Autophagy is classified as non-selective 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 STIM1, an ER Ca2+ sensor protein mediating the store-operated Ca2+ entry (SOCE) 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.

Molecular farming, also known as plant molecular farming (PMF), is a technique that involves using plants and plant cells as bioreactors to produce recombinant proteins. This is a cost-effective and sustainable way of producing large quantities of proteins for various applications, including pharmaceuticals, vaccines, and industrial enzymes. An endogenous or exogenous signal peptide (SP) is flanked at the N-terminal for recombinant protein targeting and storage. These SPs are responsible for guiding the recombinant protein products to the correct destination within the plant cell or facilitating their secretion into the extracellular space. In this chapter, we will give a brief introduction of the current PMF research outcomes supported by the basic study of vesicle trafficking and protein secretion, mainly introducing the bright yellow 2 (BY-2) cell-based secretion pathway and its associated protocols according to our study of recombinant human iduronidase.

Glucose-regulated protein 94 (Grp94) is an isoform of the heat shock protein 90 kDa (Hsp90) family of molecular chaperones. Inhibiting Grp94 has been implicated for many diseases. Co-crystal structures of two generations of Grp94 inhibitors revealed the importance of investigating the ester group, which is projected into the site 2 pocket unique to Grp94. Therefore, a series of KUNG65 benzamide analogs was designed and synthesized to evaluate their impact on the affinity and selectivity for Grp94. The data demonstrated that substituents with small and saturated ring systems that contain hydrogen bond acceptors exhibited increased affinity for Grp94, whereas larger saturated ring system manifested increased selectivity for Grp94 over Hsp90α.

Root-knot nematodes (RKNs) are microscopic parasitic worms able to infest the roots of thousands of plant species, causing massive crop yield losses worldwide. They evade the plant\'s immune system and manipulate plant cell physiology and metabolism to transform a few root cells into giant cells, which serve as feeding sites for the nematode. RKN parasitism is facilitated by the secretion in planta of effector molecules, mostly proteins that hijack host cellular processes. We describe here a conserved RKN-specific effector, effector 12 (EFF12), that is synthesized exclusively in the oesophageal glands of the nematode, and we demonstrate its function in parasitism. In the plant, MiEFF12 localizes to the endoplasmic reticulum (ER). A combination of RNA-sequencing analysis and immunity-suppression bioassays revealed the contribution of MiEFF12 to the modulation of host immunity. Yeast two-hybrid, split luciferase and co-immunoprecipitation approaches identified an essential component of the ER quality control system, the Solanum lycopersicum plant bap-like (PBL), and basic leucine zipper 60 (BZIP60) proteins as host targets of MiEFF12. Finally, silencing the PBL genes in Nicotiana benthamiana decreased susceptibility to Meloidogyne incognita infection. Our results suggest that EFF12 manipulates PBL function to modify plant immune responses to allow parasitism.

A recent study by Amankwah et al. reports how co-chaperone proteins and ATP hydrolysis fine-tune the function of endoplasmic reticulum (ER)-resident Hsp90 paralog Grp94.

Occult hepatitis B virus infection (OBI) is characterized by the presence of HBV DNA in the absence of detectable HBsAg. OBI is an important risk factor for cirrhosis and hepatocellular carcinoma, but its pathogenesis has not been fully elucidated. Mutations in the HBV preS/S genes can lead to impaired secretion of either HBsAg or S-protein resulting in the accumulation of defective viruses or S protein in cells. In our previous work, the M133S mutation was present in the HBV S gene of maintenance hemodialysis (MHD) patients with OBI. In this study, we investigated the potential role of amino acid substitutions in S proteins in S protein production and secretion through the construction of mutant S gene plasmids, structural prediction, transcriptome sequencing analysis, and in vitro functional studies. Protein structure prediction showed that the S protein M133S mutant exhibited hydrophilic modifications, with greater aggregation and accumulation of the entire structure within the membrane phospholipid bilayer. Differential gene enrichment analysis of transcriptome sequencing data showed that differentially expressed genes were mainly concentrated in protein processing in the endoplasmic reticulum (ER). The expression of heat shock family proteins and ER chaperone molecules was significantly increased in the wild-type and mutant groups, whereas the expression of mitochondria-associated proteins was decreased. Immunofluorescence staining and protein blotting showed that the endoplasmic reticulum-associated protein PDI, the autophagy marker LC3, and the lysosome-associated protein LAMP2 co-localized with the S proteins in the wild-type and mutant strains, and their expression was increased. The mitochondria-associated TOMM20 protein was also co-expressed with the S protein, but expression was significantly reduced in the mutant. The M133S mutation in the S gene is expressed as a defective and misfolded protein that accumulates in the endoplasmic reticulum causing secretion-impaired endoplasmic reticulum stress, which in turn triggers mitochondrial autophagy and recruits lysosomes to fuse with the autophagosome, leading to mitochondrial clearance. This study preliminarily demonstrated that the mutation of M133S in the S gene can cause OBI and is associated with disease progression, providing a theoretical basis for the diagnosis and treatment of OBI.

