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N-glycanase 1 (NGLY1) deficiency is an ultra-rare disease caused by autosomal recessive loss-of-function mutations in the NGLY1 gene. NGLY1 removes N-linked glycans from glycoproteins in the cytoplasm and is thought to help clear misfolded proteins from the endoplasmic reticulum (ER) through the ER associated degradation (ERAD) pathway. Despite this, the physiological significance of NGLY1 in ERAD is not understood. The best characterized substrate of NGLY1 is NRF1, a transcription factor that upregulates proteasome expression and the proteasome bounce-back response. We previously performed a genetic modifier screen using a Drosophila model of NGLY1 deficiency and identified potential modifiers that alter the lethality of the model. We identified two protein-coding variants in Hrd3 / SEL1L : S780P and Δ806–809 . Both variants are localized to the SEL1L cytoplasmic tail, an uncharacterized domain. SEL1L is a component of the ERAD complex that retrotranslocates misfolded proteins from the ER to the cytoplasm for degradation. We used CRISPR to generate fly lines carrying these SEL1L variants in a common genetic background and tested them with our model of NGLY1 deficiency. Validating our previous screen, the SEL1L S780P and SEL1L Δ806-809 variants increased the survival of the NGLY1 deficiency model, compared to the SEL1L S780 variant. To determine how these SEL1L variants were modifying lethality in NGLY1 deficiency, we interrogated the ERAD and NRF1 signaling pathways. We found that the SEL1L S780P and SEL1L Δ806-809 variants improve resistance to ER stress, with enhanced ERAD function as a likely contributing mechanism. This effect depends on NGLY1 activity, further implicating NGLY1 in general ERAD function. We also found that, in heterozygous NGLY1 null flies, these variants protect against some defects like increased lethality caused by proteasome inhibition. These results provide new insights into the role of SEL1L in the disease pathogenesis of NGLY1 deficiency. SEL1L is a strong candidate modifier gene in patients, where variability in presentation is common.

The endoplasmic-reticulum quality-control (ERQC) system shuttles misfolded proteins for degradation by the proteasome through the well-defined ER-associated degradation (ERAD) pathway. In contrast, very little is known about the role of autophagy in ERQC. Macro-autophagy, a collection of pathways that deliver proteins through autophagosomes (APs) for degradation in the lysosome (vacuole in yeast), is mediated by autophagy-specific proteins, Atgs, and regulated by Ypt/Rab GTPases. Until recently, the term ER-phagy was used to describe degradation of ER membrane and proteins in the lysosome under stress: either ER stress induced by drugs or whole-cell stress induced by starvation. These two types of stresses induce micro-ER-phagy, which does not use autophagic organelles and machinery, and non-selective autophagy. Here, we characterize the macro-ER-phagy pathway and uncover its role in ERQC. This pathway delivers 20-50% of certain ER-resident membrane proteins to the vacuole and is further induced to >90% by overexpression of a single integral-membrane protein. Even though such overexpression in cells defective in macro-ER-phagy induces the unfolded-protein response (UPR), UPR is not needed for macro-ER-phagy. We show that macro-ER-phagy is dependent on Atgs and Ypt GTPases and its cargo passes through APs. Moreover, for the first time the role of Atg9, the only integral-membrane core Atg, is uncoupled from that of other core Atgs. Finally, three sequential steps of this pathway are delineated: Atg9-dependent exit from the ER en route to autophagy, Ypt1- and core Atgs-mediated pre-autophagsomal-structure organization, and Ypt51-mediated delivery of APs to the vacuole.

Understanding the genetic causes of diseases that affect pancreatic β cells and neurons can give insights into pathways essential for both cell types. Microcephaly, epilepsy, and diabetes syndrome (MEDS) is a congenital disorder with two known etiological genes, IER3IP1 and YIPF5 . Both genes encode proteins involved in endoplasmic reticulum (ER) to Golgi trafficking. We used genome sequencing to identify 6 individuals with MEDS caused by biallelic variants in the potentially novel disease gene TMEM167A . All had neonatal diabetes (diagnosed at <6 months) and severe microcephaly, and 5 also had epilepsy. TMEM167A is highly expressed in developing and adult human pancreas and brain. To gain insights into the mechanisms leading to diabetes, we silenced TMEM167A in EndoC-βH1 cells and knocked-in one patient’s variant, p.Val59Glu, in induced pluripotent stem cells (iPSCs). Both TMEM167A depletion in EndoC-βH1 cells and the p.Val59Glu variant in iPSC-derived β cells sensitized β cells to ER stress. The p.Val59Glu variant impaired proinsulin trafficking to the Golgi and induced iPSC-β cell dysfunction. The discovery of TMEM167A variants as a genetic cause of MEDS highlights a critical role of TMEM167A in the ER to Golgi pathway in β cells and neurons.

