Unfolded protein response

Question 1

What is proteostasis?

Answer 1

The state of the dynamic equilibrium in which protein synthesis and folding is balanced with degradation, while allowing for the conformational flexibility necessary for function, thus leading to a healthy proteome

Question 2

Why does the cell experience changes in protein levels?

Answer 2

• Every cell type responds to different external stimuli often by altering the levels of different proteins and the changes in the required levels of transcription/translation/foldases/unfoldases/proteases, even the size of different loci in the cell involved with the response which means altering the number of mitochondria for example or the number of vesicles involved in secretion / endocytosis etc.
• External changes that cause “normal stress” include some of the most basic things such as metabolic changes during fed vs. fasting. Mechanisms of stress response including what is now referred to as the “Unfolded protein response” were initially identified as a result of changes in glucose levels back in the 1970s.

Question 3

What is redundancy?

Answer 3

Breakdown of any of the components of the networks maintaining proteostasis in the cell can be compensated for by upregulated another component – redundancy. External factors such as genetic traits, aging, infection, physiological stress combine to overload the system and the result is devastating: a long list of diseases ranging from neurodegenerative disease, diabetes, cancer, etc.

Question 4

What is the unfolded protein response?

Answer 4

The UPR is a signaling pathway initially characterised in the endoplasmic reticulum which leads the affected cell to respond to an overload of unfolded proteins. Similar mechanisms are being deciphered in mitochondria. It is an example of stress-sensing within cells.

Question 5

What is the effect of energetic differences between unfolded and folded?

Answer 5

Energetic differences between unfolded and folded proteins are quite small so any changes can lead to increased levels of unfolded proteins whereas DNA could be less affected by small changes. Occurs in the ER as the ER regulates more complex proteins due to modifications present in the ER.

Question 6

What is the importance of proteins being unfolded?

Answer 6

Responds to Normal fluctuations in translation / secretory activities including maintaining healthy phospholipid: cholesterol ratio. Abnormal fluctuations occur in cancer and viral infection. UPR may even regulate cell differentiation.

Question 7

What are the three levels of response in UPR mechanism?

Answer 7

1. Short term – reduce protein load
2. Medium term - increase the machinery that deals with it – this includes increasing the size of the ER and the secretory pathway organelles
3. Long term – if 1 and 2 fail, then UPR induces cell death - neurodegeneration

Question 8

What are the three classes of stress inducers?

Answer 8

IRE-1, PERK and ATF-6. These form the 3 arms of the UPR. They are sensors with domains on the ER lumen that SENSE levels of unfolded protein. They signal to other compartments to cause a response.

Question 9

What are the three parts of IRE-1?

Answer 9

lumenal sensing, transmission to cytoplasm, messages sent (the latter corresponding to 3 time steps)
1. “Sensing” unfolded protein causes activation by dimerisation of the ER domains.
2. In turn this causes specific “trans” autophosphoryllation of their cytosolic domains and activation of their specific RNase activity.
3. Messages are sent either directly via the Rnase activity of IRE-1 or indirectly (via poorly characterised mechanisms).

Question 10

What is the downstream response to IRE-1?

Answer 10

Step 1: short-term Indirect – indirect activation of a secondary RNase activity. This causes degradation of mRNAs (non-specifically) and lowers the translational load on the ER

Step 2: medium-term Direct - cleavage of mRNA encoding X-box binding protein (XBP1 mRNA), removes a small intron. The mature re-ligated mRNA has a frameshift and now codes for XBP1s a transcriptional activator. In the absence of activated IRE-1, the unspliced mRNA encodes XBP1u, an inhibitor of the UPR. The original transcriptional load is thus degraded and replaced by UPR genes, mRNA and proteins.

Step 3: long-term and if the above fail Indirect - Eventually, activation of JNK pathways (via TRAF 2 binding to IRE-1) and caspase 12 (leads to cell death).

Question 11

What is the process of PERK?

Answer 11

1. PERK senses unfolded protein in a similar way to IRE-1 (lecture 2), part of this mechanism involves dimerisation of the ER domains. The ER lumenal domains of PERK and IRE-1 are similar and can be swapped in animal models.
2. PERK dimerisation also leads to trans-autophosphoryllation of its cytoplasmic domains.
3. The activated PERK phosphoryllates the alpha domain of eIF2 (eukaryotic translation initiation factor 2) on ser 51. All of PERK’s downstream activity is then carried out via eIF2-P.
   The PERK protein is similar in the ER to IRE-1 but has distinct activities on the cytoplasmic side.

Question 12

What is the downstream response to PERK?

Answer 12

Step 1: eIF2-P inhibits eIF2B, which is a guanine exchange factor (GEF) responsible for recycling eIF2 back to its GTP-bound form. The result of this is inactivation of translation.

Step 2: Under limiting eIF2 activity, translation of the transcription factor ATF4 is increased and transcription is affected: specific UPR genes are transcribed then expressed. NFkB is activated in an unknown (but eIF2 dependent) way and this also induces transcription of specific genes.

Step 3: The gene expressing CHOP is transcribed and has several functions. It activates the transcription of the gene encoding GADD 34 which forms part of a negative feedback mechanism: it dephosphoryllates eIF2-alpha and thus slows further transcription of UPR genes including CHOP. If, despite this negative feedback mechanism, CHOP levels increase, CHOP can stimulate cell death via repression of BCL-2

Question 13

What is the process of ATF-6?

