Navigating the Endoplasmic Reticulum: New Insights and Emerging Concepts

Abstract

Endoplasmic reticulum (ER) is a membrane bound organelle adjacent to the nucleus in eukaryotic cells. It exists in the form of membranous sacs called “cisternae”. It was first discovered by Emilio Veratti in 1902 and later named as ‘Endoplasmic Reticulum’ in 1953 after visualization through electron microscopy. There are two types of endoplasmic reticulum based on the presence of ribosomes i.e., ‘rough’ ER and ‘smooth’ ER. Rough ER is the site for protein synthesis and modification by glycosylation. While the smooth ER is involved in the metabolism of lipids and carbohydrates. Recently, it has been classified on the basis of membrane structure rather than appearance. It physically interconnects with the mitochondria and these sites are named as mitochondria-associated membranes (MAMs) that are crucial for Ca+2 homeostasis. Various mechanisms of ER signaling play vital role in physiology and the onset of disease. A thorough understanding of these mechanisms and their role in physiology and pathophysiology can be applied to develop new ER-targeted therapies.

1. Introduction

Endoplasmic reticulum (ER) is a membrane bound subcellular organelle that appears as a network of tubules in the cytoplasm [1]. It was first observed in chicken fibroblast-like cells with the help of electron microscope. After 10 years, it was named by Porter in 1954 [2]. There are two types of ER based upon the presence or absence of ribosomes on its surface i.e., smooth endoplasmic reticulum SER and rough endoplasmic reticulum RER. Both of these could occur either interconnected or separately in compartments [3]. ER is a crucial site for various functions, including protein synthesis, storage and regulation of Ca²⁺ and lipid and glucose metabolism [3]. These range of functions indicate that ER plays important role in regulating metabolism and cell programming.

ER plays important role in protein synthesis and modification. After synthesis the proteins are translocated across the membrane of ER. Ribosomes attached to the RER membrane are involved in the synthesis of protein [4]. In addition, ER is also crucial for Ca²⁺ signaling and homeostasis. A storage and transport system for Ca²⁺ comprising of Ca²⁺ channels, ATPase pumps, sensors and storage proteins. This chapter highlights a new aspect for ER classification, its functions, ER phagy and role of ER signaling in health and disease [5].

2. Structure

There are two types of ER based upon the presence or absence of ribosomes on its surface i.e., smooth endoplasmic reticulum SER and rough endoplasmic reticulum RER [2]. Both of these could occur either interconnected or separately in compartments. Recently, a new classification has been introduced based on its membrane structure instead of appearance. According to this, ER carry a nuclear envelope, cisternae and three way connected tubules. The ER is interconnected with many organelles in the cell and it is connected with mitochondria through specific sites, called mitochondria-associated membranes (MAMs), that are critical for maintaining Ca²⁺ homeostasis [6]. Its interaction with the plasma membrane is regulated by stromal interaction molecule 1 a protein-like structure and Calcium channel protein 1 [7]. Moreover, SEC22b a vesicle-trafficking also involved in the maintenance of this interaction [8]. ER interaction with endosomes is stabilized by lipid transfer protein 3, the protein involved in cholesterol maintenance in endosomes [9]. ER also play role in autophagy by interacting with endolysosomal system. These intracellular interactions are crucial for the functionality of the cell (Figure 1) [10].

Figure 1.

ER molecular machines and contact sites with other organelles. The ER forms multiple membrane contact sites with other organelles, including the endosomes and lysosomes (through STARD3, STARD3NL, Mdm1; panel 2), the mitochondria (through Mfn-2, Sig-1R, PERK; panel 3), and the PM (through ORAI1, STIM1, Sec22b, VAMP7; panel 4) with various functional implications.

3. Functions

ER is a crucial site for various functions, including protein synthesis, storage and regulation of Ca²⁺ and lipid and glucose metabolism. These range of functions indicate that ER plays important role in regulating metabolism and cell programming [3].

