Structural Biochemistry/Cell Organelles/Endoplasmic Reticulum

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Function[edit]

1. Nuclear semi permeable membrane 2. Nuclear pore 3. Rough endoplasmic reticulum (REM) 4. Smooth endoplasmic reticulum 5. Ribosome attached to REM 6. Macromolecules 7. Transport vesicles 8. Golgi apparatus 9. Cis face of Golgi apparatus 10. Trans face of Golgi apparatus 11. Cisternae of Golgi apparatus 12. Secretory vesicle 13. Cell membrane 14. Fused secretory vesicle releasing contents 15. Cell cytoplasm 16. Extracellular environment

Endoplasmic Reticulum is an extensive membranous network in the eukaryotic cells that is continuous with the outer nuclear membrane and composed of ribosome-studded (rough) and ribosome-free (smooth regions.

The ER is responsible for the manufactures of the membranes and performs many other biosynethic functions.

The endoplasmic reticulum (also known as the ER) is made up of a wide system of membranes that make up over fifty percent of the total membrane in numerous eukaryotic cells, and consists of two sections that have different functions: the smooth endoplasmic reticulum and the rough endoplasmic reticulum. The endoplasmic reticulum consist of a system of membranous tubules and pouches called cisternae, which is held together by the cytoskeleton. The membrane segregates the inside section of the endoplasmic reticulum from the outside section (cytosol) . The internal section is called the lumen cavity, but is also known as the cisternal space. The name “endoplasmic” is defined as “within the cytoplasm,” and the Latin definition for the word “reticulum” is “little net.” The membrane of the endoplasmic reticulum is continuous throughout the nuclear envelope. Because of this, the area between the two membranes of the envelope is also continuous to the cisternal space.

The Endoplasmic Reticulum contains a quality control mechanism called chaperones. These special proteins attach themselves to newly synthesized proteins and assist them in folding into their native conformations. Charperones also keep mis-folded proteins in the ER to be broken down. These incorrectly folded proteins are degraded through the process of ER Associated Degradation (ERAD). These Chaperones can be affected by genetic diseases such as Cystic fibrosis. Cystic fibrosis is a genetic disease that causes the buildup of chaperones and their incorrectly folded proteins in the ER. This disease leads to mucus clogging the lungs and digestive track, which can lead to early death.

The ER Under Stress[edit]

Under stress conditions, the normal function of the ER is impaired, causing the alteration of protein homoestasis and the corresponding accumulation of misfolded proteins in the ER lumen. This condition is known as 'ER stress'. The aglomeration of misfolded and/or unfolded proteins, detonates a dynamic response process called 'unfolded protein response' (UPR), which is the combination of several intracellular signaling pathways that have as a main purpose to reduce the load of misfolded proteins in order to re-establish protein homoestasis, adjusting to the current need in a dynamic fashion. In addition, the UPR carries out the transcriptional induction of genes expressing chaperones, ER-associated degradation (ERAD) components and autophagy regulators to the cytosol and the nucleus. If the upregulation of proteins involved in folding, quality control and trafficking is not enough to balance ER homoestasis, the UPR eliminates the damaged cell by apoptosis.

UPR Sensors[edit]

The UPR (mentioned above) is activated by three stress sensors, that detect the accumulation of misfolded or unfolded proteins in the ER. These sensors are:

  • Inositol-requiring enzyme 1α (IRE1α): It's a type I transmembrane protein with an N-terminal luminal region, a cytosolic kinase and RNase domains. Its activation has been explained by two different proposed models. The first one explains that immunoglobulin-binding protein (BiP), an ER-resident chaperone, inhibits oligomerization of IRE1α by binding to it. However, the aggregation of misfolded proteins causes BiP to dissociate from IRE1α, allowing it to interact with unfolded proteins and thus, to oligomerize. The second model affirms that unfolded proteins interact directly with IRE1α's luminal domain, inducing its oligomerization. These mechanisms are still under debate, although recent research data leads to the idea that both of them operate together in the regulation of IRE1α. The oligomerization process undergone by IRE1α leads to to trans-autophosphorylation of its kinase region which in turn engages its RNase activity. Activated IRE1α launches alternative splicing of the mRNA that encodes the transcription factor X-box binding protein 1 (XBP1), leading to the translation of a more stable spliced form of XBP1 (XBP1s), which in turn upregulates certain UPR-related genes associated with folding, ERAD and quality control.
  • Protein kinase RNA-activated (PKR)-like ER kinase (PERK): It's a type I transmembrane protein that contains both a cytosolic kinase domain and an N-terminal luminal stress sensing domain. Its activation is carried out by similar mechanisms to those of IRE1α. Activated PERK contributes to reducing overload synthesis of proteins in the ER.
  • Activating transcription factor 6 (ATF6): It's is a type II transmembrane protein, which under basal conditions associates with BiP. The accumulation of unfolded proteins initiates the dissociation of ATF6 from BiP, leading to the translocation of monomeric ATF6 to the Golgi and finally to the nucleus, where it upregulates the transcription of ER chaperones and ERAD-related genes.


