Proteins are the workhorses of the cell. They catalyze important reactions, provide support and scaffolding, transport packages through the cell, signal between cells, and do many other vital functions. Proteins are made by following instructions from genes. Protein expression is very dynamic, with old or damaged proteins being replaced by new proteins on a daily or even hourly basis. This process is known as “protein turnover”.
Protein turnover is important for two reasons:
1) If the protein is involved in a signaling pathway, you don’t want it sitting around being active if its purpose is to respond to a signal. For instance, if a hormone activates expression of Gene X that makes Protein X, you wouldn’t want Protein X hanging around all the time. It should only be expressed in the presence of the hormone signal. Therefore, these types of proteins have markers on them that tell the cell to degrade the protein after a certain length of time.
2) Proteins get damaged from normal wear and tear in the cell. Damaged proteins may start acting inappropriately, so they need to be destroyed before they start running amuck. Damaged or unfolded proteins may also start binding with each other to form big aggregates that clog up the cell. Something like this is what happens in Alzheimer’s and Parkinson’s Disease.
Therefore, as molecular biology students, we learn that protein turnover is an important housecleaning procedure in the cell. Imagine my surprise, then, when I read a paper this week about extremely long-lived proteins (that’s the official scientific term and it’s abbreviated ELLP). A few ELLPs were already known, such as a protein that insulates neurons, and proteins that control how tightly DNA is packed into the nucleus. The major finding of this paper by Savas et al. was of a new group of long-lived proteins that make up the nuclear pores. They did one experiment, made one figure and blew my mind.
Pulse chase experiment
In order to look for ELLPs, the authors did a “pulse-chase” experiment. The idea behind this type of experiment is to introduce some sort of a marker that can be traced for a long time. The “pulse” is the marker that is introduced for a limited time and the “chase” is waiting to see what happens to that marker. The authors want to mark proteins that are made at the beginning of life and then see how long those original proteins last in the body.
Proteins are made of amino acids which have nitrogen in them. Therefore, in order to label proteins, the authors used an isotope of nitrogen (15N), which is heavier than normal nitrogen (14N). By using a technique called mass spectrometry, they can tell the difference between proteins made with normal nitrogen and those made with the 15N isotope. They fed newborn rats with a diet that was rich in 15N, which then became incorporated into their newly synthesized proteins. The young mice were switched off the 15N food after 6 weeks, so later in life, if a protein has 15N in it, it must have been made during the first 6 weeks of life.
They examined liver tissue from these rats 6 months later and found that only a few proteins had 15N in them. This is consistent with previous findings which show that the proteins in the liver rapidly turnover. Using another rat, they examined tissue from the brain after 12 months and found many 15N proteins. In other words, the proteins in the liver are made new all the time, whereas in the brain, some proteins that are made during the first weeks of life remain there a year later. Keep in mind that the lifespan of a lab rat is about two years, so 12 months is half of its lifetime. It’s unbelievable to me that a protein would last at least half of the lifespan of an animal. It should be noted here that they only used one rat for each time point, so their sample size is essentially one.
The authors found some well known long-lived proteins, which shows that their experimental approach was valid. They also, surprisingly, found a set of long-lived proteins that are part of the nuclear pore complex (NPC).
Nuclear Pore Complex
DNA is kept in a membrane enclosed compartment called the nucleus. The nucleus has doorways, or pores, through which proteins and other molecules can pass in and out. Small molecules can easily go through this doorway, but bigger molecules, like proteins, need an escort to get through. The escort only binds to molecules that are supposed to go through the pore. The nuclear pore complex is this door between the nucleus and the rest of the cell (called the cytoplasm). The NPC checks that everything big that is trying to get through has an appropriate escort. The NPC is analogous to a doorman at a club who will only let in people who are on the guest list. There are some things that need to stay in the nucleus (like DNA and the machinery to express it) and some things that have no business being there, so it’s important that the NPC regulates this transport process.
Electron micrographs of NPCs from cytoplasm side (left) and nuclear side (right).
One NPC is made up of 100s of proteins all bound together to create a water filled pore through the nuclear membrane. Only some of these NPC proteins were found to have the 15N isotope 12 months later, so some subunits of the NPC are rapidly turned over, like most other proteins. However, these findings imply that although individual components of the NPC may be replaced, the entire NPC as a whole is built to last for a very long time.
These findings are important, because long-lived proteins may relate to cellular aging. One can imagine that as cells age, their long-lived proteins become damaged, including those in the NPCs. In fact, this same lab has previously shown that NPCs in the brain cells of aging rats deteriorate and become increasingly more leaky. The NPCs may not work in the same way anymore and may allow toxic cytoplasmic proteins into the nucleus without the appropriate escorts. These proteins from the cytoplasm could cause damage to the DNA inside the nucleus, which would lead to more dire consequences for the cell, and may lead eventually to cell death. It is still unknown if there is any advantage to ELLPs and why they aren’t replaced like all other proteins.