Proinsulin folding and trafficking defects trigger a common pathological disturbance of endoplasmic reticulum homeostasis.

Primary defects in folding of mutant proinsulin can cause dominant-negative proinsulin accumulation in the endoplasmic reticulum (ER), impaired anterograde proinsulin trafficking, perturbed ER homeostasis, diminished insulin production, and ß-cell dysfunction. Conversely, if primary impairment of ER-to-Golgi trafficking (which also perturbs ER homeostasis) drives misfolding of nonmutant proinsulin-this might suggest bi-directional entry into a common pathological phenotype (proinsulin misfolding, perturbed ER homeostasis, and deficient ER export of proinsulin) that can culminate in diminished insulin storage and diabetes. Here, we've challenged ß-cells with conditions that impair ER-to-Golgi trafficking, and devised an accurate means to assess the relative abundance of distinct folded/misfolded forms of proinsulin using a novel nonreducing SDS-PAGE/immunoblotting protocol. We confirm abundant proinsulin misfolding upon introduction of a diabetogenic INS mutation, or in the islets of db/db mice. Whereas blockade of proinsulin trafficking in Golgi/post-Golgi compartments results in intracellular accumulation of properly-folded proinsulin (bearing native disulfide bonds), impairment of ER-to-Golgi trafficking (regardless whether such impairment is achieved by genetic or pharmacologic means) results in decreased native proinsulin with more misfolded proinsulin. Remarkably, reversible ER-to-Golgi transport defects (such as treatment with brefeldin A or cellular energy depletion) upon reversal quickly restore the ER folding environment, resulting in the disappearance of pre-existing misfolded proinsulin while preserving proinsulin bearing native disulfide bonds. Thus, proper homeostatic balance of ER-to-Golgi trafficking is linked to a more favorable proinsulin folding (as well as trafficking) outcome.

Conditional hepatocyte ablation of PDIA1 uncovers indispensable roles in both APOB and MTTP folding to support VLDL secretion.

OBJECTIVES: The assembly and secretion of hepatic very low-density lipoprotein (VLDL) plays pivotal roles in hepatic and plasma lipid homeostasis. Protein disulfide isomerase A1 (PDIA1/P4HB) is a molecular chaperone whose functions are essential for protein folding in the endoplasmic reticulum. Here we investigated the physiological requirement in vivo for PDIA1 in maintaining VLDL assembly and secretion. METHODS: Pdia1/P4hb was conditionally deleted in adult mouse hepatocytes and the phenotypes characterized. Mechanistic analyses in primary hepatocytes determined how PDIA1 ablation alters MTTP synthesis and degradation as well as altering synthesis and secretion of Apolipoprotein B (APOB), along with complementary expression of intact PDIA1 vs a catalytically inactivated PDIA1 mutant. RESULTS: Hepatocyte-specific deletion of Pdia1/P4hb inhibited hepatic MTTP expression and dramatically reduced VLDL production, leading to severe hepatic steatosis and hypolipidemia. Pdia1-deletion did not affect mRNA expression or protein stability of MTTP but rather prevented Mttp mRNA translation. We demonstrate an essential role for PDIA1 in MTTP synthesis and function and show that PDIA1 interacts with APOB in an MTTP-independent manner via its molecular chaperone function to support APOB folding and secretion. CONCLUSIONS: PDIA1 plays indispensable roles in APOB folding, MTTP synthesis and activity to support VLDL assembly. Thus, like APOB and MTTP, PDIA1 is an obligatory component of hepatic VLDL production.

Evaluating the performance of an integrated evidence-based nursing knowledge management (I-EBNKM) platform in real-world clinical environments.

