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Lecture 23

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McGill University
Biology (Sci)
BIOL 300
Siegfried Hekimi

st BIOL 300 October 31 2012 Lecture 23 Dr. Shock Unfolded Protein Response (UPR) • When there is a certain type of stress in the cell and proteins begin to be misfolded, one response would be to shut down translation (the less protein you have, the more likely they are to be folded properly) During translation: • mRNA is translated, normally, into a cytosolic protein • Other proteins are translated from mRNA directly into the ER membrane or lumen • These proteins contains a sequence which targets the ribosome and the protein being synthesized to the ER where to can either be inserted into the membrane, or into the lumen (which is then usually secreted from the cell) • These proteins are sorted to various locations by the Golgi and then end up in the plasma membrane, vesicles or secreted from the cell Various chaperones are required to catalyze the folding reaction • Whenever the cell is under stress, these chaperones are affected to regulate levels of translation and folding The event of imbalance (i.e. unfolded proteins) can be caused by: • Physiological load, in which the needs of the body force the cell to produce more proteins than it is capable of folding • A mutation in the protein, in which case it cannot be folded properly • Heat, which causes partial unfolding of most proteins This illicits an initial short term response to downregulate translation, followed by a long term response which would upregulate transcription and translation of certain genes which are able to help fold proteins correctly (i.e. chaperones, lipids, glycosylation proteins, etc.) 1 st BIOL 300 October 31 2012 Lecture 23 Dr. Shock In vitro, scientists can feed cells with certain chemicals, like tunicamycin, to induce protein unfolding to look at the mechanisms of the UPR • Tunicamycin blocks glycosylation, which is the addition of sugar side chains onto the proteins; many secretory proteins or membrane proteins require these glycosylations to be functional, without them, that can’t fold • Another chemical is called DTT, which is a reducing agent, breaking up disulfide bonds (between cystines) We will first look at the mechanism behind upregulation of gene expression (long term response), which depends on a chaperone protein known as BiP, which is necessary for folding proteins in the ER. In addition to binding unfolded ER proteins, BiP can bind to transmembrane proteins called Ire1. In the cell, there is a balance of BiP binding to either of these two proteins. • If there are a lot of unfolded proteins, very little BiP will be bound to Ire1 because they will mostly be helping fold the proteins • Ire1 will be free; this allows it to dimerize with other free Ire1 proteins which gives it an endonuclease function to “splice” certain mRNAs (doesn’t use splicing machinery) • It makes 2 cuts in an mRNA encoding a transcription factor, which is then ligated by a tRNA ligase protein • This “spliced” mRNA is able to encode a functional transcription factor called Hac1 • Hac1 then goes to the nucleus to transcribe the genes of various chaperone proteins and other components necessary for protein folding under stressful conditions • If there are very little unfolded proteins, when the cell is not under stress, BiP will be bound to most of the Ire1 and this transcription factor will not be made Note: BiP is located in the ER lumen, while Ire1 is an ER transmembrane protein with its endonuclease domain facing the cytosol. Therefore, this Ire-BiP mechanism is actually able to transduce the signal of stress across the ER membrane into the cytosol, and, through Hac1, into the nucleus. This is a long term response, because transcription and translation takes a long time. The short term response involves shutting down translation altogether, which takes place until the long- term response kicks in. 2 st BIOL 300 October 31 2012 Lecture 23 Dr. Shock BiP is once again a “sensor” protein ,which in this case is bound either to a TM protein called PERK, or unfolded proteins. • When there is a lot of unfolded proteins, PERK is left free to dimerize (like Ire1); however, PERK is a kinase, unlike Ire1, which autophosphorylates itself and then phosphorylates target proteins, eIF2, which directly downregulates translation Scientists have made mice which are mutant for PERK (i.e. PERK -/-) • As expected, under normal conditions, the cells should be fine because PERK is not active unless the cell is under stress • In order to see a phenotype is to stress the cells (by the use of tunicamycin in this case) • In this event, the cells die very rapidly because the ER proteins begin to get unfolded • This demonstrates that PERK is requ
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