3.1 Protein modification

Secretory and transmembrane proteins are synthesized in ER. Their folding, maturation, quality control and degradation also occur in this organelle. As a result, only properly folded proteins are transported to their destination [4]. Approximately, 30% proteins are cotranslationally delivered to the ER, where chaperones are involved in their folding, packaging and post-translational modification [11]. Protein modification processes includes signal sequence cleavage, formation and breakage of disulfide bonds and lipid conjugation. Misfolded proteins are damaging to normal cellular functions and are tightly monitored. Protein misfolding is a regular process but aggravate during adverse conditions. In ER several regulatory systems ensure correction of misfolded proteins. Terminally misfolded secretory proteins are removed by ER associated degradation (ERAD) process [12]. Initially, proteins are encountered by an ER resident luminal and transmembrane protein machinery, then translocated into cytosol via channel called dislocon [13].

3.2 Lipid synthesis

ER is also crucial for synthesis of membrane constituents, lipid droplets fat accumulation as energy reservoir. Lipid synthesis takes place at membrane interfaces and organelle interaction sites. The ER membrane architecture dynamically altered according to cellular lipid concentration [14]. In order to maintain the cholesterol homeostasis in the body, ER carry a family of protein sensors. It also consists of various enzymes involved in synthesis of sterols and phospholipids (Figure 1) [15].

3.3 ER export

The proteins and lipids synthesized in the ER are delivered to their destined locations through secretory pathways. The export process is tightly controlled to maintain a steady anabolic flux because anomalies in secretion could lead to detrimental consequences to ER structure and functions [16]. Synthesis of ER COPII transport vesicles is crucial to this export process. Apart from COPII mediated transport several other mechanisms have been studied such as nonvesicular. For example, large lipoprotein cargo is transported out in another vesicle or stored in lipid droplets (Figure 1) [17].

3.4 Ca²⁺ homeostasis

Ca²⁺is a metallic ion plays key role as a secondary messenger in various intracellular and extracellular signaling events such as gene expression, translation, protein trafficking, and regulation of other cellular functions [6]. ER is the main reservoir of Ca²⁺ and important for its regulation. Myriad of cellular functions are regulated by Ca²⁺-dependent way in order to maintain the calcium level of entire cell. As a result, tight regulation of both ER and cytoplasmic Ca²⁺ concentration is essential to maintain enhanced intraluminal Ca2+ concentration redox potential as compared to the cytoplasm. A variety of mechanism employed by ER to maintain the concentration of Ca²⁺ inside and outside the membrane [5]. (a) ER membrane ATP-dependent Ca²⁺ pumps for cytosol-to-lumen transport; (b) ER luminal Ca²⁺-binding chaperones for sequestering free Ca²⁺ and (c) ER membrane channels for the regulated release of Ca²⁺ into the cytosol. These mechanisms are supported by a controlled interaction between the ER and other organelles, i.e., PM and the mitochondria.

4. Perturbing ER functions

Disturbance in ER function results in condition commonly known as ‘ER stress’. In order to overcome ER stress and reestablish the homeostasis several adaptation mechanisms are activated inside the cell [18].

4.1 Intrinsic ER perturbations

In certain disease conditions such as cancer, diabetes and neurodegenerative diseases some cellular mechanisms lead to ER stress [19]. In case of cancer, rapid and uncontrolled cellular growth required high protein production that impact the ER system [20]. More specifically, in melanoma that has highest mutation rate the number of mutated proteins is increased that results in ER stress. In chronic myeloid leukemia, an oncoprotein is activated that promotes cell proliferation and disturbs Ca²⁺-dependent apoptotic response [21].

Many neurodegenerative diseases also disturb the ER homeostasis and lead to ER stress. For instance, motor neuron death is the consequence of mutations in the vesicle-associated membrane protein-associated protein B located in ER. It is mediated by the fluctuation of ER stress signaling [22, 23]. On the contrary, pancreatic beta cells involved in insulin production carry a complex and developed ER to control insulin production and use in response to high blood sugar level. Type 1 diabetes is associated with mutation induced ER stress, in this condition beta cells undergo apoptosis and insulin level reduced [24, 25]. Insulin mutation-related ER stress have also been observed in neonatal diabetes [26, 27].