BACKGROUND: For evidence-based knowledge to be applicable in clinical practice, providing support for and the management of knowledge is required to ensure the effective sharing of appropriate expertise across healthcare organizations. Knowledge management platforms can provide a wide range of benefits related to the activation and establishment of evidence-based practice (EBP) in clinical environments. OBJECTIVES: In this study, we developed an integrated evidence-based nursing knowledge management (I-EBNKM) platform and applied it in real-world clinical environments to evaluate its effectiveness. METHODS: We designed an I-EBNKM platform with three main functions: (1) clinical questioning and knowledge linkage, (2) systematic knowledge management, and (3) knowledge communication. After a two-month long application of the I-EBNKM platform in real-world clinical environments, we evaluated the changes in the levels of knowledge in EBP, attitude, practice, confidence in clinical questioning, individual innovative behavior, innovative organizational culture, and organizational knowledge management. The experimental and control groups consisted of 198 nurses, who participated in the study. RESULTS: After applying the I-EBNKM platform, the levels of EBP knowledge and skills (t = 7.16; p <.001), attitude (t = 6.30; p <.001), practice (t = 7.63; p <.001), confidence in clinical questioning (t = 4.57; p <.001), individual innovative behavior (t = 8.72; p <.001), and organizational knowledge management (t = 7.43; p <.001) differed significantly between the experimental group and the control group. CONCLUSION: The results of this study clearly indicate that the I-EBNKM platform we developed has the potential to enhance nurses' involvement in ensuring effective knowledge management in real-world clinical environments. Therefore, the provision of an innovative digital approach ensuring systematic and timely organizational support among nurses is of critical importance.

Listening to patients' voices: Applying the design-thinking method for teaching person-centered care to nursing students.

BACKGROUND: Providing a person-centered care (PCC) education program to nursing students is necessary. This study aims to determine the impact of a design-thinking based education program on how nursing students perceive PCC. METHODS: Five 2-h lessons were offered to 105 fourth-year nursing students in South Korea. Each randomly assigned group of eight or nine students was instructed to develop a plan to address the problems/dissatisfaction experienced by patients during hospitalization. The Individualized Care Scale-nurse's version was used to measure student's perception of PCC before and after the education program. RESULTS: After the program the students exhibited significant improvements in how they viewed supporting patient individuality, with that score increasing by 0.44 (from 3.64 to 4.08; p < 0.0001), and maintaining patient individuality while providing care, with that score increasing by 0.34 (from 3.71 to 4.05; p < 0.0001). Among subdomains, the most notable change was in how the students viewed the personal life situation of patients, and its impact on patients' healthcare outcomes. CONCLUSION: This education program, based on the design-thinking approach, was effective in improving the perceptions of nursing students about PCC. Expanding such PCC education programs for nursing school students should therefore be considered.

"Walking in the patient's shoes": An innovative training method using storytelling to promote knowledge transfer of patient-centered care in hospital: A quasi-experimental study.

OBJECTIVE: To evaluate an onsite patient-centered care (PCC) training program for nurses using a digital patient-storytelling approach. BACKGROUND: PCC is a dominant model for improving the quality of care. Effective strategies for providing PCC training to nurses can yield numerous benefits. DESIGN: A pretest-posttest design was used with a nonrandomized control group METHODS: PCC training program involved participants playing a patient role to experience their hospital journey. Nurses' perception of PCC, compassion and knowledge transfer were measured before (pretest) and after (posttest) PCC training (experimental group). Controls received PCC training only after pretest and posttest evaluations. RESULTS: Changes in PCC perception and compassion were significantly greater in the experimental group (n = 39) than in controls (n = 49; p = .001 and .006, respectively). PCC knowledge transfer was significantly correlated with PCC perception (r = 0.55) and compassion (r = 0.63). CONCLUSIONS: Through the PCC training program, the perceived improvements of the nurses' views on supporting patient individuality and compassion while providing care were revealed. This program is also potential for promoting PCC knowledge transfer into the daily activities of nurses. Therefore, such PCC training programs could be a good beginning in developing a patient-centered culture in healthcare systems.

Chop/Ddit3 depletion in ß cells alleviates ER stress and corrects hepatic steatosis in mice.

