I- ER and the canonical unfolded protein response (UPR)

In eukaryotic cells, most secreted and transmembrane proteins fold and mature in the lumen of the ER. Their flux is variable since it can change rapidly in response to programs of cell differentiation, environmental conditions, and the physiological state of the cell. The intracellular pathway that regulates ER homeostasis in conditions of stress is the unfolded protein response (UPR) which contains several steps (Figure 1).

Figure 1. Pathways of protein misfolding that lead to cell death. Reproduced by permission from ref 3. © Wiley-Blackwell, 2010.

Figure 1. Pathways of protein misfolding that lead to cell death. Reproduced by permission from ref 3. © Wiley-Blackwell, 2010.

First, it leads to a reduction in the protein load that enters the ER. This is a transient adaptation that is achieved by lowering protein synthesis and translocation into the ER. Second, it stimulates an increase in the capacity of the ER to handle unfolded proteins. This is a longer-term adaptation that entails transcriptional activation of UPR target genes, including those that function as part of the ER protein-folding machinery.

If homeostasis cannot be re-established, then a third mechanism, cell death, is triggered, presumably to protect the organism from rogue cells that display misfolded proteins (Figure 1).3,4 Three different classes of ER stress transducers have been identified: PERK, IRE1, and ATF6. In a well-functioning and ‘‘stress-free’’ ER,
these three transmembrane proteins are bound by a chaperone, BiP/GRP78, in their intralumenal domains and rendered inactive (Figure 2). Accumulation of improperly folded proteins and increased protein cargo in the ER results in the recruitment of BiP away from these UPR sensors which are thus activated.

Figure 2. Signaling the unfolded protein response. Reproduced by permission from ref 3. © Wiley-Blackwell, 2010.

Figure 2. Signaling the unfolded protein response. Reproduced by permission from ref 3. © Wiley-Blackwell, 2010.

FOCUS –Activation of IRE1 and ATF6: the transcriptional pathway

Activation of the ATF6 branch of the UPR requires its translocation to the Golgi apparatus where it is processed by the serine protease site-1 protease (S1P) and the metalloprotease site-2 protease (S2P) to produce an active transcription factor (Figure 2).

When activated, ATF6 moves to the nucleus to stimulate the expression of genes containing ER stress elements (ERSE-I, -II), UPR elements (UPRE), and cAMP response elements (CRE) in their promoters. The recent identification of a number of ATF6-related proteins suggests that the traditional model of a three-armed UPR may not be complete. To date, five proteins—Luman (CREB3), Oasis (CREB3L1), BBF2H7 (CREB3L2), CREBH (CREB3L3), and Tisp40 (CREB4, CREB3L4)—share a region of high sequence similarity with ATF6. However, differences in activating stimuli, tissue distribution, and response element binding indicate unique roles for each of these factors in regulating the UPR.
The oldest branch of the UPR, in an evolutionary sense, is mediated through the stress-regulated kinase and ribonuclease IRE1 (Figure 2). Periodic increases in glucose, as well as chronic hyperglycemia, activate IRE1 alpha in vivo and in isolated rat islets. The endoribonuclease activity of IRE1 alpha cleaves a 26 base-pair segment from the mRNA of the X-box binding protein-1 (XBP1), creating an alternative message that is translated into the active (or spliced) form of the transcription factor (XBP1s). Mice with XBP1 haploinsufficiency have significant increased body weight, increased phosphorylation of PERK, IRE1 alpha and also increased JNK (c-Jun N-terminal kinase) activity coupled with a loss of insulin sensitivity in their liver and adipose tissues. They succumb to ER stress and develop hyperinsulinemia, hyperglycemia, and impaired glucose and insulin tolerance compared with wild-type animals.2 Thus, XBP1s activates one of the major pathways for enhancing the folding capacity of the ER and for dealing with ER stress. Indeed, XBP1s, alone or in conjunction with ATF6, launches a transcriptional program to produce chaperones (such as Grp78) and proteins involved in ER biogenesis, phospholipid synthesis, ER associated degradation , and secretion (for example, ER degradation-enhancing a-mannosidase-like protein EDEM).