The endoplasmic reticulum (ER) is a major cellular organelle for the synthesis and folding of secretory and transmembrane proteins, whose proper function underpins organellar homeostasis, proper tissue function, and organismal physiology. Protein quality control (PQC) systems at the ER include the unfolded protein response (UPR), ER-associated degradation (ERAD), and ER-phagy, which monitor ER homeostasis and contribute to protein refolding, sequestration, or degradation. ERAD prevents the accumulation of misfolded or orphan proteins that would otherwise be toxic. By controlling the degradation of these proteins, ERAD performs a core function in governing adaptation to proteotoxic stress. ERAD also regulates the abundance of folding-competent proteins as a means to fine-tune key physiological processes. Among its complex regulatory activities, ERAD controls cellular processes such as lipid homeostasis, calcium flux, and cell fate decisions, which are all required for the maintenance of organelle homeostasis. Highlighting its importance, dysregulation of ERAD often results in devastating diseases. Here, we discuss the molecular and mechanistic understanding of protein quality and quantity control by ERAD and its interface with ER-phagy, as well as other cellular stress programs. The implications of ERAD and its associated regulatory arms for cellular homeostasis, its effects on health and disease, and current therapeutic approaches are discussed.

Nrf1-mediated proteosome activity in brown fat is required for the normal thermogenic behavior of this tissue in mice. Adipocytes possess remarkable adaptive capacity to respond to nutrient excess, fasting or cold exposure, and they are thus an important cell type for the maintenance of proper metabolic health. Although the endoplasmic reticulum (ER) is a critical organelle for cellular homeostasis, the mechanisms that mediate adaptation of the ER to metabolic challenges in adipocytes are unclear. Here we show that brown adipose tissue (BAT) thermogenic function requires an adaptive increase in proteasomal activity to secure cellular protein quality control, and we identify the ER-localized transcription factor nuclear factor erythroid 2–like 1 (Nfe2l1, also known as Nrf1) as a critical driver of this process. We show that cold adaptation induces Nrf1 in BAT to increase proteasomal activity and that this is crucial for maintaining ER homeostasis and cellular integrity, specifically when the cells are in a state of high thermogenic activity. In mice, under thermogenic conditions, brown-adipocyte-specific deletion of Nfe2l1 ( Nrf1 ) resulted in ER stress, tissue inflammation, markedly diminished mitochondrial function and whitening of the BAT. In mouse models of both genetic and dietary obesity, stimulation of proteasomal activity by exogenously expressing Nrf1 or by treatment with the proteasome activator PA28α in BAT resulted in improved insulin sensitivity. In conclusion, Nrf1 emerges as a novel guardian of brown adipocyte function, providing increased proteometabolic quality control for adapting to cold or to obesity.

The unfolded protein response (UPR) is activated to sustain cell survival by reducing misfolded protein accumulation in the endoplasmic reticulum (ER). The UPR also promotes programmed cell death (PCD) when the ER stress is severe; however, the underlying molecular mechanisms are less understood, especially in plants. Previously, two membrane-associated transcriptions factors (MTFs), bZIP28 and bZIP60, were identified as the key regulators for cell survival in the plant ER stress response. Here, we report the identification of another MTF, NAC089, as an important PCD regulator in Arabidopsis (Arabidopsis thaliana) plants. NAC089 relocates from the ER membrane to the nucleus under ER stress conditions. Inducible expression of a truncated form of NAC089, in which the transmembrane domain is deleted, induces PCD with increased caspase 3/7-like activity and DNA fragmentation. Knock-down NAC089 in Arabidopsis confers ER stress tolerance and impairs ER-stress-induced caspase-like activity. Transcriptional regulation analysis and ChIP-qPCR reveal that NAC089 plays important role in regulating downstream genes involved in PCD, such as NAC094, MC5 and BAG6. Furthermore, NAC089 is up-regulated by ER stress, which is directly controlled by bZIP28 and bZIP60. These results show that nuclear relocation of NAC089 promotes ER-stress-induced PCD, and both pro-survival and pro-death signals are elicited by bZIP28 and bZIP60 during plant ER stress response.