4.2 Extrinsic ER perturbations

4.2.1 Microenvironmental stress

Microenvironmental ER stress occurs in tumorigenic cells. These cells rapidly proliferate that leads to deprivation of nutrients and oxygen in the microenvironment, resulting in local stress accompanied with hypoxia, starvation and acidosis, consequently ER stress, perturbation of protein and lipid biogenesis [28]. Nutrient scarcity, most importantly glucose distress facilitates ER stress by perturbing glycosylation.

4.2.2 Exposure to ER stressors

ER stressors are small molecules that stimulate ER stress mediated by a number of mechanisms [29]. These include molecules such as tunicamycin [30], or 2-deoxyglucose target the N-linked glycosylation of proteins. On the other hand, dithiothreitol prevents protein disulfide bond formation [31]. While Brefeldin A inhibits the ER to-Golgi trafficking, resulting in rapid and reversible inhibition of protein secretion [32].

4.2.3 Exposure to enhancers of ER homeostasis

Some molecules have been reported to stimulate and increase ER stress, such as peptides and proteostasis regulators. A most commonly used 4-phenylbutyric acid (4-PBA) prevents the aggregation of misfolded proteins in the ER [33]. In islet cells, to reduced ER stress a bile acid called Tauroursodeoxycholic acid (TUDCA) is present [34]. FDA has approved TUDCA as a drug for patients diagnosed with primary biliary cirrhosis [35]. The precise mechanism of action of these proteostasis regulators is still unknown.

4.2.4 Temperature

Mammals normal body temperature is 36–37°C necessary for viability and normal bodily functions. Fluctuations in normal body temperature could perturb cellular homeostasis consequently protein denaturation and aggregation [36]. In addition, an acute elevation in temperature, such as heat shock leads to fragmentation of both ER and Golgi [36]. In some mammalian cells and animal models mild elevation in temperature (up to 40°C) cause the development of thermotolerance, which is linked with increased expression of heat shock proteins and ER stressors [37, 38]. Moreover, mild hypothermia (28°C) stimulates mild ER stress in human pluripotent stem cells [39].

4.2.5 Physiological ER stress signaling

In physiological stress conditions, for instance increased secretory level or pathological stress, induced by aggregation of mutant protein could lead to imbalance between requirement for folded protein and ER potential to fold protein, consequently causing ER stress [40]. In response to stress, eukaryotic cells have developed signal transduction pathways, known as unfolded protein response (UPR) (Figure 2). These pathways are regulated by a group of proteins that sense ER stress. These proteins generate stress signals that protect cell from damage or induce cell apoptosis. A strong link between UPR signaling and human diseases has been reported [41].

Figure 2.

A schematic of mammalian unfolded protein response (UPR) signaling. IRE1, PERK, and ATF-6 proteins reside at the ER membrane. In response to ER stress, they initiate a cascade of signal transduction outputs that control cell survival or death.

The main objective of the UPR is to restore homeostasis and inhibit ER stress by employing the following mechanisms: (a) increasing protein folding ability through expression of protein-folding chaperones (b) Inhibition of protein translation by downregulating the ER protein load and facilitating the denaturation of misfolded proteins. But in case of acute stress the UPR stimulates programmed cell death [40]. UPR-mediated cell death is responsible for the onset of many diseases (Table 1), including cancer, type 2 diabetes, neurodegeneration, and atherosclerosis (40).

Disease	Role of ER stress
Alzheimer’s disease	Mutant presenilin 1 induces CHOP
Parkinson’s disease	Accumulation of a substrate of Parkin in the ER activates ER stress
Amyotrophic lateral sclerosis	Mutant SOD1 aggregates and activates ER stress
Type 2 diabetes	Obesity induces ER stress; ATF6 interferes with gluconeogenesis; Free fatty acids and hyperglycemia induce beta cell death through CHOP
Atherosclerosis	Atherosclerosis-relevant stimuli induce macrophage death via CHOP; Oxidized phospholipids, hyperhomocysteinemia, and cholesterol loading induce endothelial and smooth muscle cell death via CHOP
Nonalcoholic fatty liver disease	ER stress induces SREBP-1c
HCV and HBV infection	HCV suppresses IRE1-XBP1 pathway
Alcoholic liver disease	Alcohol induces Grp78 and CHOP

Table 1.

