Macroautophagy/autophagy is a constitutively active catabolic lysosomal degradation pathway, often found dysregulated in human diseases. It is often considered to act in a cytoprotective manner and is commonly upregulated in cells undergoing stress. Its initiation is regulated at the protein level and does not require de novo protein synthesis. Historically, autophagy has been regarded as non-selective; however, it is now clear that different stimuli can lead to the selective degradation of cellular components via selective autophagy receptors (SARs). Due to its selective nature and the existence of multiple degradation pathways potentially acting in concert, monitoring of autophagy flux, i.e. selective autophagy-dependent protein degradation, should address this complexity. Here, we introduce a targeted proteomics approach monitoring abundance changes of 37 autophagy-related proteins covering process-relevant proteins such as the initiation complex and the Atg8-family protein lipidation machinery, as well as most known SARs. We show that proteins involved in autophagosome biogenesis are upregulated and spared from degradation under autophagy-inducing conditions in contrast to SARs, in a cell-line dependent manner. Classical bulk stimuli such as nutrient starvation mainly induce degradation of ubiquitin-dependent soluble SARs and not of ubiquitin-independent, membrane-bound SARs. In contrast, treatment with the iron chelator deferiprone leads to the degradation of ubiquitin-dependent and -independent SARs linked to mitophagy and reticulophagy/ER-phagy. Our approach is automatable and supports large-scale screening assays paving the way to (pre)clinical applications and monitoring of specific autophagy flux.

Autophagy is classified as nonselective or selective depending on the types of degrading substrates. Endoplasmic reticulum (ER)-phagy is a form of selective autophagy for transporting the ER-resident proteins to autolysosomes. FAM134B, a member of the family with sequence similarity 134, is a well-known ER-phagy receptor. Dysfunction of FAM134B results in several diseases including viral infection, inflammation, neurodegenerative disorder, and cancer, indicating that FAM134B has crucial roles in various kinds of intracellular functions. However, how FAM134B-mediated ER-phagy regulates intracellular functions is not well understood. In this study, we found that FAM134B knockdown in mammalian cells accelerated cell proliferation. FAM134B knockdown increased the protein amount of stromal interaction molecule 1 (STIM1), an ER Ca2+ sensor protein mediating the store-operated Ca2+ entry involved in G1 to S phase transition. FAM134B bound to STIM1 through its C-terminal cytosolic region. FAM134B knockdown reduced transport of STIM1 from the ER to autolysosomes. Finally, FAM134B knockdown accelerated G1 to S phase transition. These results suggest that FAM134B is involved in cell proliferation possibly through degradation of STIM1 via ER-phagy.

Preeclampsia (PE) is a life-threatening pregnancy-specific complication with controversial mechanisms and no effective treatment except delivery is available. Currently, increasing researchers suggested that PE shares pathophysiologic features with protein misfolding/aggregation disorders, such as Alzheimer disease (AD). Evidences have proposed defective autophagy as a potential source of protein aggregation in PE. Endoplasmic reticulum-selective autophagy (ER-phagy) plays a critical role in clearing misfolded proteins and maintaining ER homeostasis. However, its roles in the molecular pathology of PE remain unclear. We found that lncRNA DUXAP8 was upregulated in preeclamptic placentae and significantly correlated with clinical indicators. DUXAP8 specifically binds to PCBP2 and inhibits its ubiquitination-mediated degradation, and decreased levels of PCBP2 reversed the activation effect of DUXAP8 overexpression on AKT/mTOR signaling pathway. Function experiments showed that DUXAP8 overexpression inhibited trophoblastic proliferation, migration, and invasion of HTR-8/SVneo and JAR cells. Moreover, pathological accumulation of swollen and lytic ER (endoplasmic reticulum) was observed in DUXAP8-overexpressed HTR8/SVneo cells and PE placental villus trophoblast cells, which suggesting that ER clearance ability is impaired. Further studies found that DUXAP8 overexpression impaired ER-phagy and caused protein aggregation medicated by reduced FAM134B and LC3II expression (key proteins involved in ER-phagy) via activating AKT/mTOR signaling pathway. The increased level of FAM134B significantly reversed the inhibitory effect of DUXAP8 overexpression on the proliferation, migration, and invasion of trophoblasts. In vivo, DUXAP8 overexpression through tail vein injection of adenovirus induced PE-like phenotypes in pregnant rats accompanied with activated AKT/mTOR signaling, decreased expression of FAM134B and LC3-II proteins and increased protein aggregation in placental tissues. Our study reveals the important role of lncRNA DUXAP8 in regulating trophoblast biological behaviors through FAM134B-mediated ER-phagy, providing a new theoretical basis for understanding the pathogenesis of PE.