Answer 13

1. ATF6 also contains an unfolded protein sensing domain in the lumen of the ER but in this case, dimerisation does not ensue.
2. Activation of ATF6 induces transportation to the Golgi.
3. In the Golgi, 2 site-specific proteases (S1P and S2P) cleave the cytoplasmic domain of ATF6 which leads to the release of ATF6f (“f” for fragment), the DNA binding portion of ATF6.
   The structure of ATF6 is somewhat different to IRE-1 and PERK but is common to other membrane spanning proteins (e.g. CREBH) which are activated in the Golgi by proteolysis of their cytoplasmic domains.

Question 14

What is the downstream response to ATF-6?

Answer 14

(Step 1: short-term) ATF6 does not have a clear role in the suppression of normal transcription and translation of genes.

Step 2: medium-term ATF6 processing in the Golgi leads to the release of ATF6f, a transcription factor which leads to the activation of specific UPR genes in the nucleus. This is a direct effect. Similar proteins such as CREBH lead to activation of the “acute phase response”, an event which occurs in the liver and leads to inflammation via the secretion of serum proteins (referred to as acute-phase response proteins). This is part of the link between the UPR and the immune system.

(Step 3: long-term) ATF6 has no clear role here either.

Question 15

How were UPR genes identified?

Answer 15

1st observation: Pharmacological and genetic manipulations that increase unfolded protein load in the ER leads to upregulation of chaperone genes (late 1970s).
1. Treat cells with protein folding disrupting agents DTT (breaks disulfide bonds) and tunicamycin (inhibits N-linked glycosylation)
2. mRNA levels in the cell measured by high-density oligonucleotide arrays. (hybridise with mRNAs and deduce levels).
Controls: Compare cells with IRE1 deficiency. Compare new targets with known targets (make a typical or “canonical” response profile).

Question 16

How do you use mRNA extracts to find UPR genes?

Answer 16

Whole mRNA extracts from cells are purified and hybridised against an array of oligos synthesized using combinatorial chemistry methods on a glass slide. The hybridised oligos can be quantified using fluorescence labelling.
Results: New mRNA transcribed within 15 min (step 2) and still present when induction lasts >2 hours. Primes genes are involved with translocation, glycosylation, protein folding, protein degradation, vesicle trafficking, lipid transport, vacuolar protein sorting, cell wall biosynthesis.

Question 17

What is ERAD?

Answer 17

“ER associated protein degradation” and refers to a process in which proteins are exported from the ER into the cytoplasm where they are targeted for degradation by the Ubiquitin-proteasome system. This system which is often abbreviated to UPS (not to be confused with UPR!).

Question 18

How does UPR affect ERAD?

Answer 18

The unfolded protein response therefore not only encodes chaperones but upregulates the degradation of misfolded proteins through ERAD. Experiments blocking one or the other system show that efficient UPR requires ERAD and efficient ERAD requires the UPR. If both are blocked the cell dies. The coordination of these different elements reveals a new degree of regulation and control spanning different protein processing machineries in the cell.

Question 19

What does activation of UPR and ERAD do?

Answer 19

Activation of the UPR and ERAD leads to the degradation of foreign proteins and their presentation on the surface of the cell by the major histocompatibility factor 1 (MHC1). This is because one of the key roles of the proteasome is to produce antigenic peptides for MHC1.

Question 20

What is the relationship between viruses and UPR?

Answer 20

Viruses exploit the secretory system of the cell for the synthesis and secretion of their own proteins. The UPR responds by blocking transcription and translation (step 1) then by synthesizing its own proteins selectively but eventually kills infected cells. Implications for COVID-19 drug research (optional material, end of live session 2 material).

Question 21

What is the role of the UPR?

Answer 21

Part of the UPR is to activate genes involved in the synthesis of phospholipids and membrane components. It is possible that UPR components may respond directly to low levels of phospholipid, possibly high ratios of cholesterol:phospholipid.
The UPR may play a role in cell differentiation into cells with specific secretory functions. It enlarges the ER and activates the synthesis of other vesicles as part of secretory / endocytic / exocytic pathways.

Question 22

What are phenomena that influence the success of protein folding?

Answer 22

• some processes involved in protein folding are slow, such as disulphide exchange or proline isomerisation
• Incomplete domains often will not fold to stable structures, so partially synthesised proteins still attached to the ribosome are aggregation prone. Removal of 13 amino acids from C-terminus of Staph nuclease results in partly folded molecule.
• Protein may not fold stably in absence of components that make its final active state. For example
  o Multimeric proteins require many polypeptide chains to meet in order to make the complete, stable state.
  o Post translational modification
  o Non-protein binding partners
• Cellular protein concentration is of the order of 60 mg/ml in E. coli cytoplasm (1-5mM, depending on protein size). This can be much higher in eukaryotic organelles. As the protein concentration increases, the potential for non-specific, inter-molecular interactions increases. The end result is protein aggregation.
• Cellular stresses like heat shock will rapidly increase the concentration of unfolded protein increasing potential for protein aggregation.

Question 23

What can unsuccessful protein folding cause?

Answer 23

loss of protein functions, waste of energy, aggregates are associated with toxicity like prion diseases and they are very stable so it is hard to recover functional protein from them.
Sometimes protein unfolding is required for cellular function such as protein degradation or transport into organelles.