**3. Biological characterization of endoplasmic reticulum (ER) and ER stress**

#### **3.1. Endoplasmic reticulum**

synthesized as preproinsulin with a signal peptide in the ribosomes of the rough endoplas‐ mic reticulum. Preproinsulin is translocated into ER lumen by interaction of signal peptide with signal recognition particle on the ER membrane. Preproinsulin is converted to proinsu‐ lin by removing the signal peptide forming three disulfide bonds in the ER. Proinsulin is then translocated into Golgi apparatus and packaged into secretory granules that are close to the cell membrane. In the secretory granules, proinsulin is cleaved into equal amounts of insulin and C-peptide (Figure 2). Insulin is accumulated and stored in the secretory gran‐ ules. When the β cell is appropriately stimulated, insulin is secreted from the cell by exocy‐ tosis (11). As the major site for protein synthesis, ER plays an important role in insulin biosynthesis. To fulfill the requirement for secreting large amount of insulin, the pancreatic β cells are equipped with highly developed ER, leading to the vulnerability of β cell to ER stress (12). In type 1 diabetes, the loss of β cell increases the burden of insulin secretion on the residual β cells. On the on hand, this compensated action is beneficial for the control of blood glucose. On the other hand, it also increases the ER burden of residual β cells, which

**Figure 2. Biosynthesis of insulin in β cell.** In the ribosomes of rough endoplasmic reticulum, insulin is first synthe‐ sized as a precursor, preproinsulin. Preproinsulin has a signal peptide that directs it to translocate into ER lumen by interacting with signal recognition particle on the ER membrane. In ER lumen, preproinsulin is converted to proinsulin by removing the signal peptide and forming three disulfide bonds. Proinsulin is then translocated into Golgi apparatus and packaged into secretory granules where it is cleaved into equal amounts of insulin and C-peptide. After synthesis, insulin is stored in the secretory granules and secreted from the cell until the β cell is appropriately stimulated.

further exacerbates β cell death.

198 Type 1 Diabetes

Endoplasmic Reticulum (ER) is an organelle of eukaryotic cells that is responsible for the fa‐ cilitation of protein folding and assembly (13-15), manufacture of the membranes(16), bio‐ synthesis of lipid and sterol, storage of intracellular Ca2+, and transport of synthesized proteins in cisternae.It is a membranous network of tubules, vesicles, and cisternae that are interconnected by the cytoskeleton.The ER is well developed in endocrine cells such as β cell in which large amounts of secretory proteins are synthesized.

ER is categorized into two types: rough endoplasmic reticulum (RER) and smooth endoplas‐ mic reticulum (SER). As featured by its name, RER looks bumpy and rough under a micro‐ scope due to the ribosomes on the outer surfaces of the cisternae. RER is in charge for protein synthesis. The newly synthesized proteins are folded into 3-dimensional structure in RER and sent to Golgi complex or membrane via small vesicles. In contrast, SER appears to have a smooth surface under the microscope as it does not have ribosomes on its cisternae. SER is responsible for the synthesis of lipids and steroids, regulation of calcium concentra‐ tion, attachment of receptors on cell membrane proteins, and detoxification of drugs. It is found commonly in places such as in the liver and muscle. It is important for the liver to detoxify poisonous substances. Sarcoplasmic reticulum is a special type of SER. It is found in smooth and striated muscle, and is important for the regulation of calcium levels. It se‐ questers a large store of calcium and releases them when the muscle cell is stimulated.

#### **3.2. Unfolded protein response and ER stress**

ER stress is defined as the cellular responses to the disturbances of normal function of ER. The most common cause of ER stress is protein mis-folding. ER is the place where newly produced proteins fold into 3-dimensional conformation which is essential for their biologi‐ cal function. The sensitive folding environment could be disturbed by a variety of pathologi‐ cal insults like environmental toxins, viral infection, and inflammation. In addition to pathological insults, it can also be induce by many physiological processes such as overload‐ ed protein biosynthesis on ER, For example, in case of type 1 diabetes, increased insulin syn‐ thesis in residual β cell exceeds the folding capacity of ER, resulting in the accumulation of unfolded insulin. The accumulation of unfolded or mis-folded proteins in the ER leads a protective pathway to restore ER function, termed as unfolded protein response (UPR).

