Effects of ER stress on the proteome of mammalian cells

The interplay between the secretory pathway and secretory proteins could be described in terms of supply and demand. An intricate sensing and response mechanism must ensure that when demand rises (when more proteins line up for secretion), the supply increases too (the secretory machinery expands). Such sensing and response mechanisms are referred to as the Unfolded Protein Response (UPR) pathways. In response to an increase in folding load, the UPR pathways seem to have a dual objective: the increase of folding capacity and the reduction of folding load. Induction of the UPR pathways can be provoked under a variety of pathophysiological conditions such as ischemia, hypoxia or viral infection. UPR can also be raised through employment of drugs that interfere with productive folding in the ER such as tunicamycin (which prevents N-glycosylation) or DTT (which abrogates disulfide bond formation).

In yeast, the UPR is transduced via Ire1p and Hac1p. Ire1p has an ER stress ‘sensor’ domain in the ER lumen and an effector domain on the cytosolic side of the membrane. The effector domain has kinase and an endoribonuclease activity. ER stress causes Ire1p to oligomerize and to trans-autophosphorylate its effector domain. As a result, the endoribonuclease module is activated and effectuates splicing of the HAC1 mRNA. Upon religation of its transcript Hac1p is synthesized and serves as trans-activator of ER resident folding factors and components of the machinery that mediates ER associated degradation (ERAD).

The mammalian UPR is transduced along several pathways. Conserved between yeast and man is the Ire1 pathway. XBP-1 serves as downstream effector of Ire1 proteins in a similar fashion as Hac1p in yeast. Central to a second UPR pathway stand the ATF6 proteins. They consist of a ‘sensor’ domain in the ER lumen similar to Ire1, but the cytosolic domain is different. Upon ER stress, the ATF6 proteins travel to the Golgi apparatus where their cytosolic domains are cleaved off by Site 1 and Site 2 proteases. The cytosolic domains subsequently localize to the nucleus to serve as trans-activator of UPR target genes.

The key player of a third UPR pathway is PERK. Similar to Ire1, PERK has a sensor domain in the ER lumen and a kinase effector domain in the cytosol, and is activated through oligomerization and trans-autophosphorylation. Different from Ire1, PERK phosphorylates the a-subunit of eukaryotic translation initiation factor 2 (eIF2a), thereby abrogating the recruitment of initiator methionyl-tRNA to the ribosome. In that way, PERK transduces ER stress by attenuation of protein synthesis. This should relieve the ER from further accumulating unfolded load. Paradoxically, translation of transcription factor ATF4 is enhanced when eIF2a is phosphorylated, and ATF4 in turn enhances expression of the UPR target CHOP, which is a proapoptotic transcription factor that potentiates cell death when the detrimental effects of ER stress can no longer be overcome. Most likely, ATF4 also recruits other UPR targets.
Schematic overview of UPR pathways

The UPR pathways may have been unravelled to impressive detail over the past few years, but research efforts were mainly focused at the level of signal transduction and transcription. We investigate the effects of ER stress at the protein level, instead.

• The ER stress crew
  Ineke Braakman
  Eelco van Anken (alumnus)
  Viorica Lastun
  Anna Majczak
• Mariano Stornaiuolo