When you don’t want a protein you’ve already made, call in Auxin Inducible Degrons to come to your AID! Features of proteins called DEGRONS act as DEGRadatiON signals that flag unwanted proteins for tagging by ubiquitin ligases. Those ligases add chains of a little protein called Ubiquitin (Ub) that act as “shipping labels” for a trip to the proteasome (protein version of a paper shredder). Our cells use degrons all the time – and so do plants – it’s how they’re able to regulate a bunch of proteins at once to grow when the time’s right. And scientists can “add on” plant degrons to non-plant proteins to flip protein switches in cells and organisms in the lab. Here’s how it works. 

Your cells have a protein shipping system that’s actually called the UPS – but in this case, it stands for the Ubiquitin-Proteasome System, not the United Postal Service – & it takes all its packages (unwanted proteins) to the same location, the proteasome, that’s like a paper shredder for proteins – it “scans” the shipping label and if all checks out, it unfolds the protein, pulls it in, & cuts it up into smaller pieces that can be recycled. 

It knows what packages to take because they have a “shipping label” in the form of a polyubiquitin chain. Ubiquitin is a small (76 amino acid letter) protein that your cells have a ton of (you might even say it’s ubiquitous…) that can be placed on proteins that aren’t wanted in order to target them for degradation. Lots more here: https://bit.ly/2XQhpPF

These unwanted proteins can be “retiring” proteins that already served their function; damaged or only partially-made proteins that can no longer function; misfolded proteins that are causing mischief by clumping up (aggregating); regulatory proteins whose time is up, etc. Bottom line – any type of protein your cell doesn’t need it doesn’t want, but it wants its parts, so it needs a system that’s generic enough to ship any package, but specific enough that you can control what packages you ship & when.

The proteasome part’s “generic” in that it mainly just cares that the package has the right shipping label. So you need specificity in the labeling part. The labeling part is carried out by a series of 3 protein enzymes (reaction mediators) – 1 of each of the E1, E2, and E3 families  – that function in concert. 

E1 (ubiqutin-activating enzyme) spends ATP energy money to buy a Ubiquitin (Ub). Then it passes the Ub off to an E2 (ubiquitin-conjugating enzyme). Then that E2 puts the Ub on the protein package. But it needs the package to be taken to it, and that’s the role of E3 (ubiquitin ligase).

That described order’s for the “RING ligases” – there are 2 other families of ligases, HECT & RBR ligases, and those have an extra transfer – they first transfer Ub from E2 to E3 and then E3 puts it on the protein. 

Either way, E2 has to bring the Ub label in, so it (or copies of it) comes and goes bringing in more labels. But you want to keep the protein ready at the post office, so you need to keep it stuck to E3, & this is where the degron shines.

DEGRONS are features of protein “packages” that act as “degradation signals” that flirt with the E3 post office’s desk worker (recognin) to keep the package there there while E2 does its work. “Recognin” is just the part of E3 that “recognizes” the degron on the unwanted protein “package” and binds it – the stronger the degron-recognin attraction, the longer the package will stay there and more Ubs that can be added. This is important because you need at least 4 Ubs for the proteasome to accept it and other enzymes called deubiquitinating enzymes (DUBs) serve as label removers and can erase them.

E3s are the main source of specificity – there are about 600 of these and they recognize different features, including the degrons. We call things that recognize degrons recognins. There are different types of degrons and they can be in different places in the protein. You don’t always want them visible to E3 or else the protein wouldn’t have time to work, so degrons are often hidden (such as being sheltered by another protein) or require further modification. 

The labels get added to lysine residues on the protein. These don’t have to be in the degron, but proteins that are under strong regulation by the UPS often have these lysines in places that are easy for E2 to find. Speaking of degrons, a quick jargon not: when degrons are at the start of the protein we call them N-degrons (cuz they’re at the “N terminus” and they travel through “shipping routes” called N-DEGRON PATHWAYS that follow “N-end rules” of the road. I’ll talk more about these later.

But how do you prevent proteins from just always being degraded? Firstly, as I will talk more about later, proteins often require modification to make them have a degree, so you can regulate when you generate the degron (like when you cleave, acetylate, arginate, etc.) And secondly, it’s important to keep in mind though that, even if a protein has an degron, it might not be degraded because the degron has to be found. If it’s “taken” by another protein that hides it, it’s safe from degradation. If that partner decides it’s had enough and releases the protein, it’s now vulnerable. This can serve as a mechanism to selectively get rid of certain subunits of multi-subunit complexes, such as those that need to bind one thing at one point in the cell cycle and a different thing at another time. Degrons can also serve as a sort of quality control, being hidden if a protein is folded correctly but visible if the protein misfolds.

The degron functions in the tagging part but then its job’s done – anything with the right shipping label can be taken to the proteasome, where it binds through ubiquitin-binding domains (UBDs) on ubiquitin receptors – proteins acting as “security guards” at the entrance to the proteasome shredder.

A cool thing about this is that you can artificially add a “portable degron” onto any protein to give it a trip to the proteasome. And this can be used to see when proteins get desequestered or stuff. Or, it can be used to degrade proteins “on demand” – a common way to do this is with an Auxin Inducible Degron (AID) system. Auxin is a small molecule plants use as a chemical messenger (hormone). “Auxin” is actually an “umbrella term” for a family of hormones, including indole-3-acetic acid (IAA; a natural auxin) and 1-naphthaleneacetic acid (NAA; a synthetic auxin).