Selective autophagy of the endoplasmic reticulum (ER-phagy) is a mechanism that is necessary for degrading damaged ER components and preventing cells from experiencing ER stress. Various ER-phagy receptors orchestrate this process by building protein assemblies with dedicated functions. In order to understand the molecular building principles of ER-phagy, it is important to reveal the assembly of ER-phagy receptors in a temporal and functional context. However, direct visualization is hampered by the diffraction limit in light microscopy. Super-resolution microscopy (SRM) can bypass this limitation and resolve single proteins and nanoscale protein clusters in cells. In particular, DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) is a powerful technology that can resolve individual protein clusters in cells and provide information on their molecular composition. Here, we report a step-by-step protocol on how to utilize DNA-PAINT to perform super-resolution imaging of ER-phagy receptors in fixed cells. In addition, we provide a detailed explanation of image generation, cluster analysis, and molecular quantification.

The endoplasmic reticulum (ER) serves as a central hub for protein synthesis, folding, and lipid biosynthesis in eukaryotic cells. Maintaining ER homeostasis is essential for optimal cellular function, and one mechanism that has garnered attention is endoplasmic reticulum-specific autophagy, or ER-phagy. ER-phagy selectively removes specific ER portions, playing a pivotal role in cellular health and adaptation to environmental stressors. ER-phagy can be induced by diverse cellular conditions such as amino acid starvation, disruption of ER quality control mechanisms, and accumulation of misfolded ER protein, highlighting cellular adaptability and the significance of ER-phagy in stress responses. Clinically relevant mutations in ER-phagy receptors are implicated in various diseases, underlining the fundamental importance of ER-phagy in ER homeostasis. Here, we provide comprehensive protocols and general considerations while investigating ER-phagy using three fundamental techniques-Western blotting, immunofluorescence, and flow cytometry-commonly used in ER-phagy detection and quantitation.

Protein biogenesis within the endoplasmic reticulum (ER) is crucial for organismal function. Errors during protein folding necessitate the removal of faulty products. ER-associated protein degradation and ER-phagy target misfolded proteins for proteasomal and lysosomal degradation. The mechanisms initiating ER-phagy in response to ER proteostasis defects are not well understood. By studying mouse primary cells and patient samples as a model of ER storage disorders (ERSDs), we show that accumulation of faulty products within the ER triggers a response involving SESTRIN2, a nutrient sensor controlling mTORC1 signaling. SESTRIN2 induction by XBP1 inhibits mTORC1\'s phosphorylation of TFEB/TFE3, allowing these transcription factors to enter the nucleus and upregulate the ER-phagy receptor FAM134B along with lysosomal genes. This response promotes ER-phagy of misfolded proteins via FAM134B-Calnexin complex. Pharmacological induction of FAM134B improves clearance of misfolded proteins in ERSDs. Our study identifies the interplay between nutrient signaling and ER quality control, suggesting therapeutic strategies for ERSDs.

As an efficient alternative copper (Cu) source, copper nanoparticles (nano-Cu) have been widely supplemented into animal-producing food. Therefore, it is necessary to assess the effect of nano-Cu exposure on the biological health risk. Recently, the toxic effects of nano-Cu have been confirmed but the underlying mechanism remains unclear. This study reveals the impact of nano-Cu on endoplasmic reticulum autophagy (ER-phagy) in chicken hepatocytes and further identifies Drp1 and its downstream gene FAM134B as crucial regulators of nano-Cu-induced hepatotoxicity. Nano-Cu exposure can induce Cu ion overaccumulation and pathological injury in the liver, trigger excessive mitochondrial fission and mitochondria-associated membrane (MAM) integrity damage, and activate ER-phagy in vivo and in vitro. Interestingly, the knockdown of Drp1 markedly decreases the expression of FAM134B induced by nano-Cu. Furthermore, the expression levels of ATL3, CCPG1, SEC62, TEX264, and LC3II/LC3I induced by nano-Cu exposure are decreased by inhibiting the expression of Drp1. Simultaneously, the inhibition of FAM134B effectively alleviates nano-Cu-induced ER-phagy by downregulating the expression of ATL3, CCPG1, SEC62, TEX264, and LC3II/LC3I. Overall, these results suggest that Drp1-mediated impairment of MAM integrity leads to ER-phagy as a novel molecular mechanism involved in the regulation of nano-Cu-induced hepatotoxicity. These findings provide new ideas for future research on the mechanism of nano-Cu-induced hepatotoxicity.

