Damián Gatica

Endoplasmic reticulum and the unfolded protein response: dynamics and metabolic integration.

The endoplasmic reticulum (ER) is a dynamic intracellular organelle with multiple functions essential for cellular homeostasis, development, and stress responsiveness. In response to cellular stress, a well-established signaling cascade, the unfolded protein response (UPR), is activated. This intricate mechanism is an important means of re-establishing cellular homeostasis and alleviating the inciting stress. Now, emerging evidence has demonstrated that the UPR influences cellular metabolism through diverse mechanisms, including calcium and lipid transfer, raising the prospect of involvement of these processes in the pathogenesis of disease, including neurodegeneration, cancer, diabetes mellitus and cardiovascular disease. Here, we review the distinct functions of the ER and UPR from a metabolic point of view, highlighting their association with prevalent pathologies.

Endoplasmic reticulum: ER stress regulates mitochondrial bioenergetics

Endoplasmic reticulum (ER) stress activates an adaptive unfolded protein response (UPR) that facilitates cellular repair, however, under prolonged ER stress, the UPR can ultimately trigger apoptosis thereby terminating damaged cells. The molecular mechanisms responsible for execution of the cell death program are relatively well characterized, but the metabolic events taking place during the adaptive phase of ER stress remain largely undefined. Here we discuss emerging evidence regarding the metabolic changes that occur during the onset of ER stress and how ER influences mitochondrial function through mechanisms involving calcium transfer, thereby facilitating cellular adaptation. Finally, we highlight how dysregulation of ER-mitochondrial calcium homeostasis during prolonged ER stress is emerging as a novel mechanism implicated in the onset of metabolic disorders.

Dexamethasone-induced autophagy mediates muscle atrophy through mitochondrial clearance

Glucocorticoids, such as dexamethasone, enhance protein breakdown via ubiquitin–proteasome system. However, the role of autophagy in organelle and protein turnover in the glucocorticoid-dependent atrophy program remains unknown. Here, we show that dexamethasone stimulates an early activation of autophagy in L6 myotubes depending on protein kinase, AMPK, and glucocorticoid receptor activity. Dexamethasone increases expression of several autophagy genes, including ATG5, LC3, BECN1, and SQSTM1 and triggers AMPK-dependent mitochondrial fragmentation associated with increased DNM1L protein levels. This process is required for mitophagy induced by dexamethasone. Inhibition of mitochondrial fragmentation by Mdivi-1 results in disrupted dexamethasone-induced autophagy/mitophagy. Furthermore, Mdivi-1 increases the expression of genes associated with the atrophy program, suggesting that mitophagy may serve as part of the quality control process in dexamethasone-treated L6 myotubes. Collectively, these data suggest a novel role for dexamethasone-induced autophagy/mitophagy in the regulation of the muscle atrophy program.

Cardiomyocyte ryanodine receptor degradation by chaperone-mediated autophagy

AIMS Chaperone-mediated autophagy (CMA) is a selective mechanism for the degradation of soluble cytosolic proteins bearing the sequence KFERQ. These proteins are targeted by chaperones and delivered to lysosomes where they are translocated into the lysosomal lumen and degraded via the lysosome-associated membrane protein type 2A (LAMP-2A). Mutations in LAMP2 that inhibit autophagy result in Danon disease characterized by hypertrophic cardiomyopathy. The ryanodine receptor type 2 (RyR2) plays a key role in cardiomyocyte excitation-contraction and its dysfunction can lead to cardiac failure. Whether RyR2 is degraded by CMA is unknown. METHODS AND RESULTS To induce CMA, cultured neonatal rat cardiomyocytes were treated with geldanamycin (GA) to promote protein degradation through this pathway. GA increased LAMP-2A levels together with its redistribution and colocalization with Hsc70 in the perinuclear region, changes indicative of CMA activation. The inhibition of lysosomes but not proteasomes prevented the loss of RyR2. The recovery of RyR2 content after incubation with GA by siRNA targeting LAMP-2A suggests that RyR2 is degraded via CMA. In silico analysis also revealed that the RyR2 sequence harbours six KFERQ motifs which are required for the recognition Hsc70 and its degradation via CMA. Our data suggest that presenilins are involved in RyR2 degradation by CMA. CONCLUSION These findings are consistent with a model in which oxidative damage of the RyR2 targets it for turnover by presenilins and CMA, which could lead to removal of damaged or leaky RyR2 channels.

Cargo recognition and degradation by selective autophagy

Macroautophagy, initially described as a non-selective nutrient recycling process, is essential for the removal of multiple cellular components. In the past three decades, selective autophagy has been characterized as a highly regulated and specific degradation pathway for removal of unwanted cytosolic components and damaged and/or superfluous organelles. Here, we discuss different types of selective autophagy, emphasizing the role of ligand receptors and scaffold proteins in providing cargo specificity, and highlight unanswered questions in the field.In this Review Article, Klionsky and co-authors discuss selective autophagy pathways that degrade unwanted cytosolic components and organelles, and how these pathways require ligand receptors and scaffold proteins for cargo specificity.

