agents in the treatment of DME as well as the difference in response between patient groups with different baseline visual acuities