Sensing Unfolded Proteins

Do cells take joy in tidying? Absolutely! But how can they tell where the messes are?

Any household knows that without cleaning and disposal, nothing else is possible. Things pile up, messes accumulate, everything comes to a standstill. Cells are the same, needing a constant flow of new materials in, trash out, recycling, and cleanups. One of the major kinds of mess in cells is unfolded proteins, which when they accumulate can cause aggregations and diseases like Huntington's, other dementias, atherosclerosis, and inflammation generally. While there are a lot of mechanisms (chaperones, chaperonin cage complexes, proteasomes) dedicated to proper folding, sometimes they are not enough, and excess junk in the form of unfolded proteins builds up. One major response this is the unfolded protein response, or UPR, which is centered at the endoplasmic reticulum (ER).

Why the ER? This is where countless ribosomes dock to feed in nascent proteins destined for secretion, the plasma membrane, and many locations inside the cell other than the cytoplasm. So it is a key organelle for protein synthesis and distribution, and thus for sensing how cellular proteins are doing. This sensing, the UPR, starts with a sensor protein, called IRE1 (also called ERN1), which sits on the inside face of the endoplasmic membrane. IRE1 binds to unfolded proteins inside the ER, and when the concentration rises high enough, it gathers into condensates of its own that trigger a variety of responses that include, in the extreme, cell death. Yes, lack of timely tidying can have serious consequences!

IRE1 is multifunctional, containing a kinase that activates AMPK and JNK, proteins which influence metabolism and inflammation. It also contains an RNAse enzymatic activity, which activates a whole other program by altering the mRNA of XBP1, which then becomes active and produces a transcription regulator that starts building more ER, more chaperones, and more protein folding capacity generally. The RNAse also digests other mRNAs indiscriminately, lightening the load of translation and thus incoming nascent proteins. So IRE1 is a key homeostatic regulator that adjusts the size of the ER to cellular needs, and informs the rest of the cell how things are going in the ER. 

One structure of IRE1, luminal domain only. This is a dimer, with halves shown symmetrically across the middle. How does this bind unfolded proteins?

But how does IRE1 sense unfolded proteins? A recent paper went through a modeling study to show how IRE1 operates. Sitting in the ER membrane, the enzymatic, signaling parts of the protein are outside, while the sensor is inside, called the core luminal domain (cLD). The authors demonstrate that this domain binds unfolded proteins directly, using them as bridging elements to bring multiple IRE1 proteins together and form the glob that nudges aside the inhibitory protein BiP and promotes the phosphorylation activity and thus activation, of IRE1. BiP also binds to unfolded proteins itself, so forms another sensor that indirectly activates IRE1 under stress conditions. 

Firstly, IRE1 is a dimer, and the luminal domain is where it dimerizes. The structure (just of the luminal domain, above) shows a beta ribbon core and peripheral unstructured areas and alpha helices. After running the crystal structure (shown) though very brief molecular dynamics simulations, the unstructured areas flop around dramatically, while the core and dimeric structure remain stable. Intriguingly, the beta stranded center of the dimer resembles (between the blue helices, above) the peptide binding cleft of MHC molecules- those that present random antigens to T-cell receptors of the immune system. Yet the authors show that unfolded proteins bind differently to IRE1. They flop across the whole central region, attracted by negative charge as well as the open hydrophobic surface. The next image shows molecular dynamics before/after shots of various unfolded peptides known to bind IRE1 in vitro, showing how after starting straight across the central region, they each find different comfort zones, mostly over this central region, but some off to a side. 

Collection of IRE1 structures (gray) with unstructured peptides of various kinds bound to the luminal face. t=0 is the starting point for the molecular dynamics simulation, which went for one microsecond. End points are below. In each case, the unfolded peptide stayed bound, and settled into some comfortable position, based on the hydrophobic and negatively charged landscape of the IRE1 face. 

The intriguing thing about this binding is that the structure of IRE1 itself is unaffected. The dimer interface remains stable and open, leading to the hypothesis that these peptides form bridges by becoming the meat of a two-dimer IRE1 sandwich. And it is this that then displaces the inhibitory protein BiP and leads to condensation and activation, by bringing together multiple IRE1 kinase domains (on the outside of the ER), and activating their kinase/signaling functions, turning on the various downstream pathways.

A helpful model from another lab, describing how, once the unfolded proteins are bound inside the ER (by mechanisms unknown at the time), the outside domains would gather, trans-phosphorylate, and activate downstream pathways. 

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