Kinetics of the thapsigargin-induced Ca mobilisation: A quantitative analysis in the HEK-293 cell line.

Thapsigargin (TG) inhibits the sarco/endoplasmic reticulum Ca ATPase (SERCA) pump and, when applied acutely, it initiates a Ca mobilisation that begins with the loss of Ca from the endoplasmic reticulum (ER) and culminates with store-operated Ca entry (SOCE) from the extracellular space. Using the popular model cell line HEK-293, we quantified TG-induced changes in cytosolic and ER Ca levels using FURA-2 and the FRET-based ER Ca sensor D1ER, respectively. Our analysis predicts an ER Ca leak of 5-6 µM⋅s for the typical basal ER Ca level of 335-407 µM in HEK-293 cells.

Antagonizing Sec62 function in intracellular Ca homeostasis represents a novel therapeutic strategy for head and neck cancer.

Various cancer types including head and neck squamous cell carcinomas (HNSCC) show a frequent amplification of chromosomal region 3q26 that encodes, among others, for the gene. Located in the ER membrane, this translocation protein is known to play a critical role as a potential driver oncogene in cancer development. High expression levels were observed in various cancer entities and were associated with a poor outcome and increased metastatic burden.

Mycolactone enhances the Ca2+ leak from endoplasmic reticulum by trapping Sec61 translocons in a Ca2+ permeable state.

The Mycobacterium ulcerans exotoxin, mycolactone, is an inhibitor of co-translational translocation via the Sec61 complex. Mycolactone has previously been shown to bind to, and alter the structure of the major translocon subunit Sec61α, and change its interaction with ribosome nascent chain complexes. In addition to its function in protein translocation into the ER, Sec61 also plays a key role in cellular Ca2+ homeostasis, acting as a leak channel between the endoplasmic reticulum (ER) and cytosol.

Remodelling of Ca homeostasis is linked to enlarged endoplasmic reticulum in secretory cells.

The endoplasmic reticulum (ER) is extensively remodelled during the development of professional secretory cells to cope with high protein production. Since ER is the principal Ca store in the cell, we characterised the Ca homeostasis in NALM-6 and RPMI 8226 cells, which are commonly used as human pre-B and antibody secreting plasma cell models, respectively. Expression levels of Sec61 translocons and the corresponding Sec61-mediated Ca leak from ER, Ca storage capacity and store-operated Ca entry were significantly enlarged in the secretory RPMI 8226 cell line.

Eeyarestatin 24 impairs SecYEG-dependent protein trafficking and inhibits growth of clinically relevant pathogens.

Eeyarestatin 1 (ES1) is an inhibitor of endoplasmic reticulum (ER) associated protein degradation, Sec61-dependent Ca homeostasis and protein translocation into the ER. Recently, evidence was presented showing that a smaller analog of ES1, ES24, targets the Sec61-translocon, and captures it in an open conformation that is translocation-incompetent. We now show that ES24 impairs protein secretion and membrane protein insertion in Escherichia coli via the homologous SecYEG-translocon.