EDEM1 Drives Misfolded Protein Degradation via ERAD and Exploits ER-Phagy as Back-Up Mechanism When ERAD Is Impaired.

Endoplasmic reticulum (ER)-associated degradation (ERAD) is the main mechanism of targeting ER proteins for degradation to maintain homeostasis, and perturbations of ERAD lead to pathological conditions. ER-degradation enhancing α-mannosidase-like (EDEM1) was proposed to extract terminally misfolded proteins from the calnexin folding cycle and target them for degradation by ERAD. Here, using mass-spectrometry and biochemical methods, we show that EDEM1 is found in auto-regulatory complexes with ERAD components. Moreover, the N-terminal disordered region of EDEM1 mediates protein-protein interaction with misfolded proteins, whilst the absence of this domain significantly impairs their degradation. We also determined that overexpression of EDEM1 can induce degradation, even when proteasomal activity is severely impaired, by promoting the formation of aggregates, which can be further degraded by autophagy. Therefore, we propose that EDEM1 maintains ER homeostasis and mediates ERAD client degradation via autophagy when either dislocation or proteasomal degradation are impaired.

Estrogen enhances secretion of apolipoprotein B-100 containing lipoproteins by BeWo cells.

Although the early human embryo is capable of covering its cholesterol demand by endogenous synthesis, during later stages of development the fetus may become dependent on transplacental cholesterol transport. On one hand, this conclusion is based on the severe developmental abnormalities of embryos with mutations in the gene specifying the enzyme catalyzing the last step of cholesterol synthesis, 7-dehydrocholesterol reductase, causing Smith-Lemli-Opitz Syndrome. On the other hand, increased total maternal plasma cholesterol levels may reflect the requirement by the growing fetus and/or the placenta for cholesterol. Various molecules and complexes must cross the placental barrier consisting of trophoblasts and fetal endothelial cells to reach the fetal circulation. The de novo synthesis of apolipoprotein B (apoB)-containing lipoproteins coupled to secretion from trophoblasts towards the fetal side is one efficient pathway for cholesterol supply. ApoB and the microsomal triglyceride transfer protein (MTP) are essential components for the assembly of apoB-containing lipoproteins. The aim of this study was to evaluate functional properties of the human placental cell line BeWo as an in vitro model for placental synthesis of apoB-containing lipoproteins by focusing on components required for lipoprotein assembly and secretion. We demonstrate mRNA and protein production of apoB-100, MTP, and protein disulfide isomerase (PDI) in BeWo cells. In addition, metabolic radiolabeling and apoB-immunoprecipitation of cell extracts and media revealed that synthesis and secretion of apoB-containing lipoproteins are enhanced by estrogen. The expression of apoB-100, MTP, and PDI, and the estrogen-stimulated lipoprotein secretion by BeWo cells suggest that these cells are a useful system to study aspects of lipoprotein metabolism at the placental barrier.

Expression of microsomal triglyceride transfer protein in lipoprotein-synthesizing tissues of the developing chicken embryo.

In contrast to mammals, in the chicken major sites of lipoprotein synthesis and secretion are not only the liver and intestine, but also the kidney and the embryonic yolk sac. Two key components in the assembly of triglyceride-rich lipoproteins are the microsomal triglyceride transfer protein (MTP) and apolipoprotein B (apoB). We have analyzed the expression of MTP in the embryonic liver, small intestine, and kidney, and have studied the expression of MTP in, and the secretion of apoB from, the developing yolk sac (YS). Transcript and protein levels of MTP increase during embryogenesis in YS, liver, kidney, and small intestine, and decrease in YS, embryonic liver, and kidney after hatching. In small intestine, the MTP mRNA level rises sharply during the last trimester of embryo development (after day 15), while MTP protein is detectable only after hatching (day 21). In the YS of 15- and 20-day old embryos, apoB secretion was detected by pulse-chase metabolic radiolabeling experiments and subsequent immunoprecipitation. Taken together, our data reveal the importance of coordinated production of MTP and apoB in chicken tissues capable of secreting triglyceride-rich lipoproteins even before hatching.

Molecular cloning, expression, and hormonal regulation of the chicken microsomal triglyceride transfer protein.