Type 2 diabetes (T2D) is a metabolic disorder characterized by hyperglycemia, hyperinsulinemia, and insulin resistance (IR). During the early phase of T2D, insulin synthesis and secretion by pancreatic ß cells is enhanced, which can lead to proinsulin misfolding that aggravates endoplasmic reticulum (ER) protein homeostasis in ß cells. Moreover, increased circulating insulin may contribute to fatty liver disease. Medical interventions aimed at alleviating ER stress in ß cells while maintaining optimal insulin secretion are therefore an attractive therapeutic strategy for T2D. Previously, we demonstrated that germline Chop gene deletion preserved ß cells in high-fat diet (HFD)-fed mice and in leptin receptor-deficient db/db mice. In the current study, we further investigated whether targeting Chop/Ddit3 specifically in murine ß cells conferred therapeutic benefits. First, we showed that Chop deletion in ß cells alleviated ß cell ER stress and delayed glucose-stimulated insulin secretion (GSIS) in HFD-fed mice. Second, ß cell-specific Chop deletion prevented liver steatosis and hepatomegaly in aged HFD-fed mice without affecting basal glucose homeostasis. Third, we provide mechanistic evidence that Chop depletion reduces ER Ca2+ buffering capacity and modulates glucose-induced islet Ca2+ oscillations, leading to transcriptional changes of ER chaperone profile ("ER remodeling"). Last, we demonstrated that a GLP1-conjugated Chop antisense oligonucleotide strategy recapitulated the reduction in liver triglycerides and pancreatic insulin content. In summary, our results demonstrate that Chop depletion in ß cells provides a therapeutic strategy to alleviate dysregulated insulin secretion and consequent fatty liver disease in T2D.

Defects in Protein Folding and/or Quality Control Cause Protein Aggregation in the Endoplasmic Reticulum.

Protein aggregation is now a common hallmark of numerous human diseases, most of which involve cytosolic aggregates including Aß (AD) and ⍺-synuclein (PD) in Alzheimer's disease and Parkinson's disease. However, it is also evident that protein aggregation can also occur in the lumen of the endoplasmic reticulum (ER) that leads to specific diseases due to loss of protein function or detrimental effects on the host cell, the former is inherited in a recessive manner where the latter are dominantly inherited. However, the mechanisms of protein aggregation, disaggregation and degradation in the ER are not well understood. Here we provide an overview of factors that cause protein aggregation in the ER and how the ER handles aggregated proteins. Protein aggregation in the ER can result from intrinsic properties of the protein (hydrophobic residues in the ER), oxidative stress or nutrient depletion. The ER has quality control mechanisms [chaperone functions, ER-associated protein degradation (ERAD) and autophagy] to ensure only correctly folded proteins exit the ER and enter the cis-Golgi compartment. Perturbation of protein folding in the ER activates the unfolded protein response (UPR) that evolved to increase ER protein folding capacity and efficiency and degrade misfolded proteins. Accumulation of misfolded proteins in the ER to a level that exceeds the ER-chaperone folding capacity is a major factor that exacerbates protein aggregation. The most significant ER resident protein that prevents protein aggregation in the ER is the heat shock protein 70 (HSP70) homologue, BiP/GRP78, which is a peptide-dependent ATPase that binds unfolded/misfolded proteins and releases them upon ATP binding. Since exogenous factors can also reduce protein misfolding and aggregation in the ER, such as chemical chaperones and antioxidants, these treatments have potential therapeutic benefit for ER protein aggregation-associated diseases.

Unbiased Profiling of the Human Proinsulin Biosynthetic Interaction Network Reveals a Role for Peroxiredoxin 4 in Proinsulin Folding.

The ß-cell protein synthetic machinery is dedicated to the production of mature insulin, which requires the proper folding and trafficking of its precursor, proinsulin. The complete network of proteins that mediate proinsulin folding and advancement through the secretory pathway, however, remains poorly defined. Here we used affinity purification and mass spectrometry to identify, for the first time, the proinsulin biosynthetic interaction network in human islets. Stringent analysis established a central node of proinsulin interactions with endoplasmic reticulum (ER) folding factors, including chaperones and oxidoreductases, that is remarkably conserved in both sexes and across three ethnicities. The ER-localized peroxiredoxin PRDX4 was identified as a prominent proinsulin-interacting protein. In ß-cells, gene silencing of PRDX4 rendered proinsulin susceptible to misfolding, particularly in response to oxidative stress, while exogenous PRDX4 improved proinsulin folding. Moreover, proinsulin misfolding induced by oxidative stress or high glucose was accompanied by sulfonylation of PRDX4, a modification known to inactivate peroxiredoxins. Notably, islets from patients with type 2 diabetes (T2D) exhibited significantly higher levels of sulfonylated PRDX4 than islets from healthy individuals. In conclusion, we have generated the first reference map of the human proinsulin interactome to identify critical factors controlling insulin biosynthesis, ß-cell function, and T2D.

PDIA1/P4HB is required for efficient proinsulin maturation and ß cell health in response to diet induced obesity.