Diseases related to endoplasmic reticulum (ER) stress.

4.2.6 Role of ER in metabolism

The ER plays crucial role in the regulation of metabolic reactions. More specifically, the UPR pathway is involved in the regulation of glycolysis and it was recently reported that a regulatory protein mediates a metabolic decrease upon decrease in glucose level in neurons, suggesting an important role for the UPR as an adaptive response mechanism in relation to energy metabolism [42]. In addition, another signaling molecule known as mTOR maintains protein synthesis.

To maintain lipid content in the body ER plays an important role. Hepatocytes, liver cells carry SER in abundance, because along with protein synthesis, these cells also produce bile acids, cholesterol and phospholipids. Lipid accumulation leads to lipotoxicity, which is the fundamental cause of metabolic diseases. ER carry various enzymes that are crucial for lipid metabolism. Cellular cholesterol level is regulated via signaling pathways. One of these pathways is SCAP/SREBP2, which converts cholesterol into oxysterols and eventually to bile acids. Similarly, level of intracellular fatty acid is controlled by ER by a bunch of enzymes including, desaturases, elongases, and beta oxidation cycles [43].

The UPR has also been reported important for amino acid metabolism. Amino acid synthesis is closely linked with demand for protein biogenesis during ER stress. In response to ER stress, amino acid biosynthetic genes are expressed.

4.2.7 Role of ER stress in age-related diseases

According to current studies, the process of aging and age-related disorders are interlinked with ER stress response [44]. With aging, the normal cellular functions are declined, particularly slow degradation of chaperones, which results in increased aggregation of misfolded proteins [45]. These misfolded proteins accumulated in various organs of the body, such as in case of Alzheimer’s disease (AD), an inflammatory neurodegenerative disease. In AD brains, ER stress responses have been observed, because ER is the site for synthesis of secretory and membranous proteins [46].

Aging disrupts the balance between UPR and pro-apoptotic signaling, leading to reduced protective response against ER-stress signaling [47]. Essential chaperons and enzymes, required for protein folding are functionally damaged with aging [48]. ER structure is also altered with aging. Hinds and McNelly reported dispersion in the highly organized ER cisternae [49].

Autophagy, a process activated by UPR system, remove the aggregation of misfolded proteins. Nevertheless, this process slows down with age, leading to neurodegeneration [50]. ER stress responses have been associated with certain neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, ALS and Huntington’s disease. In neurodegenerative disorders, UPR activity is sustained, while apoptotic pathways are upregulated, encompassed by accumulation of aggregated proteins, hence neuronal death in old age [51].

It has been postulated that ER stress also plays role in the onset of metabolic disorders. For instance, in Type 2 Diabetes, a metabolic disease, caused by insulin resistance is regulated by various ER stress response mechanisms. The two main mechanisms that disrupt insulin activity are interlinked with ER stress [52].

5. Conclusion

ER is a complex and well-organized organelle, crucial for various cellular metabolic functions. ER homeostasis is maintained by a network of signaling pathways, collectively known as ER stress response, in order to deal with genetic, infectious and inflammatory stressors. With age, UPR, ER stress response mechanism, lost its activity thus less efficiently respond to these stressors. This results in onset of various age-related metabolic and neurodegenerative diseases. Advent of ER stress targeted therapeutics, particularly those improving protein folding and efficiency of associated regulatory mechanisms, promoting early detection of misfolded proteins could be useful in preventing and treating age-related disorders discussed in this chapter. Moreover, detection of anomalies in the ER stress response may led to development of therapeutics that could maintain ER homeostasis. This represents so far unexplored approach for disease prevention.

Conflict of interest

The authors declare no conflict of interest.