Four Steps: Adaptation to Apoptosis[edit]

The activation of the UPR Sensors is followed by a Four Step process in which the cell first attempts to repair the stressed ER. If this fails the cell will set itself on a path to apoptosis.

Step One[edit]

The First Step of the process attempts to decrease the amount of unfolded proteins on the ER. This is done by activating the previously introduced Protein kinase RNA-activated (PKR)-like ER kinase, or PERK. PERK directly decreases the amount of all protein synthesis in an effort to halt the production of proteins that are difficult to fold. Additionally Inositol-requiring enzyme 1α (IRE1α) plays a role in this stage by initiating macroautophagy, a process to destroy misfolded proteins to relieve stress on the ER.

Step Two[edit]

The Second Step consists of a focus on the increase in quality of proteins being produced and restoring homeostasis to the ER. This is accomplished by two methods in no particular order. First, transcription factors XBP1s, ATF6f, ATF4, AXP1s, and ATF6f collectively work to folding and degradation capacity by upregulating chaperone activity,protein modification enzymes and the size of both the ER and Golgi. The second part of this step is ATF4 upregulating genes that change the redox state of the ER to provide a better environment for the folding of proteins. Both of these steps work to reduce the amount of misfolding proteins and increasing the precision with which new protiens are constructed to avoid producing more misfolded and unfolded proteins.

Step Three[edit]

The Third Step begins to transition the cell to a state where apoptosis is more likely. This step has two contributing factors, both influenced by a protein called C/EBP homologous protein or CHOP. CHOP, acting downstream of AFT4 changes the redox state of the entire cell so that the cell is out of its natural state and therefore has trouble functioning. Additionally, again after attempts to slow protein synthesis, CHOP increases the protein synthesis of the cell further stressing it. Both of these factors will sensitize the cell to apoptosis.

Step Four[edit]

The Fourth Step is the apoptosis of the cell. When ER stress results in apoptosis, the BCL2 protein family acts to bring the cell to apoptosis; all pro-apoptotic members of this family contain BH1, BH2, and BH3. Specifically, BH3 acts to trigger the activation of BAX and BAK. These then permeablilize the membrane of the cells mitrochonira and release cytochrome c and apoptosome. This moves the cell into a stage of apoptosis.

ER Stress and Illness[edit]

When discussing ER stress and human disease, it is important to recognize that nearly all data that suggests a linkage between ER stress is either correlative or based on in vitro evidence. Correlative evidence is flawed as it is unwise to assume that correlation always implies causation, and in vitro data is flawed as it is an isolated study rather than a comprehensive view of the organism in question.

Tumors and Cancer[edit]

Tumors often cause hypoxia in the tissues they affect. When this occurs, the ER begins a stress response to adapt to the new conditions. Under normal conditions the efforts of the ER stress response would be beneficial to the body, however, the result of the adaptation is that the tumorous cells are allowed to replicate, driving tumor growth rather than the death that would have occurred had ER stress not took place. Scientists believe that the IRE1α-XBP1 pathway involved in this process is a promising target for cancer treatment.

Diabetes[edit]

When glucose levels become excessive, an ER stress response is initiated. This response is thought to suppress insulin gene expression via IRE1α degradation of insulin mRNA. This downregulation of insulin is characteristic in type two diabetes.