Regulated proinsulin biosynthesis, disulfide bond formation and ER redox homeostasis are essential to prevent Type two diabetes. In ß cells, protein disulfide isomerase A1 (PDIA1/P4HB), the most abundant ER oxidoreductase of over 17 members, can interact with proinsulin to influence disulfide maturation. Here we find Pdia1 is required for optimal insulin production under metabolic stress in vivo. ß cell-specific Pdia1 deletion in young high-fat diet fed mice or aged mice exacerbated glucose intolerance with inadequate insulinemia and increased the proinsulin/insulin ratio in both serum and islets compared to wildtype mice. Ultrastructural abnormalities in Pdia1-null ß cells include diminished insulin granule content, ER vesiculation and distention, mitochondrial swelling and nuclear condensation. Furthermore, Pdia1 deletion increased accumulation of disulfide-linked high molecular weight proinsulin complexes and islet vulnerability to oxidative stress. These findings demonstrate that PDIA1 contributes to oxidative maturation of proinsulin in the ER to support insulin production and ß cell health.

Lipotoxicity induces hepatic protein inclusions through TANK binding kinase 1-mediated p62/sequestosome 1 phosphorylation.

Obesity commonly leads to hepatic steatosis, which often provokes lipotoxic injuries to hepatocytes that cause nonalcoholic steatohepatitis (NASH). NASH, in turn, is associated with the accumulation of insoluble protein aggregates that are composed of ubiquitinated proteins and ubiquitin adaptor p62/sequestosome 1 (SQSTM1). Formation of p62 inclusions in hepatocytes is the critical marker that distinguishes simple fatty liver from NASH and predicts a poor prognostic outcome for subsequent liver carcinogenesis. However, the molecular mechanism by which lipotoxicity induces protein aggregation is currently unknown. Here, we show that, upon saturated fatty acid-induced lipotoxicity, TANK binding kinase 1 (TBK1) is activated and phosphorylates p62. TBK1-mediated p62 phosphorylation is important for lipotoxicity-induced aggregation of ubiquitinated proteins and formation of large protein inclusions in hepatocytes. In addition, cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING), upstream regulators of TBK1, are involved in lipotoxic activation of TBK1 and subsequent p62 phosphorylation in hepatocytes. Furthermore, TBK1 inhibition prevented formation of ubiquitin-p62 aggregates not only in cultured hepatocytes, but also in mouse models of obesity and NASH. CONCLUSION: These results suggest that lipotoxic activation of TBK1 and subsequent p62 phosphorylation are critical steps in the NASH pathology of protein inclusion accumulation in hepatocytes. This mechanism can provide an explanation for how hypernutrition and obesity promote the development of severe liver pathologies, such as steatohepatitis and liver cancer, by facilitating the formation of p62 inclusions. (Hepatology 2018).

TRIM31 promotes Atg5/Atg7-independent autophagy in intestinal cells.

Autophagy is responsible for the bulk degradation of cytosolic constituents and plays an essential role in the intestinal epithelium by controlling beneficial host-bacterial relationships. Atg5 and Atg7 are thought to be critical for autophagy. However, Atg5- or Atg7-deficient cells still form autophagosomes and autolysosomes, and are capable of removing proteins or bacteria. Here, we report that human TRIM31 (tripartite motif), an intestine-specific protein localized in mitochondria, is essential for promoting lipopolysaccharide-induced Atg5/Atg7-independent autophagy. TRIM31 directly interacts with phosphatidylethanolamine in a palmitoylation-dependent manner, leading to induction of autolysosome formation. Depletion of endogenous TRIM31 significantly increases the number of intestinal epithelial cells containing invasive bacteria. Crohn's disease patients display TRIM31 downregulation. Human cytomegalovirus-infected intestinal cells show a decrease in TRIM31 expression as well as a significant increase in bacterial load, reversible by the introduction of wild-type TRIM31. We provide insight into an alternative autophagy pathway that protects against intestinal pathogenic bacterial infection.

Tumor suppressive role of sestrin2 during colitis and colon carcinogenesis.