Protein folding requires a serial of ER-resident protein folding machinery. A special type of proteins called chaperones is used as a quality control mechanism in the ER. As the major mechanisms to promote protein folding, chaperones assist protein folding by interacting with the newly synthesized proteins.In addition,chaperones also help to break down un‐ folded or incorrectly folded proteins in the ER via a process called ER associated degrada‐ tion.The monitoring mechanism ensures the correct protein folding in the ER. The unfolded proteins usually have a higher number of hydrophobic surface patches than that of proteins with native conformation (17). Thus, unfolded proteins are prone to aggregate with each other in a crowed environment and directed to degradative pathway (18). Molecular chaper‐ ones in the ER preferentially interact with hydrophobic surface patches on unfolded pro‐ teins and create a private folding environment by preventing unfolded proteins from interaction and aggregation with other unfolded proteins. In addition, the concentration of Ca2+ in ER also impairs protein folding by inhibiting the activity of ER-resident chaperones and foldases (19-22). ER is the major site for Ca2+ storage in mammalian cells. The concentra‐ tion of Ca2+ in ER is thousands times higher than that in the cytosol (23). Most chaperones and foldases in ER are vigorous Ca2+ binding proteins. Their activity, therefore, is affected by the concentration of Ca2+ in ER.

sol and N-terminal in the ER lumen. The N-terminal is usually engaged by an ER resident chaperone BiP (Grp78) to avoid aggregation. When unfolded proteins accumulate in ER, chaperons are occupied by unfolded proteins and release those transmembrane signaling proteins. There are three axes of signals that are initiated by the pancreatic endoplasmic re‐ ticulum kinase (PERK), the inositol-requiring enzyme 1 (IRE1), and the activating transcrip‐ tion factor 6 (ATF6) respectively. The release of these proteins from BiP triggers UPR and ER

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**Figure 3. UPR signal pathways.** Under normal condition, PERK, IRE1, and ATF6 binding to the ER chaperone BiP to remain inactive state. Upon the accumulation of unfolded proteins, BiP preferentially binds to the unfolded proteins, leading to the release of PERK, IRE1, and ATF6. PERK becomes oligomerized and activated once released from BiP, and subsequently phosphorylates eIF2α. The phosphorylation of eIF2α results in the suppression of the overall transcrip‐ tion of mRNAs and selectively enhanced transcription of genes implicated in UPR such as the ATF4 mRNA. Similar to PERK, IRE1 is dimerized and activated after released from BiP. Activated IRE1 induces XBP-1 by enhancing the splicing of its mRNA. XBP-1 enhances UPR by regulating the transcription of its target genes. The detachment of ATF6 from BiP results in the translocation of ATF6 to the Golgi apparatus and cleavage of ATF6. Cleaved ATF6 then translocates into

*PERK/eIF2α/ATF4 axis:* PERK is a type I transmembrane Ser/Thr protein kinase uniquely present in ER. In response to ER stress, the binding of unfolded proteins to BiP leads to the

the nucleus and initiates the transcription of target genes.

stress (Figure 3).

