EDEM1 Regulates Amyloid Precursor Protein (APP) Metabolism and Amyloid-ß Production.

Endoplasmic reticulum (ER) degradation-enhancing α-mannosidase-like protein 1 (EDEM1) is a quality control factor directly involved in the endoplasmic reticulum-associated degradation (ERAD) process. It recognizes terminally misfolded proteins and directs them to retrotranslocation which is followed by proteasomal degradation in the cytosol. The amyloid-ß precursor protein (APP) is synthesized and N-glycosylated in the ER and transported to the Golgi for maturation before being delivered to the cell surface. The amyloidogenic cleavage pathway of APP leads to production of amyloid-ß (Aß), deposited in the brains of Alzheimer's disease (AD) patients. Here, using biochemical methods applied to human embryonic kidney, HEK293, and SH-SY5Y neuroblastoma cells, we show that EDEM1 is an important regulatory factor involved in APP metabolism. We find that APP cellular levels are significantly reduced after EDEM1 overproduction and are increased in cells with downregulated EDEM1. We also report on EDEM1-dependent transport of APP from the ER to the cytosol that leads to proteasomal degradation of APP. EDEM1 directly interacts with APP. Furthermore, overproduction of EDEM1 results in decreased Aß40 and Aß42 secretion. These findings indicate that EDEM1 is a novel regulator of APP metabolism through ERAD.

Trehalose protects Escherichia coli against carbon stress manifested by protein acetylation and aggregation.

The disaccharide trehalose is widely distributed in nature and can serve as a carbon reservoir, a signaling molecule for controlling glucose metabolism and a stress protectant. We demonstrated that in Escherichia coli ΔotsA cells, which are unable to synthesize trehalose, the aggregation of endogenous proteins during the stationary phase was increased in comparison to wild-type cells. The lack of trehalose synthesis boosted Nε-lysine acetylation of proteins, which in turn enhanced their hydrophobicity and aggregation. This increased Nε-lysine acetylation could result from carbon overflow and the accumulation of acetyl phosphate caused by the ΔotsA mutation. These findings provide a better understanding of the previously reported protective functions of trehalose in protein stabilization and the prevention of protein aggregation. Our results indicate that trehalose may participate in proteostasis not only as a chemical chaperone but also as a metabolite that indirectly counteracts detrimental protein acetylation. We propose that trehalose protects E. coli against carbon stress - the synthesis and storage of trehalose can prevent carbon overflow, which otherwise is manifested by protein acetylation and aggregation.

Toxins Utilize the Endoplasmic Reticulum-Associated Protein Degradation Pathway in Their Intoxication Process.

Several bacterial and plant AB-toxins are delivered by retrograde vesicular transport to the endoplasmic reticulum (ER), where the enzymatically active A subunit is disassembled from the holotoxin and transported to the cytosol. In this process, toxins subvert the ER-associated degradation (ERAD) pathway. ERAD is an important part of cellular regulatory mechanism that targets misfolded proteins to the ER channels, prior to their retrotranslocation to the cytosol, ubiquitination and subsequent degradation by a protein-degrading complex, the proteasome. In this article, we present an overview of current understanding of the ERAD-dependent transport of AB-toxins to the cytosol. We describe important components of ERAD and discuss their significance for toxin transport. Toxin recognition and disassembly in the ER, transport through ER translocons and finally cytosolic events that instead of overall proteasomal degradation provide proper folding and cytotoxic activity of AB-toxins are discussed as well. We also comment on recent reports presenting medical applications for toxin transport through the ER channels.

Intracellular Transport and Cytotoxicity of the Protein Toxin Ricin.

Ricin can be isolated from the seeds of the castor bean plant (Ricinus communis). It belongs to the ribosome-inactivating protein (RIP) family of toxins classified as a bio-threat agent due to its high toxicity, stability and availability. Ricin is a typical A-B toxin consisting of a single enzymatic A subunit (RTA) and a binding B subunit (RTB) joined by a single disulfide bond. RTA possesses an RNA N-glycosidase activity; it cleaves ribosomal RNA leading to the inhibition of protein synthesis. However, the mechanism of ricin-mediated cell death is quite complex, as a growing number of studies demonstrate that the inhibition of protein synthesis is not always correlated with long term ricin toxicity. To exert its cytotoxic effect, ricin A-chain has to be transported to the cytosol of the host cell. This translocation is preceded by endocytic uptake of the toxin and retrograde traffic through the trans-Golgi network (TGN) and the endoplasmic reticulum (ER). In this article, we describe intracellular trafficking of ricin with particular emphasis on host cell factors that facilitate this transport and contribute to ricin cytotoxicity in mammalian and yeast cells. The current understanding of the mechanisms of ricin-mediated cell death is discussed as well. We also comment on recent reports presenting medical applications for ricin and progress associated with the development of vaccines against this toxin.