The mTOR complex 1 (mTORC1) and endoplasmic reticulum (ER) stress pathways are critical regulators of intestinal inflammation and colon cancer growth. Sestrins are stress-inducible proteins, which suppress both mTORC1 and ER stress; however, the role of Sestrins in colon physiology and tumorigenesis has been elusive due to the lack of studies in human tissues or in appropriate animal models. In this study, we show that human SESN2 expression is elevated in the colon of ulcerative colitis patients but is lost upon p53 inactivation during colon carcinogenesis. In mouse colon, Sestrin2 was critical for limiting ER stress and promoting the recovery of epithelial cells after inflammatory injury. During colitis-promoted tumorigenesis, Sestrin2 was shown to be an important mediator of p53's control over mTORC1 signaling and tumor cell growth. These results highlight Sestrin2 as a novel tumor suppressor, whose downregulation can accelerate both colitis and colon carcinogenesis.

O-GlcNAcylation of eIF2α regulates the phospho-eIF2α-mediated ER stress response.

O-GlcNAcylation is highly involved in cellular stress responses including the endoplasmic reticulum (ER) stress response. For example, glucosamine-induced flux through the hexosamine biosynthetic pathway can promote ER stress and ER stress inducers can change the total cellular level of O-GlcNAcylation. However, it is largely unknown which component(s) of the unfolded protein response (UPR) is directly regulated by O-GlcNAcylation. In this study, eukaryotic translation initiation factor 2α (eIF2α), a major branch of the UPR, was O-GlcNAcylated at Ser 219, Thr 239, and Thr 241. Upon ER stress, eIF2α is phosphorylated at Ser 51 by phosphorylated PKR-like ER kinase and this inhibits global translation initiation, except for that of specific mRNAs, including activating transcription factor 4, that induce stress-responsive genes such as C/EBP homologous protein (CHOP). Hyper-O-GlcNAcylation induced by O-GlcNAcase inhibitor (thiamet-G) treatment or O-GlcNAc transferase (OGT) overexpression hindered phosphorylation of eIF2α at Ser 51. The level of O-GlcNAcylation of eIF2α was changed by dithiothreitol treatment dependent on its phosphorylation at Ser 51. Point mutation of the O-GlcNAcylation sites of eIF2α increased its phosphorylation at Ser 51 and CHOP expression and resulted in increased apoptosis upon ER stress. These results suggest that O-GlcNAcylation of eIF2α affects its phosphorylation at Ser 51 and influences CHOP-mediated cell death. This O-GlcNAcylation of eIF2α was reproduced in thiamet-G-injected mouse liver. In conclusion, proper regulation of O-GlcNAcylation and phosphorylation of eIF2α is important to maintain cellular homeostasis upon ER stress.

Pharmacological correction of obesity-induced autophagy arrest using calcium channel blockers.

Autophagy deregulation during obesity contributes to the pathogenesis of diverse metabolic disorders. However, without understanding the molecular mechanism of obesity interference in autophagy, development of therapeutic strategies for correcting such defects in obese individuals is challenging. Here we show that a chronic increase of the cytosolic calcium concentration in hepatocytes during obesity and lipotoxicity attenuates autophagic flux by preventing the fusion between autophagosomes and lysosomes. As a pharmacological approach to restore cytosolic calcium homeostasis in vivo, we administered the clinically approved calcium channel blocker verapamil to obese mice. Such treatment successfully increases autophagosome-lysosome fusion in liver, preventing accumulation of protein inclusions and lipid droplets and suppressing inflammation and insulin resistance. As calcium channel blockers have been safely used in clinics for the treatment of hypertension for more than 30 years, our results suggest they may be a safe therapeutic option for restoring autophagic flux and treating metabolic pathologies in obese patients.

Hepatoprotective role of Sestrin2 against chronic ER stress.

Upon prolonged endoplasmic reticulum (ER) stress, cells attenuate protein translation to prevent accumulation of unfolded proteins. Here we show that Sestrin2 is critical for this process. Sestrin2 expression is induced by an ER stress-activated transcription factor CCAAT-enhancer-binding protein beta (c/EBPß). Once induced, Sestrin2 halts protein synthesis by inhibiting mammalian target of rapamycin complex 1 (mTORC1). As Sestrin2-deficient cells continue to translate a large amount of proteins during ER stress, they are highly susceptible to ER stress-associated cell death. Accordingly, dietary or genetically induced obesity, which does not lead to any pathological indication other than simple fat accumulation in the liver of wild-type (WT) mice, can provoke Sestrin2-deficient mice to develop severe ER stress-associated liver pathologies such as extensive liver damage, steatohepatitis and fibrosis. These pathologies are suppressed by liver-specific Sestrin2 reconstitution, mTORC1 inhibition or chemical chaperone administration. The Sestrin2-mediated unfolded protein response (UPR) may be a general protective mechanism against ER stress-associated diseases.