Exhaustion of the protein folding machineries or insufficient energy supply increases the ac‐ cumulation of unfolded or mis-folded proteins in ER, which is responsible for the activation of UPR. UPR is a protective mechanism by which it monitors and maintains the homeostasis of ER. Various physiological and pathological insults such as increased protein synthesis, failure of posttranslational modifications, nutrient/glucose starvation, hypoxia, and altera‐ tions in calcium homeostasis, can result in the accumulation of unfolded or mis-folded pro‐ teins in ER which further causes ER stress (24).For example, altered expression of antithrombin III (25;26) or blood coagulation factor VIII (27;28), may result in the exhaustion of protein folding machinery and thus induces UPR. Some physiological processes such as the differentiation of B lymphocytes into plasma cells along with the development of highly specialized secretory capacity can also cause unfolded protein accumulation and activate UPR (29-31). In response to those physiological and pathological insults, cells initiate UPR process to get rid of the unfolded or mis-folded proteins. For instance, UPR can increase the folding capacity by up-regulating ER chaperones and foldases, as well as attenuate the bio‐ synthetic burden through down-regulating the expression of secreted proteins (32-34). In addition, UPR also eliminates unfolded or mis-folded proteins by activating ER associated degradation process (35-37). However, once the stress is beyond the compensatory capacity of UPR, the cells would undergo apoptosis. As such, UPR and ER stress are reported to be implicated in a variety of pathological processes, including diabetes, neurodegenerative dis‐ eases, pathogenic infections, atherosclerosis, and ischemia (24;38).

In addition to protein folding, a variety of post-translational modifications including Nlinked glycosylation, disulfide bond formation, lipidation, hydroxylation, and oligomeriza‐ tion, occur in ER. Disruption of those post-translational modifications can also result in the accumulation of incorrectly folded proteins and thereby induce UPR or ER stress. For exam‐ ple, glucose deprivation impairs the process for N-linked protein glycosylation and thus leads to ER stress (39).

#### **3.3. ER stress pathways**

As a protective mechanism during ER stress, UPR initiates a variety of process to ensure the homeostasis of ER. UPR can be mediated by three major pathways, which are initiated by the three transmembrane signaling proteins located on the ER membrane. Those transmem‐ brane proteins function as a bridge linking cytosol and ER with their C-terminal in the cyto‐ sol and N-terminal in the ER lumen. The N-terminal is usually engaged by an ER resident chaperone BiP (Grp78) to avoid aggregation. When unfolded proteins accumulate in ER, chaperons are occupied by unfolded proteins and release those transmembrane signaling proteins. There are three axes of signals that are initiated by the pancreatic endoplasmic re‐ ticulum kinase (PERK), the inositol-requiring enzyme 1 (IRE1), and the activating transcrip‐ tion factor 6 (ATF6) respectively. The release of these proteins from BiP triggers UPR and ER stress (Figure 3).

with native conformation (17). Thus, unfolded proteins are prone to aggregate with each other in a crowed environment and directed to degradative pathway (18). Molecular chaper‐ ones in the ER preferentially interact with hydrophobic surface patches on unfolded pro‐ teins and create a private folding environment by preventing unfolded proteins from interaction and aggregation with other unfolded proteins. In addition, the concentration of Ca2+ in ER also impairs protein folding by inhibiting the activity of ER-resident chaperones and foldases (19-22). ER is the major site for Ca2+ storage in mammalian cells. The concentra‐ tion of Ca2+ in ER is thousands times higher than that in the cytosol (23). Most chaperones and foldases in ER are vigorous Ca2+ binding proteins. Their activity, therefore, is affected by

Exhaustion of the protein folding machineries or insufficient energy supply increases the ac‐ cumulation of unfolded or mis-folded proteins in ER, which is responsible for the activation of UPR. UPR is a protective mechanism by which it monitors and maintains the homeostasis of ER. Various physiological and pathological insults such as increased protein synthesis, failure of posttranslational modifications, nutrient/glucose starvation, hypoxia, and altera‐ tions in calcium homeostasis, can result in the accumulation of unfolded or mis-folded pro‐ teins in ER which further causes ER stress (24).For example, altered expression of antithrombin III (25;26) or blood coagulation factor VIII (27;28), may result in the exhaustion of protein folding machinery and thus induces UPR. Some physiological processes such as the differentiation of B lymphocytes into plasma cells along with the development of highly specialized secretory capacity can also cause unfolded protein accumulation and activate UPR (29-31). In response to those physiological and pathological insults, cells initiate UPR process to get rid of the unfolded or mis-folded proteins. For instance, UPR can increase the folding capacity by up-regulating ER chaperones and foldases, as well as attenuate the bio‐ synthetic burden through down-regulating the expression of secreted proteins (32-34). In addition, UPR also eliminates unfolded or mis-folded proteins by activating ER associated degradation process (35-37). However, once the stress is beyond the compensatory capacity of UPR, the cells would undergo apoptosis. As such, UPR and ER stress are reported to be implicated in a variety of pathological processes, including diabetes, neurodegenerative dis‐