The article "Endoplasmic Reticulum Stress and Type 2 Diabetes" by Sung Hoon Back and Randal J. Kaufman, they discuss how, due to certain lifestyles now, type 2 diabetes has vastly increased over the years. Type 2 diabetes is "characterized by increased levels of blood glucose owing to insulin resistance in adipose tissue, muscle, and liver and/or to impaired insulin secretion from pancreatic [beta]-cells." They discuss how beta-cells increase in response to obesity and insulin resistance, but it doesn't last forever and normally begins to fail. It is possible that beta-cell loss could be due to endoplasmic reticulum (ER) stress. Pancreatic beta-cells secrete insulin, which is a "blood glucose-lowering hormone." Proinsulin is synthesized in the ER and then stored in beta-cells until glucose levels increase and then insulin is released. The ER is in charge of many things and requires a certain environment, so when the environment changes, there are several stress responses that occur to try to keep the ER functioning properly. The main stress response is called the unfolded protein response (UPR). UPR includes "expansion of ER size, enhanced folding capacity, reduced protein synthesis through transcriptional and translational controls, and increased clearance of unfolded or misfolded proteins." IF this does not work and the ER does not return to homeostasis, then cell death is normally activated. It is believed that the ER sends signals for apoptosis is the stress is severe and unresolved by UPR. IRE1α can also mediate cell death pathways. ER stress can increase activation of IRE1α which leads to increased splicing of genes leading to cellular dysfunction and beta-cell death.

Neurodegenerative Diseases[edit]

Correlative evidence shows that ER stress markers are present in deceased persons that suffered from Alzheimer's, Parkinson's, ALS, Huntington's, and Creutzfedt-Jabob disease. Scientists recognize however, that the role of ER response will be neurodegenerative diseases will be very difficult to determine.

Final Remarks[edit]

The detailed study of persistent ER stress and the UPR is essential for the complete understanding of certain diseases, such as cancer, diabetes, neurodegenerative diseases (including AAlzheimer’s, Parkinson’s and amyotrophic lateral sclerosis) and inflammation-related diseases. What such diseases have in common is the accumulation of abnormal intracellular and/or extracellular protein aggregations and inclusions. Therefore, by the meticulous study of the enzymatic pathways followed by the UPR stress sensors, new pharmachological interventions could be developed for the mentioned illnesses.

Rough Endoplasmic Reticulum (RER)[edit]

ER NIH.jpg

The description of endoplasmic reticulum (ER) being "rough" comes from the organelle being studded with ribosomes. These ribosomes on the rough ER, are used to synthesize proteins which are destined to either be inserted into the membrane of the cell or transported out of the cell. Differences between ribosomes lying on the ER and those lying in the cytosol stem mainly from the destination the protein that is synthesized by the ribosome. With protein synthesis via ER ribosomes, proteins are synthesized as they normally would through translation of mRNA. After the protein is synthesized, called "posttranslation", it then enters the lumen of the endoplasmic reticulum, the inner compartment of the organelle. Although the secondary structure (i.e. beta sheets and alpha helixes) of the newly-synthesized peptide forms almost instantaneously as it is translated,its tertiary structure does not begin to form until it is in the lumen, where it will begin to fold into its specific 3-D conformation with the aid of chaperone proteins [1]. It is inside the lumen of the rough endoplasmic reticulum that N- and O- linked glycosidic bonds are formed between amino acids and sugars, thus this process is called "glycosylation" and the newly formed protein is called "glycoprotein".

Vesicle Transport[edit]

The endoplasmic reticulum transports the proteins through the use of vesicles. Vesicles are membrane enclosed packages that contain protein. On the surface of the vesicles, there are coating proteins called COPI and COPII. The protein COPII allows the vesicles to be sent the Golgi and the COPII protein allow the vesicles to come back to the Rough ER. From inside the Golgi, proteins are again packaged into vesicles and transported to the cell membrane. Once at the membrane, the vesicle itself merges with and become part of the membrane as it is composed of the same lipid bi-layer. Its protein content either will remain on the membrane if it is to function as a membrane protein or be secreted outside of the cell [1].

Smooth Endoplasmic Reticulum (SER)[edit]

The smooth endoplasmic reticulum, unlike its "rough" counterpart, does not package protein. Instead it carries out various metabolic reactions within the confines of its membrane. Functions range from carbohydrate metabolism, lipid synthesis, and even drug detoxification. Drug detoxification is usually accomplished by adding hydroxyl groups to drug molecules. This makes them more soluble in water and therefore easier to be flushed from the body. Another important function of the smooth endoplasmic reticulum is the storage of calcium ions. This is imperative for muscle contraction. When a muscle cell is stimulated by a nerve impulse, the calcium ions rush into the cytosol and trigger muscle contraction.

Structurally the smooth ER is not continuous with the cell's nuclear envelope. The smooth ER is involved in phospholipid synthesis.

1. http://www.annualreviews.org/doi/full/10.1146/annurev-biochem-062209-093836?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%3dpubmed

Interesting Facts about the Endoplasmic Reticulum[edit]

1. Proteins are folded and modified in this organelle of the cell.

2. The internal volume of lumen in the endoplasmic reticulum (ER) is almost equal to the outside of the cell. This supports the idea that the ER evolved from the cellular membrane.