Herp depletion protects from protein aggregation by up-regulating autophagy

Herp is an endoplasmic reticulum (ER) stress inducible protein that participates in the ER-associated protein degradation (ERAD) pathway. However, the contribution of Herp to other protein degradation pathways like autophagy and its connection to other types of stress responses remain unknown. Here we report that Herp regulates autophagy to clear poly-ubiquitin (poly-Ub) protein aggregates. Proteasome inhibition and glucose starvation (GS) led to a high level of poly-Ub protein aggregation that was drastically reduced by stably knocking down Herp (shHerp cells). The enhanced removal of poly-Ub inclusions protected cells from death caused by glucose starvation. Under basal conditions and increasingly after stress, higher LC3-II levels and GFP-LC3 puncta were observed in shHerp cells compared to control cells. Herp knockout cells displayed basal up-regulation of two essential autophagy regulators-Atg5 and Beclin-1, leading to increased autophagic flux. Beclin-1 up-regulation was due to a reduction in Hrd1 dependent proteasomal degradation, and not at transcriptional level. The consequent higher autophagic flux was necessary for the clearance of aggregates and for cell survival. We conclude that Herp operates as a relevant factor in the defense against glucose starvation by modulating autophagy levels. These data may have important implications due to the known up-regulation of Herp in pathological states such as brain and heart ischemia, both conditions associated to acute nutritional stress.

HERPUD1 protects against oxidative stress-induced apoptosis through downregulation of the inositol 1,4,5-trisphosphate receptor.

Homocysteine-inducible, endoplasmic reticulum (ER) stress-inducible, ubiquitin-like domain member 1 (HERPUD1), an ER resident protein, is upregulated in response to ER stress and Ca(2+) homeostasis deregulation. HERPUD1 exerts cytoprotective effects in various models, but its role during oxidative insult remains unknown. The aim of this study was to investigate whether HERPUD1 contributes to cytoprotection in response to redox stress and participates in mediating stress-dependent signaling pathways. Our data showed that HERPUD1 protein levels increased in HeLa cells treated for 30 min with H2O2 or angiotensin II and in aortic tissue isolated from mice treated with angiotensin II for 3 weeks. Cell death was higher in HERPUD1 knockdown (sh-HERPUD1) HeLa cells treated with H2O2 in comparison with control (sh-Luc) HeLa cells. This effect was abolished by the intracellular Ca(2+) chelating agent BAPTA-AM or the inositol 1,4,5-trisphosphate receptor (ITPR) antagonist xestospongin B, suggesting that the response to H2O2 was dependent on intracellular Ca(2+) stores and the ITPR. Ca(2+) kinetics showed that sh-HERPUD1 HeLa cells exhibited greater and more sustained cytosolic and mitochondrial Ca(2+) increases than sh-Luc HeLa cells. This higher sensitivity of sh-HERPUD1 HeLa cells to H2O2 was prevented with the mitochondrial permeability transition pore inhibitor cyclosporine A. We concluded that the HERPUD1-mediated cytoprotective effect against oxidative stress depends on the ITPR and Ca(2+) transfer from the ER to mitochondria.

New insights into MTORC1 amino acid sensing and activation

The mechanistic target of rapamycin complex 1 (MTORC1) is an evolutionarily conserved eukaryotic Ser/Thr kinase that works as a master transducer of multiple cellular inputs involving growth factor sensing, and amino acid and ATP availability. Nutrient-rich conditions promote MTORC1 activation, which in turn leads to the stimulation of various anabolic processes such as protein and lipid synthesis, and cellular growth. Similarly, once activated MTORC1 also inhibits certain catabolic pathways such as macroautophagy/autophagy. In this regard, it is no surprise that a common feature in many cancer types is the deregulation of MTORC1 signaling (1). The close relationship between MTORC1 and cancer highlights the importance for a better understanding of the regulatory pathways by which MTORC1 is activated.

TOS-sing aside the glycolytic role of HK2/hexokinase-II to activate autophagy

Hexokinase is the first enzyme in the glycolytic pathway catalyzing the reaction in which glucose is phosphorylated into glucose-6-phosphate. Mammals possess 4 isoforms of hexokinase; HK2 (hexokinase 2) is the predominant form in insulin-sensitive tissues such as adipocytes, as well as skeletal and cardiac muscle. In addition to its function in glucose metabolism, HK2 is associated with cardiomyocyte protection against mitochondrial-mediated apoptotic cell death; whether or not HK2 played a role in cardioprotective autophagy was yet to be discovered. However, in a recent study highlighted by a punctum in this issue of Autophagy, Roberts et al. addressed this possibility, uncovering a direct link between HK2, TORC1, and autophagy regulation.