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DNA & Protein Synthesis: Unfolded protein response

What is the unfolded protein response (UPR)?

 

The unfolded protein response (UPR) is a feedback mechanism which prevents unfolded or misfolded proteins accumulating in the lumen of the endoplasmic reticulum (ER). Some misfolding of proteins is normal and the ER can usually identify this, attempt to repair it and if this fails remove the protein through endoplasmic reticulum associated degradation (ERAD). Misfolded protein only activates UPR when it is so widespread that there is a risk of proteins aggregating (sticking together in large clumps).

 

The first resort of the UPR is to try and restore normal cell function. It does this by slowing protein translation to allow time to fix the damage, then it increases production of chaperone proteins which assist in protein folding. If this is unsuccessful the UPR will then activate apoptosis as a last resort causing the cell to die in a programmed way.

 

    Where and when does the UPR happen?

     

    The UPR occurs in the lumen of the endoplasmic reticulum where proteins destined for the secretory pathway are folded and processed. It is a stress response which activates when there is a prolonged production of misfolded and unfolded proteins and the levels of these in the ER accumulate. This is usually due to either mutations in the proteins synthesised, exposure to toxins or deprivation of essential nutrients in the endoplasmic reticulum.

       

          What are the aims of the UPR?

           

          1. To control the production of molecular chaperones e.g. BiP. Molecular chaperones are proteins which aid the correct folding of other proteins.

               

              2. To increase the rate of endoplasmic reticulum associated degradation (ERAD) by activating transcription of genes that encode ERAD associated proteins. As a result, more unfolded or misfolded proteins can be moved out of the ER and into the cytosol where they are degraded by proteosomes.

               

              3. To reduce the translation of proteins via the PERK protein kinase pathway giving the ER time to remove the buildup of misfolded proteins.

               



              IRE1, PERK and ATF6

               

              The three major transducers of ER stress are IRE1, PERK and ATF6. They are controlled by a molecular chaperone BiP, discussed below.

               

              IRE1: A transmembrane protein which is an RNase and a kinase. When ER stress occurs it phosphorylates itself which activates its RNAse activity. This processes mRNA (e.g. XBP1 in mammals to the mature form). XBP1 is a transcription factor which regulates response to ER stress at the level of gene expression. 

               

              PERK: Also an ER transmembrane protein kinase, PERK phosphorylates eIF2α. This causes less translation initiation complexes to form in the cell so less RNA is translated into protein. This reduces the "workload" for the ER. 

               

              ATF6: ATF6 is a transcription factor which in normal conditions is an ER transmembrane protein. When ER stress occurs it is translocated to the Golgi where it is cleaved to give a cytosolic fragment. Like other transcription factors, this is able to translocate to the nucleus and regulate gene transcription. 

               

              1. Controlling the production of molecular chaperones e.g. BiP

                 

                • BiP (binding immunoglobulin protein) is located in the lumen of the ER
                • It binds to newly synthesised proteins to inhibit interactions which would cause them to fold improperly therefore, preventing the proteins from becoming misfolded permanently
                • BiP and other molecular chaperones retain proteins in the ER until they are folded properly. However, it is important to understand that not all proteins fold properly and these proteins have to be degraded outside of the ER (see 2. increasing ERAD)
                • BiP binds to three major ER stress transducers IRE1, PERK and ATF6 which are needed to initiate UPR and keeps them in an inactive state.
                • One theory is that when there are too many misfolded proteins in the ER, BiP dissociates from these receptor proteins and binds to misfolded proteins instead. This leaves the receptor proteins free to signal and initiate UPR.
                • BiP has to be tightly regulated as too much in the ER can cause a decrease in the unfolded protein response and too little of it can cause an abnormally long unfolded protein response
                • The cell is capable of controlling BiP production by upregulating its production at the transcriptional level when too many misfolded proteins have accumulated in the ER causing ER stress. 

                 



                  2. Increasing the rate of ERAD by activating the transcription of relevant genes

                     

                    • Endoplasmic reticulum associated degradation (ERAD) is the process in which proteins which have been misfolded permanently in the ER are translocated to the cytosol where they are degraded by proteasomes.
                    • The misfolded proteins are translocated across a protein channel called Sec61 (see 1 on the diagram).
                    • Ubiquitination then takes place. Ubiquitination is the process by which ubiquitin molecules bind to lysine residues on the misfolded protein.
                    • Many ubiquitin molecules bind to a single protein producing a polyubiquitin complex. This process requires energy from ATP (see 2 on the diagram).
                    • Poly-ubiquitination tags the misfolded protein for degradation.
                    • This complex then finds a 26S proteasome and binds to it forming a ubiquitin-proteasome complex (see 3 on diagram).
                    • Proteolytic enzymes in the proteasome break down the protein. This requires energy in the form of ATP (see 4 on diagram).
                    • Small peptide fragments are produced.
                    • The UPR upregulates components of the ERAD pathway to increase the rate of ERAD and remove unfolded proteins faster.

                     



                      3. Reducing the translation of proteins via the PERK pathway

                       

                      • The UPR reduces the translation of proteins and inhibits protein synthesis in general.
                      • When there is an accumulation of misfolded proteins in the ER and the UPR is activated, the activity of PERK is increased.
                      • PERK phosphorylates the alpha subunit of eIF2alpha which is a translation initiation factor
                      • Phosphorylation of this factor inactivates it resulting in a reduction in the rate of translation.
                      • As a consequence, less proteins will be synthesised reducing ER stress.

                       

                        Apoptosis

                         

                        When the above mechanisms fail to decrease the levels of misfolded proteins in the ER, apoptosis is triggered as the last resort of action. Apoptosis is a type of programmed cell death. It is generally preferable for an organism for cells to die in a controlled rather than an uncontrolled way. This is because it means signalling molecules are not released into the surrounding tissues in an uncontrolled way and phagocytosis by other cells can enable the retrieval of some of the dying cells components and stored energy.

                         

                        As a consequence of ER stress remaining unresolved, apoptosis is triggered and the cell dies in a controlled way before protein aggregation in the ER causes death or abnormal cell behaviour. PERK and IRE1 are both involved in this process. PERK promotes apoptosis by inducing transcription of CHOP (a pro-apoptotic transcription factor) and arrests the cell cycle by repressing translation of cyclins. IRE1 initiates a series signals which lead to activation of the caspase cascade, leading to apoptosis.

                         

                        References

                         

                        1. Liu, C. and Kaufman, R. (2003) The unfolded protein response. Journal of Cell Science. 116:1861-1862.
                        2. Jenner, E. (2012) Peptide folding. Fastbleep.com, Biochemistry Chapter.
                        3. O'Kane, E. (2012) Protein trafficking in eukaryotic cells. Fastbleep.com, Biochemistry Chapter.
                        4. Tsai, Y. and Weissman, A. (2010) The unfolded protein response, degradation from the endoplasmic reticulum and cancer. Genes and Cancer. 1: 764-778.
                        5. Alberts, B. et al (2008) Molecular Biology of the Cell 5th Edition. Garland Science Publishing
                        6. Campbell, N. and Reece, J. (2005) Biology 7th Edition. Pearson Education Inc.
                        7. Schweitzer-Stenner, R. and Uversky, V. (2012) Peptide folding, misfolding and non-folding. Wiley Science Publishing.
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