Estrogens promote misfolded proinsulin degradation to protect insulin production and delay diabetes

Summary: Conjugated estrogens (CE) delay the onset of type 2 diabetes (T2D) in postmenopausal women, but the mechanism is unclear. In T2D, the endoplasmic reticulum (ER) fails to promote proinsulin folding and, in failing to do so, promotes ER stress and β cell dysfunction. We show that CE prevent insulin-deficient diabetes in male and in female Akita mice using a model of misfolded proinsulin. CE stabilize the ER-associated protein degradation (ERAD) system and promote misfolded proinsulin proteasomal degradation. This involves activation of nuclear and membrane estrogen receptor-α (ERα), promoting transcriptional repression and proteasomal degradation of the ubiquitin-conjugating enzyme and ERAD degrader, UBC6e. The selective ERα modulator bazedoxifene mimics CE protection of β cells in females but not in males. : Estrogens prevent diabetes in women, but the mechanism is poorly understood. Xu et al. report that estrogens activate the endoplasmic-reticulum-associated protein degradation pathway, which promotes misfolded proinsulin degradation, suppresses endoplasmic reticulum stress, and protects insulin secretion in mice and in human pancreatic β cells. Keywords: estrogens, beta cell, islet, endoplasmic reticulum stress, proinsulin misfolding, diabetes, bazedoxifene, sex dimorphism, ERAD, SER

Ricin B chain targeted to the endoplasmic reticulum of tobacco protoplasts is degraded by a CDC48- and vacuole-independent mechanism

The B chain of ricin was expressed and delivered to the endoplasmic reticulum of tobacco protoplasts where it disappeared with time in a manner consistent with degradation. This turnover did not occur in the vacuoles or upon secretion. Indeed, several lines of evidence indicate that, in contrast to the turnover of endoplasmic reticulum-targeted ricin A chain in the cytosol, the bulk of expressed ricin B chain was degraded in the secretory pathway

Retrograde trafficking of Argonaute 2 acts as a rate-limiting step for de novo miRNP formation on endoplasmic reticulum–attached polysomes in mammalian cells

microRNAs are short regulatory RNAs in metazoan cells. Regulation of miRNA activity and abundance is evident in human cells where availability of target messages can influence miRNA biogenesis by augmenting the Dicer1-dependent processing of precursors to mature microRNAs. Requirement of subcellular compartmentalization of Ago2, the key component of miRNA repression machineries, for the controlled biogenesis of miRNPs is reported here. The process predominantly happens on the polysomes attached with the endoplasmic reticulum for which the subcellular Ago2 trafficking is found to be essential. Mitochondrial tethering of endoplasmic reticulum and its interaction with endosomes controls Ago2 availability. In cells with depolarized mitochondria, miRNA biogenesis gets impaired, which results in lowering of de novo–formed mature miRNA levels and accumulation of miRNA-free Ago2 on endosomes that fails to interact with Dicer1 and to traffic back to endoplasmic reticulum for de novo miRNA loading. Thus, mitochondria by sensing the cellular context regulates Ago2 trafficking at the subcellular level, which acts as a rate-limiting step in miRNA biogenesis process in mammalian cells

Cytosolic redox components regulate protein homeostasis via additional localisation in the mitochondrial intermembrane space

Oxidative protein folding is confined to the bacterial periplasm, endoplasmic reticulum and the mitochondrial intermembrane space. Maintaining a redox balance requires the presence of reductive pathways. The major thiol-reducing pathways engage the thioredoxin and the glutaredoxin systems which are involved in removal of oxidants, protein proofreading and folding. Alterations in redox balance likely affect the flux of these redox pathways and are related to ageing and diseases such as neurodegenerative disorders and cancer. Here, we first review the well-studied oxidative and reductive processes in the bacterial periplasm and the endoplasmic reticulum, and then discuss the less understood process in the mitochondrial intermembrane space, highlighting its importance for the proper function of the cell

Uterine Endoplasmic Reticulum Stress and Its Unfolded Protein Response May Regulate Caspase 3 Activation in the Pregnant Mouse Uterus

We have previously proposed that uterine caspase-3 may modulate uterine contractility in a gestationally regulated fashion. The objective of this study was to determine the mechanism by which uterine caspase-3 is activated and consequently controlled in the pregnant uterus across gestation. Utilizing the mouse uterus as our gestational model we examined the intrinsic and extrinsic apoptotic signaling pathways and the endoplasmic reticulum stress response as potential activators of uterine caspase-3 at the transcriptional and translational level. Our study revealed robust activation of the uterine myocyte endoplasmic reticulum stress response and its adaptive unfolded protein response during pregnancy coinciding respectively with increased uterine caspase-3 activity and its withdrawal to term. In contrast the intrinsic and extrinsic apoptotic signaling pathways remained inactive across gestation. We speculate that physiological stimuli experienced by the pregnant uterus likely potentiates the uterine myocyte endoplasmic reticulum stress response resulting in elevated caspase-3 activation, which is isolated to the pregnant mouse myometrium. However as term approaches, activation of an elevated adaptive unfolded protein response acts to limit the endoplasmic reticulum stress response inhibiting caspase-3 resulting in its decline towards term. We speculate that these events have the capacity to regulate gestational length in a caspase-3 dependent manne