The endoplasmic reticulum (ER) produces proteins destined to organelles of the endocytic and secretory pathways, the plasma membrane, and the extracellular space. While native proteins are transported to their intra- or extracellular site of activity, folding-defective polypeptides are retro-translocated across the ER membrane into the cytoplasm, poly-ubiquitylated and degraded by 26 S proteasomes in a process called ER-associated degradation (ERAD). Large misfolded polypeptides, such as polymers of alpha1 antitrypsin Z (ATZ) or mutant procollagens, fail to be dislocated across the ER membrane and instead enter ER-to-lysosome-associated degradation (ERLAD) pathways. Here, we show that pharmacological or genetic inhibition of ERAD components, such as the α1,2-mannosidase EDEM1 or the OS9 ERAD lectins triggers the delivery of the canonical ERAD clients Null Hong Kong (NHK) and BACE457Δ to degradative endolysosomes under control of the ER-phagy receptor FAM134B and the LC3 lipidation machinery. Our results reveal that ERAD dysfunction is compensated by the activation of FAM134B-driven ERLAD pathways that ensure efficient lysosomal clearance of orphan ERAD clients.

Stasimon/Tmem41b is a transmembrane protein with phospholipid scrambling activity that resides in the endoplasmic reticulum and has been implicated in autophagy, lipid metabolism, and viral replication. Stasimon/Tmem41b has also been linked to the function of sensory-motor circuits and the pathogenesis of spinal muscular atrophy. However, the early embryonic lethality of constitutive knockout in mice has hindered the analysis of spatial and temporal requirements of Stasimon/Tmem41b in vivo. To address this, we developed a novel mouse line harboring a conditional knockout allele of the Stasimon/Tmem41b gene in which exon 4 has been flanked by loxP sites (Stas/Tmem41bCKO). Cre-mediated recombination of Stas/Tmem41bCKO generates a functionally null allele (Stas/Tmem41bΔ4) resulting in loss of protein expression and embryonic lethality in the homozygous mouse mutant. Here, using a ubiquitously expressed, tamoxifen inducible Cre recombinase in the homozygous Stas/Tmem41bCKO mice, we demonstrate that postnatal depletion of Stasimon/Tmem41b rapidly arrests weight gain in adult mice and causes motor dysfunction and death approximately three weeks after tamoxifen treatment. Moreover, we show that depletion of Stasimon/Tmem41b severely affects cell proliferation in mouse embryonic fibroblasts. This study provides new insights into the essential requirement of Stasimon/Tmem41b for cellular and organismal fitness and expands the experimental toolkit to investigate its functions in the mammalian system.

Graves\' ophthalmopathy (GO), or thyroid eye disease (TED), is the most frequent extrathyroidal manifestation of Graves\' disease (GD). Inflammation and subsequent aberrant tissue remodeling with fibrosis are important pathogenesis. There are many proposed mechanisms and molecular pathways contributing to tissue remodeling and fibrosis in GO, including adipogenesis, fibroblast proliferation and myofibroblasts differentiation, oxidative stress, endoplasmic reticulum (ER) stress, hyaluronan (HA) and glycosaminoglycans (GAGs) accumulation in the extracellular matrix (ECM) and new concepts of epigenetics modification, such as histone modification, DNA methylation, non-coding RNAs, and gut microbiome. This review summarizes the current understanding of ECM proteins and associated tissue remodeling in the pathogenesis and potential mediators for the treatment of GO.

In this book chapter, we present a method for microsome isolation from the hypocotyl tissue of dark-grown Arabidopsis thaliana. Microsomes are heterogeneous, vesicle-like membranes, which are, not exclusively, derived but enriched with membranes of the endoplasmic reticulum (ER). Here, we describe the experimental setup, including sample preparation, homogenization, differential centrifugation steps, and quality control measures after microsome isolation.