In addition to protein folding, a variety of post-translational modifications including Nlinked glycosylation, disulfide bond formation, lipidation, hydroxylation, and oligomeriza‐ tion, occur in ER. Disruption of those post-translational modifications can also result in the accumulation of incorrectly folded proteins and thereby induce UPR or ER stress. For exam‐ ple, glucose deprivation impairs the process for N-linked protein glycosylation and thus

As a protective mechanism during ER stress, UPR initiates a variety of process to ensure the homeostasis of ER. UPR can be mediated by three major pathways, which are initiated by the three transmembrane signaling proteins located on the ER membrane. Those transmem‐ brane proteins function as a bridge linking cytosol and ER with their C-terminal in the cyto‐

eases, pathogenic infections, atherosclerosis, and ischemia (24;38).

the concentration of Ca2+ in ER.

200 Type 1 Diabetes

leads to ER stress (39).

**3.3. ER stress pathways**

**Figure 3. UPR signal pathways.** Under normal condition, PERK, IRE1, and ATF6 binding to the ER chaperone BiP to remain inactive state. Upon the accumulation of unfolded proteins, BiP preferentially binds to the unfolded proteins, leading to the release of PERK, IRE1, and ATF6. PERK becomes oligomerized and activated once released from BiP, and subsequently phosphorylates eIF2α. The phosphorylation of eIF2α results in the suppression of the overall transcrip‐ tion of mRNAs and selectively enhanced transcription of genes implicated in UPR such as the ATF4 mRNA. Similar to PERK, IRE1 is dimerized and activated after released from BiP. Activated IRE1 induces XBP-1 by enhancing the splicing of its mRNA. XBP-1 enhances UPR by regulating the transcription of its target genes. The detachment of ATF6 from BiP results in the translocation of ATF6 to the Golgi apparatus and cleavage of ATF6. Cleaved ATF6 then translocates into the nucleus and initiates the transcription of target genes.

*PERK/eIF2α/ATF4 axis:* PERK is a type I transmembrane Ser/Thr protein kinase uniquely present in ER. In response to ER stress, the binding of unfolded proteins to BiP leads to the release of PERK from BiP. Once released from BiP, PERK becomes oligomerized and auto‐ phosphorylated. As a result, PERK inactivates eukaryotic initiation factor 2α (eIF2α) by the phosphorylation of Ser51 to inhibit mRNA translation and protein load on ER (34;40). In ad‐ dition, phosphorylated eIF2α also promotes the expression of stress-induced genes includ‐ ing the transcription factors ATF4 and CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) (41). Deficiency of PERK results in an abnormally elevated protein synthesis in response to the accumulation of unfolded proteins in ER.

protein, the two critical factors implicated in innate immune responses. Like ATF6, CREBH is an ER-membrane-bound protein. In response to ER stress, CREBH release an N-terminal fragment and transit to nucleus to regulate the expression of target genes. Innate immune response, in turn, regulates the expression of CREBH through inflammatory cytokines such as IL-1β and IL-6 (60). The development of dendritic cells, the major innate immune cells, is also regulated by ER stress response (61). High levels of mRNA splicing for XBP-1 are found in dendritic cell, and mice deficient in XBP-1 show defective differentiation of dendritic cell.