Regulation of podocyte survival and endoplasmic reticulum stress by fatty acids and its modification by Stearoyl-CoA desaturases and cyclic AMP

Podocyte apoptosis is a hallmark in the development and progression of diabetic nephropathy (DN). Several factors of the diabetic milieu are known to induce podocyte apoptosis. Currently, the role of free fatty acids (FFAs) for podocytopathy and podocyte cell death is unknown, although FFAs are considered to be crucially involved in the development of diabetes mellitus type II. It is well known that FFAs are toxic to several cell types including pancreatic § cells and they may contribute to the development of insulin resistance. The aims of this study were to elucidate the role of the saturated palmitic acid and the monounsaturated palmitoleic and oleic acid on podocyte cell death and endoplasmic reticulum (ER)-stress, to investigate more specifically the impact of ER-stress on podocyte survival as well as to elaborate strategies to protect podocytes from lipotoxicity. The present study uncovered that palmitic acid induces podocyte apoptosis and necrosis and leads to ER-stress as reflected by induction of the unfolded protein response (UPR), i.e. upregulation of the ER chaperone immunoglobulin heavy chain binding protein (BiP), X-box protein 1 (XBP-1) mRNA splicing, and a strong upregulation of the proapoptotic transcription factor C/EBP homologous protein (CHOP). Gene silencing experiments of CHOP support a crucial involvment of CHOP and ER-stress in mediating the proapoptotic effect of palmitic acid in podocytes. Contrariwise, monounsaturated FFAs (MUFAs) such as palmitoleic and oleic acid prevent palmitic acid-induced podocyte death and attenuate ER-stress. This study further revealed that the liver X receptor (LXR) agonist TO901317 (TO) ameliorates survival of palmitic acid-treated podocytes. Mechanistically, this beneficial effect can be explained mainly by the induction of stearoyl-CoA desaturase (SCD-) 1 and 2 as shown by gene silencing experiments and further supported from overexpression studies of SCD-1. Moreover, palmitic acid tracing experiments revealed a higher incorporation of palmitic acid into the triglyceride (TG) fraction in podocytes treated with TO or oleic acid, which is at least compatible with a benefit of increased fatty acid storage, by TO, i.e. SCDs, and MUFAs, respectively. In addition, this study provides some preliminary data that adenylate cyclases (AC) may be an interesting target to protect podocytes from ER-stress in general and in particular from palmitic acid-induced podocytopathy and cell death. Experiments with forskolin, a specific AC agonist, and cyclic AMP (cAMP) analogons protect from palmitic acid-induced podocyte lipotoxicity. The effect cannot be explained by an involvment of PKA-CREB signaling as overexpression of a dominant negative CREB mutant could not abrogate the protective effect of forskolin. Furthermore, the beneficial impact of forskolin is not influencing the intrinisic (mitochondrial) apoptotic pathway. However, in addition to the protection from palmitic acid-induced cell death, forskolin is suppressing podocyte death caused by other independent ER-stressors such as tunicamycin and thapsigargin. These findings suggest a direct role of forskolin and increased cAMP levels for a protection from ER-stress in podocytes. In summary, this study unveiled antagonistic effects of palmitic acid versus monounsaturated FFAs for podocyte survival, ER-stress and the UPR. They support an important role of CHOP in the regulation of podocyte death by FFAs. Similarly to exogenous MUFAs, induction of SCDs partially protects podocytes from palmitic acid-induced ER-stress and podocyte death. The protective effect of MUFAs may be related to increased incorporation of palmitic acid into TGs. Additional, preliminary data indicate that AC agonists such as forskolin may be interesting compounds to protect podocytes from ER-stress and from the toxic effects of FFAs. The results of this study offer a rationale for interventional studies aimed at testing whether dietary shifting of the FFA balance toward MUFAs, or tissue- (podocyte-) specific stimulation or overexpression of SCDs can delay the progression of DN. Similarly, the results of this study should encourage more studies to evaluate the therapeutic potential of AC agonists or phosphodiesterase inhibitors for the prevention and treatment of DN

Regulated Inositol‐Requiring Protein 1‐Dependent Decay as a Mechanism of Corin RNA and Protein Deficiency in Advanced Human Systolic Heart Failure