For the past several years, fundamental research on Sigma-1R (S1R) protein has unveiled its necessity for maintaining proper cellular homeostasis through modulation of calcium and lipid exchange between the endoplasmic reticulum (ER) and mitochondria, ER-stress response, and many other mechanisms. Most of these processes, such as ER-stress response and autophagy, have been associated with neuroprotective roles. In fact, improving these mechanisms using S1R agonists was beneficial in several brain disorders including neurodegenerative diseases. In this review, we will examine S1R subcellular localization and describe S1R-associated biological activity within these specific compartments, i.e., the Mitochondrion-Associated ER Membrane (MAM), ER–Lipid Droplet (ER–LD) interface, ER–Plasma Membreane (ER–PM) interface, and the Nuclear Envelope (NE). We also discussed how the dysregulation of these pathways contributes to neurodegenerative diseases, while highlighting the cellular mechanisms and key binding partners engaged in these processes.

Emerging evidence has established primary nephrotic syndrome (NS), including focal segmental glomerulosclerosis (FSGS), as a primary podocytopathy. Despite the underlying importance of podocyte endoplasmic reticulum (ER) stress in the pathogenesis of NS, no treatment currently targets the podocyte ER. In our monogenic podocyte ER stress-induced NS/FSGS mouse model, the podocyte type 2 ryanodine receptor (RyR2)/calcium release channel on the ER was phosphorylated, resulting in ER calcium leak and cytosolic calcium elevation. The altered intracellular calcium homeostasis led to activation of calcium-dependent cytosolic protease calpain 2 and cleavage of its important downstream substrates, including the apoptotic molecule procaspase 12 and podocyte cytoskeletal protein talin 1. Importantly, a chemical compound, K201, can block RyR2-Ser2808 phosphorylation-mediated ER calcium depletion and podocyte injury in ER-stressed podocytes, as well as inhibit albuminuria in our NS model. In addition, we discovered that mesencephalic astrocyte-derived neurotrophic factor (MANF) can revert defective RyR2-induced ER calcium leak, a bioactivity for this ER stress-responsive protein. Thus, podocyte RyR2 remodeling contributes to ER stress-induced podocyte injury. K201 and MANF could be promising therapies for the treatment of podocyte ER stress-induced NS/FSGS.

Over the past few decades, understandings and evidences concerning the role of endoplasmic reticulum (ER) stress in deciding the cell fate have been constantly growing. Generally, during ER stress, the signal transductions are mainly conducted by three ER stress transducers: protein kinase R-like endoplasmic reticulum kinase (PERK), inositol-requiring kinase 1 (IRE1) and activating transcription factor 6 (ATF6). Consequently, the harmful stimuli from the ER stress transducers induce apoptosis and autophagy, which share several crosstalks and eventually decide the cell fate. The dominance of apoptosis or autophagy induced by ER stress depends on the type and degree of the stimuli. When ER stress is too severe and prolonged, apoptosis is induced to eliminate the damaged cells; however, when stimuli are mild, cell survival is promoted to maintain normal physiological functions by inducing autophagy. Although all the three pathways participate in ER stress-induced apoptosis and autophagy, PERK shows several unique characteristics by interacting with some specific downstream effectors. Notably, there are some preliminary findings on PERK-dependent mechanisms switching autophagy and apoptosis. In this review, we particularly focused on the novel, intriguing and complicated role of PERK in ER stress-decided cell fate, and also discussed more roles of PERK in restoring cellular homeostasis. However, more in-depth knowledge of PERK in the future would facilitate our understanding about many human diseases and benefit in searching for new molecular therapeutic targets.

The Unfolded Protein Response (UPR) maintains homeostasis in the endoplasmic reticulum (ER) and defends against ER stress, an underlying factor in various human diseases. During the UPR, numerous genes are activated that sustain and protect the ER. These responses are known to involve the canonical UPR transcription factors XBP1, ATF4, and ATF6. Here, we show in C. elegans that the conserved stress defense factor SKN-1/Nrf plays a central and essential role in the transcriptional UPR. While SKN-1/Nrf has a well-established function in protection against oxidative and xenobiotic stress, we find that it also mobilizes an overlapping but distinct response to ER stress. SKN-1/Nrf is regulated by the UPR, directly controls UPR signaling and transcription factor genes, binds to common downstream targets with XBP-1 and ATF-6, and is present at the ER. SKN-1/Nrf is also essential for resistance to ER stress, including reductive stress. Remarkably, SKN-1/Nrf-mediated responses to oxidative stress depend upon signaling from the ER. We conclude that SKN-1/Nrf plays a critical role in the UPR, but orchestrates a distinct oxidative stress response that is licensed by ER signaling. Regulatory integration through SKN-1/Nrf may coordinate ER and cytoplasmic homeostasis.