During an egg-laying cycle, oviparous animals transfer massive amounts of triglycerides, the major lipid component of very low density lipoprotein (VLDL), from the liver to the developing oocytes. A major stimulus for this process is the rise in estrogen associated with the onset of an egg-laying cycle. In mammals, the microsomal triglyceride transfer protein (MTP) is required for VLDL assembly and secretion. To enable studies to determine if MTP plays a role in basal and estrogen-stimulated VLDL assembly and secretion in an oviparous vertebrate, we have cloned and sequenced the chicken MTP cDNA. This cDNA encodes a protein of 893 amino acids with an N-terminal signal sequence. The primary sequence of chicken MTP is, on average, 65% identical to that of mammalian homologs, and 23% identical to the Drosophila melanogaster protein. We have obtained a clone of chicken embryo fibroblast cells that stably express the avian MTP cDNA and show that these cells display MTP activity as measured by the transfer of a fluorescently labeled neutral lipid. As in mammals, chicken MTP is localized to the endoplasmic reticulum as revealed by indirect immunofluorescence and by the fact that its N-linked oligosaccharide moiety remains sensitive to endoglycosidase H. Endogenous, enzymatically active MTP is also expressed in an estrogen receptor-expressing chicken hepatoma cell line that secretes apolipoprotein B-containing lipoproteins. In this cell line and in vivo, the expression and activity of MTP are not influenced by estrogen. Therefore, up-regulation of MTP in the liver is not required for the increased VLDL assembly during egg production in the chicken. This indicates that MTP is not rate-limiting, even for the massive estrogen-induced secretion of VLDL accompanying an egg-laying cycle.

Processing of N-linked glycans during endoplasmic-reticulum-associated degradation of a short-lived variant of ribophorin I.

Recently, the role of N-linked glycans in the process of ERAD (endoplasmic reticulum-associated degradation) of proteins has been widely recognized. In the present study, we attempted to delineate further the sequence of events leading from a fully glycosylated soluble protein to its deglycosylated form. Degradation intermediates of a truncated form of ribophorin I, namely RI(332), which contains a single N-linked oligosaccharide and is a substrate for the ERAD/ubiquitin-proteasome pathway, were characterized in HeLa cells under conditions blocking proteasomal degradation. The action of a deoxymannojirimycin- and kifunensine-sensitive alpha1,2-mannosidase was shown here to be required for both further glycan processing and progression of RI(332) in the ERAD pathway. In a first step, the Man(8) isomer B, generated by ER mannosidase I, appears to be the major oligomannoside structure associated with RI(332) intermediates. Some other trimmed N-glycan species, in particular Glc(1)Man(7)GlcNAc(2), were also found on the protein, indicating that several mannosidases might be implicated in the initial trimming of the oligomannoside. Secondly, another intermediate of degradation of RI(332) accumulated after proteasome inhibition. We demonstrated that this completely deglycosylated form arose from the action of an N-glycanase closely linked to the ER membrane. Indeed, the deglycosylated form of the protein remained membrane-associated, while being accessible from the cytoplasm to ubiquitinating enzymes and to added protease. Our results indicate that deglycosylation of a soluble ERAD substrate glycoprotein occurs in at least two distinct steps and is coupled with the retro-translocation of the protein preceding its proteasomal degradation.

Regulation by estrogen of synthesis and secretion of apolipoprotein A-I in the chicken hepatoma cell line, LMH-2A.

The synthesis and secretion of apolipoprotein A-I (apoA-I) in response to the treatment with estrogen were investigated in the chicken hepatoma cell line, LMH-2A. Exposure of these cells to exogenous estrogen for up to 48 h results in a decrease of apoA-I production, as evident from Western blotting, immunoprecipitation, and immunofluorescence experiments. Likewise, the secretion of apoA-I is also decreased in estrogen-treated cells when compared to controls. However, under both conditions, the disappearance of the apoprotein from the cells occurs very rapidly and with similar kinetics. The bulk of apoA-I secreted from LMH-2A cells is recovered on lipoprotein particles with a buoyant density of > or =1.10 g/ml, corresponding to HDL and heavy LDL. Interestingly, apoA-I is detectable on apoB-containing lipoproteins by sequential immunoprecipitation, suggesting that the two apoproteins co-reside at least on a subfraction of the secreted particles, or that apoB- and apoA-I-containing particles interact. These interactions are more pronounced in estrogen-treated cells, most likely due to the dramatic estrogen-mediated induction of apoB synthesis and secretion.