BACKGROUND: The compensatory actions of the endogenous natriuretic peptide system require adequate processing of natriuretic peptide pro‐hormones into biologically active, carboxyl‐terminal fragments. Natriuretic peptide pro‐peptide processing is accomplished by corin, a transmembrane serine protease expressed by cardiomyocytes. Brain natriuretic peptide (BNP) processing is inadequate in advanced heart failure and is independently associated with adverse outcomes; however, the molecular mechanisms causing impaired BNP processing are not understood. We hypothesized that the development of endoplasmic reticulum stress in cardiomyocytes in advanced heart failure triggers inositol‐requiring protein 1 (IRE1)‐dependent corin mRNA decay, which would favor a molecular substrate favoring impaired natriuretic peptide pro‐peptide processing. METHODS AND RESULTS: Two independent samples of hearts obtained from patients with advanced heart failure at transplant demonstrated that corin RNA was reduced as Atrial natriuretic peptide (ANP)/BNP RNA increased. Increases in spliced X‐box protein 1, a marker for IRE1‐endoribonuclease activity, were associated with decreased corin RNA. Moreover, ≈50% of the hearts demonstrated significant reductions in corin RNA and protein as compared to the nonfailing control sample. In vitro experiments demonstrated that induction of endoplasmic reticulum stress in cultured cardiomyocytes with thapsigargin activated IRE1s endoribonuclease activity and time‐dependent reductions in corin mRNA. In HL‐1 cells, overexpression of IRE1 activated IRE1 endoribonuclease activity and caused corin mRNA decay, whereas IRE1‐RNA interference with shRNA attenuated corin mRNA decay after induction of endoplasmic reticulum stress with thapsigargin. Pre‐treatment of cells with Actinomycin D to inhibit transcription did not alter the magnitude or time course of thapsigargin‐induced corin mRNA decline, supporting the hypothesis that this was the result of IRE1‐mediated corin mRNA degradation. CONCLUSIONS: These data support the hypothesis that endoplasmic reticulum stress‐mediated, IRE1‐dependent targeted corin mRNA decay is a mechanism leading to corin mRNA resulting in corresponding corin protein deficiency may contribute to the pathophysiology of impaired natriuretic peptide pro‐hormone processing in humans processing in humans with advanced systolic heart failure

IRE1 phosphatase PP2Ce regulates adaptive ER stress response in the postpartum mammary gland.

We recently reported that the PPM1l gene encodes an endoplasmic reticulum (ER) membrane targeted protein phosphatase (named PP2Ce) with highly specific activity towards Inositol-requiring protein-1 (IRE1) and regulates the functional outcome of ER stress. In the present report, we found that the PP2Ce protein is highly expressed in lactating epithelium of the mammary gland. Loss of PP2Ce in vivo impairs physiological unfolded protein response (UPR) and induces stress kinase activation, resulting in loss of milk production and induction of epithelial apoptosis in the lactating mammary gland. This study provides the first in vivo evidence that PP2Ce is an essential regulator of normal lactation, possibly involving IRE1 signaling and ER stress regulation in mammary epithelium

Targeting of the prion protein to the cytosol: mechanisms and consequences

Prion diseases are characterized by the conformational transition of the cellular prion protein (PrPC) into an aberrant protein conformer, designated scrapie-prion protein (PrPSc). A causal link between protein misfolding and neurodegeneration has been established for a variety of neurodegenerative disease, such as Alzheimer's disease, Parkinson's disease and polyglutamine diseases, but there is an ongoing debate about the nature of the neurotoxic species and how non-native conformers can damage neuronal populations. PrP is normally imported into the endoplasmic reticulum (ER) and targeted to the outer leaflet of the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. However, several conditions, such as ER stress or some pathogenic mutations in the PrP gene, can induce the mislocalization of PrP in the cytosol, where it has a neurotoxic potential as demonstrated in cell culture and transgenic mouse models. In this review we focus on intrinsic factors and cellular pathways implicated in the import of PrP into the ER and its mistargeting to the cytosol. The findings summarized here not only reveal a complex regulation of the biogenesis of PrP, but also provide interesting new insight into toxic activities of pathogenic protein conformers and quality control pathways of ER-targeted proteins

Cytosolic chaperones influence the fate of a toxin dislocated from the endoplasmic reticulum

The plant cytotoxin ricin enters target mammalian cells by receptor-mediated endocytosis and undergoes retrograde transport to the endoplasmic reticulum (ER). Here, its catalytic A chain (RTA) is reductively separated from the cell-binding B chain, and free RTA enters the cytosol where it inactivates ribosomes. Cytosolic entry requires unfolding of RTA and dislocation across the ER membrane such that it arrives in the cytosol in a vulnerable, nonnative conformation. Clearly, for such a dislocated toxin to become active, it must avoid degradation and fold to a catalytic conformation. Here, we show that, in vitro, Hsc70 prevents aggregation of heat-treated RTA, and that RTA catalytic activity is recovered after chaperone treatment. A combination of pharmacological inhibition and cochaperone expression reveals that, in vivo, cytosolic RTA is scrutinized sequentially by the Hsc70 and Hsp90 cytosolic chaperone machineries, and that its eventual fate is determined by the balance of activities of cochaperones that regulate Hsc70 and Hsp90 functions. Cytotoxic activity follows Hsc70-mediated escape of RTA from an otherwise destructive pathway facilitated by Hsp90. We demonstrate a role for cytosolic chaperones, proteins typically associated with folding nascent proteins, assembling multimolecular protein complexes and degrading cytosolic and stalled, cotranslocational clients, in a toxin triage, in which both toxin folding and degradation are initiated from chaperone-bound states