Toxoplasma gondii, a widespread parasite, has the ability to infect nearly any nucleated cell in warm-blooded vertebrates. It is estimated that around 2 billion people globally have been infected by this pathogen. Although most healthy individuals can effectively control parasite replication, certain parasites may evade the immune response, establishing cysts in the brain that are refractory to the immune system and resistant to available drugs. For its chronic persistence in the brain, the parasite relies on host cells\' nutrients, particularly amino acids and lipids. Therefore, understanding how latent parasites persist in the brain is crucial for identifying potential drug targets against chronic forms. While shielded within parasitophorous vacuoles (PVs) or cysts, Toxoplasma exploits the host endoplasmic reticulum (ER) metabolism to sustain its persistence in the brain, resulting in host neurological alterations. In this study, we demonstrate that T. gondii disrupts the host ER homeostasis, resulting in the accumulation of unfolded protein within the host ER. The host counters this stress by initiating an autophagic pathway known as ER-phagy, which breaks down unfolded proteins into amino acids, promoting their recycling. Our findings unveil the underlying mechanisms employed by T. gondii to exploit host ER and lysosomal pathways, enhancing nutrient levels during infection. These insights provide new strategies for the treatment of toxoplasmosis.
OBJECTIVE: Intracellular parasites employ several mechanisms to manipulate the cellular environment, enabling them to persist in the host. Toxoplasma gondii, a single-celled parasite, possesses the ability to infect virtually any nucleated cell of warm-blooded vertebrates, including nearly 2 billion people worldwide. Unfortunately, existing treatments and immune responses are not entirely effective in eliminating the chronic persisting forms of the parasite. This study reveals that T. gondii induces the host\'s autophagic pathway to boost amino acid levels in infected cells. The depletion of amino acids, in turn, influences the persistence of the parasite\'s chronic forms. Significantly, our investigation establishes the crucial role of host endoplasmic reticulum (ER)-phagy in the parasite\'s persistence within the host during latent infection.

The endoplasmic reticulum (ER) is a crucial organelle that orchestrates key cellular functions like protein folding and lipid biosynthesis. However, it is highly sensitive to disturbances that lead to ER stress. In response, the unfolded protein response (UPR) activates to restore ER homeostasis, primarily through three sensors: IRE1, ATF6, and PERK. ERAD and autophagy are crucial in mitigating ER stress, yet their dysregulation can lead to the accumulation of misfolded proteins. Cisplatin, a commonly used chemotherapy drug, induces ER stress in tumor cells, activating complex signaling pathways. Resistance to cisplatin stems from reduced drug accumulation, activation of DNA repair, and anti-apoptotic mechanisms. Notably, cisplatin-induced ER stress can dualistically affect tumor cells, promoting either survival or apoptosis, depending on the context. ERAD is crucial for degrading misfolded proteins, whereas autophagy can protect cells from apoptosis or enhance ER stress-induced apoptosis. The complex interaction between ER stress, cisplatin resistance, ERAD, and autophagy opens new avenues for cancer treatment. Understanding these processes could lead to innovative strategies that overcome chemoresistance, potentially improving outcomes of cisplatin-based cancer treatments. This comprehensive review provides a multifaceted perspective on the complex mechanisms of ER stress, cisplatin resistance, and their implications in cancer therapy.

OBJECTIVE: Rapid proliferation and nutrition starvation in the tumor microenvironment pose significant challenges to cellular protein homeostasis. The accumulation of misfolded proteins in the endoplasmic reticulum lumen induces stress on cells and causes irreversible damage to cells if unresolved. Emerging reports emphasize the influence of the tumor microenvironment on therapeutic molecule efficacy and treatment outcomes. Hence, we aimed to understand the influence of tamoxifen on the cellular adaptation to endoplasmic reticulum stress during metabolic stress in breast cancer cells.
METHODS: Nutrition deprivation induces endoplasmic reticulum stress (ER stress), and the unfolded protein response (UPR) in breast cancer cells was confirmed by a Thioflavin B assay and western blotting. Tamoxifen-indued ER-phagy was studied using an MCD assay, confocal microscopy, and western blotting.
RESULTS: Nutrition deprivation induces ER stress in breast cancer cells. Interestingly, tamoxifen modulates the nutrition deprivation-induced endoplasmic reticulum stress through enhancing the selective ER-phagy, a specialized autophagy. The tamoxifen-induced ER-phagy is mediated by AMPK activation. The pharmacological inhibition of AMPK blocks tamoxifen-induced ER-phagy and tamoxifen modulatory effect on ER stress during nutrition deprivation.
CONCLUSIONS: Tamoxifen modulates ER stress by inducing ER-phagy through AMPK, thereby, may support breast cancer cell survival during nutrition deprivation conditions.