Plants have to do a lot of coordinated stuff in order to grow at the right time and this involves turning on the production of multiple proteins “on demand” when the time’s right. To keep these proteins from being expressed too early, transcriptional repressors sit on those protein’s genes and prevent them from being copied into messenger RNA (mRNA) (a process called transcription) which can be translated into protein. 

So, in order to express those genes, you need to get rid of the repressor. And this is where auxin inducible degrons come in. Those repressors contain an auxin recognition element which binds to auxin and a ubiquitin ligase subunit called an F-box protein. 

F-box proteins serve as part of an “SCF” ubiquitin ligase complex consisting of a scaffolding protein called Cul1 holding onto a protein called Skp1, which binds to F-box proteins (FBPs). The F-box protein is the only part that specifically binds the thing to be tagged (substrate), so it decides what gets tagged and what doesn’t. Cells use a bunch of different F-box proteins that can be “swapped out” so that the same SCF complex can be used to ubiquitinate a bunch of different things. The ubiquitinating is done by an E2 ligase, which binds to SCF with the help of a RING protein like Rbx1. 

So you have Cul1 bound on one end to Skp1 which binds the FFB which binds the substrate. And then on Cul1’s other end you have the RING protein which binds the E2 which does the ubiquitinating. 

Animal cells don’t have one of those auxin responsive F-box proteins like TIR1, but we do have the SCF, RING, and E2 parts. And the F-box proteins are interchangeable. So if you get a cell to express the F-box protein that recognizes auxin – and then you stick the auxin-recognizable degree onto a protein you want to degrade on demand you can get that cell to degrade the protein when you add auxin. (To add this tag you have to alter the genetic instructions of the protein to contain a few more letters at the end).

Before you add auxin, the protein’s made normally, so this allows you to precisely control when in a cell or organism’s life you shut it off, which can be especially useful if you’re studying an “essential” protein that a cell can’t grow/live without. 

When you add auxin, it allows TIR1, attached to the SCF, to bind to the substrate. This SCF-TIR1 serves as an E3 ligase – it recruits an E2 ligase which brings in the ubiquitin and adds it. And this earns the protein a trip to the shredder (proteasome). 

Although the individual protein copies that got sent to the shredder (proteasome) are goners, once you stop adding auxin, and the remaining auxin gets cleared out, new copies of the protein will be safe, even though they have the degron. So you can turn that protein “back on”

Plants use this AID system naturally, but our cells don’t. But don’t take that to mean we don’t use degrons! We definitely do! Commonly through “N-degron pathways.” The “N” refers to the N-terminus of the protein. Proteins are made up of chains of amino acids by linking them amino group to carboxyl group to form peptide bonds. The starting end has a free amino group & is called the N-terminus & the ending end has a free carboxyl group & is called the C-terminus. 

Scientists found that proteins with certain amino acids at their N-end were less stable than others. They had to do some cleaver trickery to figure this out because all proteins are made starting with methionine (Met, M) because its codon also serves as a start codon. But this methionine (alone or with a whole chunk of protein) can get removed, giving the protein a new N-terminal residue (Nt).

Some of these new ends are unstable as is (primary degrons) – these can be divided into 2 types : type I positively-charged (arginine (Arg, R), lysine (Lys, K), histidine (His, H)) & type II big/bulky hydrophobic (water-avoiding) (phenylalanine (Phe, F), tyrosine (Tyr, Y), tryptophan  (Trp, W), leucine (Leu, L), isoleucine (Ile, I)) 

Other ends aren’t unstable as is, but can be made unstable – we call these pro-N-degrons – and they can be “secondary” or “tertiary”

some pro-N-degrons can be made into N-degrons in a single step (secondary): Asp & Glu can have Arg added onto them. This is unlike usual peptide bond formation because this Arg is being added to the “front end” of the protein, so it can’t use the same enzyme – it needs different ones. Those different ones are arginyl-tRNA transferases (ATES) & they can only add it to these particular amino acids.

Asp & Glu are really similar to Asn & Gln, so you can interconvert them by You can turn Asn & Gln into Asp & Glu respectively by deamidating them with specific Nt amidases (NTAN/NTAQ). Since this requires a 3rd step, we call them “tertiary”

Another tertiary one’s cysteine (Cys, C). It can get oxidized by oxygen or nitric oxide (NO) to produce cysteine-sulfinic or cys-sulfonic acid – looks like Asp so arginylated. This is cool cuz it lets it act as a sensor of NO levels

All that’s for the Arg/N-end rule pathway. That’s the “classical” pathway that was first discovered & characterized. But there are 2 other (known) N-degron pathways in mammals. The Ac/N-end rule pathway recognizes certain acetylated Nts & the Pro/N-end rule pathway recognizes Proline (Pro, P) in certain contexts. 

All in all, there’s basically a way to turn any end into an N-degron. You can also create a new end by cutting the protein, and enzymes called proteases can do this (e.g. separases, caspases, and calpains).

link to helpful video: http://bit.ly/2ILKdDN and review article:  http://bit.ly/2DDW0zN

more about all sorts of stuff: #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0 

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