Sestrin2 inhibits uncoupling protein 1 expression through suppressing reactive oxygen species.

Uncoupling protein 1 (Ucp1), which is localized in the mitochondrial inner membrane of mammalian brown adipose tissue (BAT), generates heat by uncoupling oxidative phosphorylation. Upon cold exposure or nutritional abundance, sympathetic neurons stimulate BAT to express Ucp1 to induce energy dissipation and thermogenesis. Accordingly, increased Ucp1 expression reduces obesity in mice and is correlated with leanness in humans. Despite this significance, there is currently a limited understanding of how Ucp1 expression is physiologically regulated at the molecular level. Here, we describe the involvement of Sestrin2 and reactive oxygen species (ROS) in regulation of Ucp1 expression. Transgenic overexpression of Sestrin2 in adipose tissues inhibited both basal and cold-induced Ucp1 expression in interscapular BAT, culminating in decreased thermogenesis and increased fat accumulation. Endogenous Sestrin2 is also important for suppressing Ucp1 expression because BAT from Sestrin2(-/-) mice exhibited a highly elevated level of Ucp1 expression. The redox-inactive mutant of Sestrin2 was incapable of regulating Ucp1 expression, suggesting that Sestrin2 inhibits Ucp1 expression primarily through reducing ROS accumulation. Consistently, ROS-suppressing antioxidant chemicals, such as butylated hydroxyanisole and N-acetylcysteine, inhibited cold- or cAMP-induced Ucp1 expression as well. p38 MAPK, a signaling mediator required for cAMP-induced Ucp1 expression, was inhibited by either Sestrin2 overexpression or antioxidant treatments. Taken together, these results suggest that Sestrin2 and antioxidants inhibit Ucp1 expression through suppressing ROS-mediated p38 MAPK activation, implying a critical role of ROS in proper BAT metabolism.

ERADication of EDEM1 occurs by selective autophagy and requires deglycosylation by cytoplasmic peptide N-glycanase.

ER degradation-enhancing α-mannosidase-like 1 protein (EDEM1) is involved in the routing of misfolded glycoproteins for degradation in the cytoplasm. Previously, we reported that EDEM1 leaves the endoplasmic reticulum via non-COPII vesicles (Zuber et al. in Proc Natl Acad Sci USA 104:4407-4412, 2007) and becomes degraded by basal autophagy (Le Fourn et al. in Cell Mol Life Sci 66:1434-1445, 2009). However, it is unknown which type of autophagy is involved. Likewise, how EDEM1 is targeted to autophagosomes remains elusive. We now show that EDEM1 is degraded by selective autophagy. It colocalizes with the selective autophagy cargo receptors p62/SQSTM1, neighbor of BRCA1 gene 1 (NBR1) and autophagy-linked FYVE (Alfy) protein, and becomes engulfed by autophagic isolation membranes. The interaction with p62/SQSTM1 and NBR1 is required for routing of EDEM1 to autophagosomes since it can be blocked by short inhibitory RNA knockdown of the cargo receptors. Furthermore, p62/SQSTM1 interacts only with deglycosylated EDEM1 that is also ubiquitinated. The deglycosylation of EDEM1 occurs by the cytosolic peptide N-glycanase and is a prerequisite for interaction and aggregate formation with p62/SQSTM1 as demonstrated by the effect of peptide N-glycanase inhibitors on the formation of protein aggregates. Conversely, aggregation of p62/SQSTM1 and EDEM1 occurs independent of cytoplasmic histone deacetylase. These data provide novel insight into the mechanism of autophagic degradation of the ER-associated protein degradation (ERAD) component EDEM1 and disclose hitherto unknown parallels with the clearance of cytoplasmic aggregates of misfolded proteins by selective autophagy.

Genetic ablation and short-duration inhibition of lipoxygenase results in increased macroautophagy.