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 CD11c+ ) are decreased by >50%. Dendritic cells deficient for XBP-1 are vulnerable to ER stress-in‐ duced apoptosis (61). Moreover, the secretion of inflammatory cytokine IL-23 by dendritic cell also involves ER stress response. CHOP, a UPR mediator, can directly bind to the *IL-23* gene and regulate its transcription. ER stress combined with Toll-like receptor (TLR) ago‐ nists was found to markedly increase the mRNA of IL-23 p19 subunit and the secretion of IL-23, while knockdown of CHOP suppressed the induction of IL-23 by ER stress and TLR

The association of ER stress with innate immune response is confirmed in many disease models. Richardson and coworkers reported that innate immune response induced by *P. aeruginosa* infection causes ER stress in *C. elegans*, and loss-of-function mutations of XBP-1 lead to larval lethality (63). In consistent with that, polymorphisms of *XBP-1* gene were found to be associated with Crohn's disease and ulcerative colitis in humans (64), the two autoimmune diseases share similar properties with type 1 diabetes. Lack of XBP-1 in intesti‐ nal epithelial cells may induce Paneth cell dysfunction which further results in impaired

In addition to IRE1/XBP-1 axis, PERK/eIF2α/ATF4 axis of UPR is also associated with innate response. TLR signaling, the most important innate signaling pathway, can induce selective suppression of the PERK/eIF2α/ATF-4/CHOP axis of UPR pathway (65). The activation of TLR decreases eIF2α-induced ATF4 translation. For instance, pretreatment of LPS, an ago‐ nist for TLR4, attenuated ATF4/CHOP signaling and prevented systemic ER stress-induced apoptosis in macrophages, renal tubule cells, and hepatocytes (65). In contrast, loss of Toll-IL-1R-containing adaptor inducing IFN-β (TRIF), an important adapter for TLR signaling, abrogated the protective effect of LPS on renal dysfunction and hepatosteatosis induced by ER stress, suggesting that TLR signaling suppresses ATF4/CHOP via a TRIF-dependent

The presence of β cell specific autoantibodies is a marker for autoimmune diabetes (66). IRE1/XBP1 axis is required for the differentiation of antibody-producing B lymphocytes. IRE1 is necessary for the Ig gene rearrangement, production of B cell receptors, and lympho‐ poiesis. The expression multiple UPR components including BiP, GRP94, and XBP-1 is up-

mucosal defense to *Listeria monocytogenes* and increased sensitivity to colitis (64).

**4.2. ER stress and adaptive immune response**

Both conventional (CD11b+

signaling (62).

pathway (65).

) and plasmacytoid dendritic cells (B220+

CD11c+

*IRE1/XBP-1 axis:* IRE1 is another axis of signal involved in UPR. There are 2 isoforms of IRE1: IRE1α and IRE1β. IRE1α is expressed in most cells and tissues, while IRE1β is restrict‐ ed in intestinal epithelial cells (42;43). Once disassociated with BiP, IRE1 becomes activated. Activated IRE1 possesses endoribonuclease activity and cleaves 26 nucleotides from the mRNA encoding X-box binding protein-1 (XBP-1), resulting in the increased production of XBP-1 (44). XBP-1 is a transcriptional factor belonging to basic leucine zipper transcription factorfamily. It heterodimerizes with NF-Y and enhances gene transcription by binding to the ER stress enhancer and unfolded protein response element in the promoters of targeted genes involved in ER expansion, protein maturation, folding and export from the ER, and degradation of mis-folded proteins (44-49). In addition, IRE1α also mediates the degradation of ER-targeted mRNAs, thus decreasing the ER burden (50).

*ATF6 axis:* The third axis of ER stress signal is mediated by ATF6. Unlike PERK and IRE1 which oligomerize upon UPR, ATF6 translocates into the Golgi apparatus after released from BiP. The transmembrane domain is then cleaved in the Golgi apparatus (51). The 50 kDa cleaved ATF6 is relocated into the nucleus where it binds to the ER stress response ele‐ ment CCAAT(N)9CCACG to regulate the expression of targeted genes. For example, once released from the ER membrane, ATF6 enhances the transcription of XBP-1 mRNA which is further regulated by IRE1 (44). In addition, ATF6 also increases the expression of the two major chaperon systems in the ER: calnexin/calreticulin and BiP/GRP94 (44;52;53).