1. Activation of PERK: the translational pathway

When beta-cells are exposed to hyperglycemia, insulin is secreted from a readily available pool and simultaneously proinsulin biosynthesis is activated. In the ER, proinsulin undergoes protein folding (Sitia Lecture),5 then is delivered to the Golgi apparatus and packaged into secretory granules where converted to insulin before exocytosis (Figure 3).6 In this context of elevated glucose concentrations the increased demand for insulin production leads to ER stress and UPR activation in beta-cells.

Figure 3. Glucose stimulated insulin biosynthesis and secretion in pancreatic -cells. Reprinted by permission from ref 15. © The American Physiological Society, 2009.

Figure 3. Glucose stimulated insulin biosynthesis and secretion in pancreatic beta-cells. Reprinted by permission from ref 15. © The American Physiological Society, 2009.

PERK (Figure 2) is chronologically the first UPR activated pathway. It results in phosphorylation of eIF2 alpha at Ser51, which causes reduced global protein synthesis and a subsequent reduction in the workload of the ER. This branch of the UPR is also linked to broad transcriptional regulation through several distinct mechanisms, including the transcriptional regulation of ribosomal RNA. This results in activation of ATF4 and NF-kB , a master transcription factor with numerous functions including regulation of the inflammatory response. ATF4 is produced through alternative translation and induces expression of genes involved in apoptosis (CHOP, C/EBP homologous protein), ER redox control (ERO1), the negative feedback release of eIF2α inhibition and glucose metabolism (fructose 1,6-bisphosphate; glucokinase, and phosphoenolpyruvate carboxykinase).

Mice and humans harboring loss-of-function mutations in PERK develop diabetes. In mice with conditional homozygous eIF2 alpha Ser51/Ala mutation which prevents eIF2 alpha phosphorylation, mRNA translation is not repressed by ER stress and causes oxidative damage. When a loxP-flanked wild-type eIF2 alpha transgene is expressed into these mice except in pancreatic Beta-cells to prevent eIF2 alpha phosphorylation, a severe diabetic phenotype
is observed. This suggests that phosphorylation of eIF2 alpha coordinately attenuates mRNA translation, prevents oxidative stress, and optimizes ER protein folding to support insulin production in the Beta-cell (Kaufman, Lecture).7
The performance of a model UPR has been compared, both with and without a translation attenuation mechanism, by monitoring 2 variables: (i) the maximal increase in ER unfolded proteins during a response, and (ii) the accumulation of chaperones between two consecutive pulses of stress. It has been evidenced that the translation attenuation mechanism is important for minimizing these two variables when the ER is repeatedly subjected to transient unfolded protein stresses and when it sustains a large flux of secretory pathway proteins which are both conditions encountered physiologically by pancreatic Beta-cells (Tang, Lecture).8

2. The ER-associated degradation pathway

Proteins that fail to achieve their correct conformation may be retained in the ER and degraded by the cytoplasmic proteasome via a process referred to as ER-associated degradation (Figure 1). Soluble ER-associated degradation substrates are retrotranslocated from the ER to the cytosol, or dislocated after selection, and are easily ubiquitinated
and degradated by the 26S proteasome. Membrane ER-associated degradation substrates are likely to involve more elaborate machineries. The ERassociated degradation of two membrane proteins Ste6p* and the epithelial sodium channel (ENaC), are described in Nakatsukasa et al (2008)9 and Buck et al (2010)10 respectively (Brodsky, Lecture). It has been suggested that beta-cells must likely upregulate ER-associated degradation components during ER stress for maintaining normal insulin biosynthesis. Indeed, proteasome inhibition reduces proinsulin biosynthesis.
Furthermore, in the Akita diabetic mouse – characterized by a dominant cysteine96-to-tyrosine missense mutation in the Ins2 gene that leads to disruption of disulfide bond formation between the A and B chain of proinsulin, causing insulin to misfold and accumulate in the ER of the beta-cell – expression of ER-associated degradation components is induced to degrade the misfolded mutant insulin.
Because an ever-increasing number ER-associated degradation substrates have been identified that are associated with diseases, a better definition of the requirements for the ER-associated degradation of these and other substrates may lead to novel opportunities for therapeutic intervention.

“The Stressed Beta-Cell”
I- ER and the canonical unfolded protein response (UPR)
II- When UPR leads to cell death
III- Cellular stress in type 2 diabetes
IV- The mitochondria and cellular stress
V- Therapeutic targeting of ER dysfunction
VI- Conclusions