12/15-lipoxygenase (12/15-LOX) is involved in organelle homeostasis by degrading mitochondria in maturing red blood cells and by eliminating excess peroxisomes in liver. Furthermore, 12/15-LOX contributes to diseases by exacerbating oxidative stress-related injury, notably in stroke. Nonetheless, it is unclear what the consequences are of abolishing 12/15-LOX activity. Mice in which the alox15 gene has been ablated do not show an obvious phenotype, and LOX enzyme inhibition is not overtly detrimental. We show here that liver histology is also unremarkable. However, electron microscopy demonstrated that 12/15-LOX knockout surprisingly leads to increased macroautophagy in the liver. Not only macroautophagy but also mitophagy and pexophagy were increased in hepatocytes, which otherwise showed unaltered fine structure and organelle morphology. These findings were substantiated by immunofluorescence showing significantly increased number of LC3 puncta and by Western blotting demonstrating a significant increase for LC3-II protein in both liver and brain homogenates of 12/15-LOX knockout mice. Inhibition of 12/15-LOX activity by treatment with four structurally different inhibitors had similar effects in cultured HepG2 hepatoma cells and SH-SY5Y neuroblastoma cells with significantly increased autophagy discernable already after 2 hours. Hence, our study reveals a link between ablation or inhibition of 12/15-LOX and stimulation of macroautophagy. The enhanced macroautophagy may be related to the known tissue-protective effects of LOX ablation or inhibition under various diseased conditions caused by oxidative stress and ischemia. This could provide an important cleaning mechanism of cells and tissues to prevent accumulation of damaged mitochondria and other cellular components.

Large protein complexes retained in the ER are dislocated by non-COPII vesicles and degraded by selective autophagy.

Multisubunit protein complexes are assembled in the endoplasmic reticulum (ER). Existing pools of single subunits and assembly intermediates ensure the efficient and rapid formation of complete complexes. While being kinetically beneficial, surplus components must be eliminated to prevent potentially harmful accumulation in the ER. Surplus single chains are cleared by the ubiquitin-proteasome system. However, the fate of not secreted assembly intermediates of multisubunit proteins remains elusive. Here we show by high-resolution double-label confocal immunofluorescence and immunogold electron microscopy that naturally occurring surplus fibrinogen Aα-γ assembly intermediates in HepG2 cells are dislocated together with EDEM1 from the ER to the cytoplasm in ER-derived vesicles not corresponding to COPII-coated vesicles originating from the transitional ER. This route corresponds to the novel ER exit path we have previously identified for EDEM1 (Zuber et al. Proc Natl Acad Sci USA 104:4407-4412, 2007). In the cytoplasm, detergent-insoluble aggregates of fibrinogen Aα-γ dimers develop that are targeted by the selective autophagy cargo receptors p62/SQSTM1 and NBR1. These aggregates are degraded by selective autophagy as directly demonstrated by high-resolution microscopy as well as biochemical analysis and inhibition of autophagy by siRNA and kinase inhibitors. Our findings demonstrate that different pathways exist in parallel for ER-to-cytoplasm dislocation and subsequent proteolytic degradation of large luminal protein complexes and of surplus luminal single-chain proteins. This implies that ER-associated protein degradation (ERAD) has a broader function in ER proteostasis and is not limited to the elimination of misfolded glycoproteins.

Maintenance of metabolic homeostasis by Sestrin2 and Sestrin3.

Chronic activation of mammalian target of rapamycin complex 1 (mTORC1) and p70 S6 kinase (S6K) in response to hypernutrition contributes to obesity-associated metabolic pathologies, including hepatosteatosis and insulin resistance. Sestrins are stress-inducible proteins that activate AMP-activated protein kinase (AMPK) and suppress mTORC1-S6K activity, but their role in mammalian physiology and metabolism has not been investigated. We show that Sestrin2--encoded by the Sesn2 locus, whose expression is induced upon hypernutrition--maintains metabolic homeostasis in liver of obese mice. Sesn2 ablation exacerbates obesity-induced mTORC1-S6K activation, glucose intolerance, insulin resistance, and hepatosteatosis, all of which are reversed by AMPK activation. Furthermore, concomitant ablation of Sesn2 and Sesn3 provokes hepatic mTORC1-S6K activation and insulin resistance even in the absence of nutritional overload and obesity. These results demonstrate an important homeostatic function for the stress-inducible Sestrin protein family in the control of mammalian lipid and glucose metabolism.