3.The ER contains an environment that is distinctly different from that of the cytosol in terms of solute and protein composition. This is because this environment is conducive to the ER’s role as a “staging post”; proteins stop at the this organelle to be packaged and modified before exiting the cell. The ER is also used as a checkpoint before the proteins are secreted, checking the protein quality and accuracy in terms of folding and modification before the protein is allowed to exit the cell.

4. How proteins are folded and modified within the ER: When a protein first enters the ER, it is recognized by a signal sequence typically found at the N terminus of the protein. This sequence is recognized by a receptor located on the surface of the ER membrane called signal recognition particular (SPR). The molecule is then direted towards a pore, called the Sec61 translocon, in the membrane of the ER where it is allowed to pass through and into the ER. One interesting fact about protein folding is that when some proteins are passed through the ER, before entering the ER lumen, they are mere polypeptide chains. The folding of the protein actually sometimes happens entirely within the ER lumen! It is therefore important to note that when the polypeptide sequence enters the lumen of the ER and are subject to the modifications of the sidechains of its amino acids, this can alter the way the protein would fold. Thus, these modifications influence folding, and folding sometimes also influences modifications too. There are some exceptions to this, such as transmembrane proteins that have large cytosolic domains. These proteins fold within the lumen and within the cytosol of the cell. Once the protein is fully translocated into the ER lumen, it was finish the folding and modification that it needs. If the protein is part of a multisubunit complex, then the ER will assemble the protein into its native oligomeric structure. Modifications catalyzed by enzymes in the lumen of the ER greatly aid the maturation of the protein and bring it closer to its final desired outcome. Once this is complete, the finished protein is then put into a transport vesicle, which buds off from the ER and continues on its journey to either the golgi apparatus or for secretion. Some proteins, which do not end up being modified correctly can be consequently targeted for destruction and may leave the ER to be degraded by enzymes in the cytosol.

5. The UPR (mentioned above) is activated by three stress sensors, that detect the accumulation of misfolded or unfolded proteins in the ER. These sensors are:

  • Inositol-requiring enzyme 1α (IRE1α): It's a type I transmembrane protein with an N-terminal luminal region, a cytosolic kinase and RNase domains. Its activation has been explained by two different proposed models. The first one explains that immunoglobulin-binding protein (BiP), an ER-resident chaperone, inhibits oligomerization of IRE1α by binding to it. However, the aggregation of misfolded proteins causes BiP to dissociate from IRE1α, allowing it to interact with unfolded proteins and thus, to oligomerize. The second model affirms that unfolded proteins interact directly with IRE1α's luminal domain, inducing its oligomerization. These mechanisms are still under debate, although recent research data leads to the idea that both of them operate together in the regulation of IRE1α. The oligomerization process undergone by IRE1α leads to to trans-autophosphorylation of its kinase region which in turn engages its RNase activity. Activated IRE1α launches alternative splicing of the mRNA that encodes the transcription factor X-box binding protein 1 (XBP1), leading to the translation of a more stable spliced form of XBP1 (XBP1s), which in turn upregulates certain UPR-related genes associated with folding, ERAD and quality control.
  • Protein kinase RNA-activated (PKR)-like ER kinase (PERK): It's a type I transmembrane protein that contains both a cytosolic kinase domain and an N-terminal luminal stress sensing domain. Its activation is carried out by similar mechanisms to those of IRE1α. Activated PERK contributes to reducing overload synthesis of proteins in the ER.
  • Activating transcription factor 6 (ATF6): It's is a type II transmembrane protein, which under basal conditions associates with BiP. The accumulation of unfolded proteins initiates the dissociation of ATF6 from BiP, leading to the translocation of monomeric ATF6 to the Golgi and finally to the nucleus, where it upregulates the transcription of ER chaperones and ERAD-related genes.

References[edit]

Braakman, Ineke, and Neil J. Bulleid. "Protein Folding and Modification in the Mammalian Endoplasmic Reticulum." Annual Review of Biochemistry 80.1 (2010): 110301095147058. Print.

Woehlbier, U., Hetz, C. Modulating stress responses by the UPRosome: A matter of life and death. Trends in Biochemical Sciences, June 2011, Vol. 36, No. 6.

Back, Sung H., Kaufman, Randal J. "Endoplasmic Reticulum Stress and Type 2 Diabetes." Annu. Rev. Biochem. 2012. 81:767–93