Molecular mechanism of ER stress

Molecular mechanism of ER stress-induced cell death

Eva Szegezdi

The endoplasmic reticulum (ER) is a central organelle of each eukaryotic cell as the place of lipid synthesis, protein folding and protein maturation. Proteins of the plasma membrane, secreted proteins as well as proteins of the Golgi apparatus and lysosomes fold into their tertiary and quaternary structure in the ER. The ER is the major signal transducing organelle that senses and responds to changes of the homeostasis. Conditions interfering with the function of ER are collectively called ER stress. ER stress is induced by accumulation of unfolded protein aggregates (unfolded protein response, UPR) or by excessive protein traffic usually caused by viral infection (ER overload response, EOR).

The ER stress possesses its own signalling pathways. UPR - caused by formation of unfolded protein aggregates - uses an evolutionarily conserved signalling pathway during which the signal of unfolded proteins activates a set of ER-located sensors:

1. The PERK kinase (or eIF2 kinase): it phosphorylates the a subunit of the eIF2 (eukaryotic initiation factor 2) that leads to attenuation of general protein synthesis.

2. A special kinase family, carrying an endonuclease activity (involving Ire1a and b) is also activated and in mammals it initiates gene expressional changes by processing the mRNA of the transcription factor XBP1. XBP1 then translocates to the nucleus and induces a wide range of ER located chaperones, involving the members of the Grp (glucose regulated protein) family (Grp78, Grp94). These chaperones play a role in protecting the cell probably by:

   • preventing protein aggregation (keeping the unfolded proteins in a folding-competent state)

   • binding and keeping Ca ² in the ER lumen.

3. Beside PERK and Ire1, there is a third ER-stress sensor molecule, called ATF6. ATF6 is a transcription factor and it is essential for XBP1 mRNA expression and chaperone induction upon ER stress.

In summary, stress conditions lead to decreased rate of protein translation to prevent further accumulation of unfolded proteins. Simultaneously, transcription factors are activated in order to induce the expression of ER-resident chaperones to deal with accumulated protein aggregates. Furthermore, the ER-specific protein-degrading apparatus becomes activated and eliminates denatured proteins. This co-ordinated response halts the build up of proteins, allows time for the elimination of unfolded proteins, and re-establishes cellular homeostasis. However, if the stress cannot be resolved, the cell dies by apoptosis.

 At present, the exact signalling mechanism underlying ER stress-induced apoptosis is poorly understood. Although, it is clear that two main pathways, a transcription factor and a caspase-dependent one, are activated. In the former, Ire1 is thought to serve a pro-apoptotic function possibly through upregulation of the transcription factor, GADD153/CHOP, which may in turn amplify the pro-apoptotic signal by altering the balance between Bcl-2 and Bax.

ER stress can also initiate cell death through activation of caspases. This pathway is independent from mitochondria and death receptors and thought to be mediated by caspase-12. In murine cells several studies examined the activation and function of caspase-12 in response to ER stress-mediated apoptosis. Caspase-12 ^-/- mice were found to be resistant to Ab peptide-induced apoptosis in an Alzheimer’s disease model. The report of Morishima et al. suggested that upon activation, caspase-12 translocates from the ER to the cytosol where it directly cleaves pro-caspase-9 which in turn activates the effector caspase, caspase-3.

Although many steps of the signalling pathway have been elucidated, several questions still arise:

1. The exact inhibitory mechanism by which Grp78 and Grp94 can protect cells, is a matter of debate. There is accumulating information about the interaction between these stress proteins and signalling molecules of the ER stress. In resting cells the Grp78/Bip chaperone protein is thought to be associated with ER stress sensors, such as PERK and Ire1 and prevents their activation.

Our aim is to further investigate the roles of Grp78 and Grp94 in ER stress and to decide whether Grp78 and Grp94 protect against ER stress induced cell death of cardiac myocytes. It is most likely that these stress proteins play a significant role during UPR but the signalling events that enable their protective effect are still to be further examined. Another important question is which ER proteins interact with Grp78 and Grp94 and what type of interaction it is. Our special focus is on cardiac myocytes with which we plan a series of experiments to disclose the connections and the similarities between ER-stress induced apoptosis and clinically important disorders that may trigger similar mechanisms of action in cardiac myocytes e.g. ischemia.

1. The mechanism of pro-caspase-12 activation is questioned and its substrates have not been identified. Therefore, it is a matter of debate, whether caspase-12 is an initiator of an ER stress-specific novel caspase cascade and if yes, what other caspases are involved.

Our goal is to delineate the molecular events of ER stress induced cell death. Our studies involve the mechanism of pro-caspase-12 activation, particularly the involvement of the Ca ² activated cystein protease calpains, and also initiator and effector caspases. We also examine the downstream events of caspase-12 activation, and the necessity and sufficiency of caspase-12 activation to complete the ER stress-induced death programme. We aim to describe the caspase cascade activated by caspase-12 and to elucidate whether ER stress mediated processes can converge into the intrinsic, mitochondrial pathway of apoptosis.

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