Mutation Explained by Daniel H. Lin and André Hoelz, Caltech

Grab a cup of coffee and learn all about mutation from CalTech Professor. A chance at some free education information.

Why are mutations in nucleoporins associated with human disease?

To commemorate this year’s ANE awareness day, we have been asked to write a bit about our research on the nuclear pore complex, the cellular machine that RanBP2 (also known as Nucleoporin 358 or Nup358) is a component of. Our research is sometimes referred to as “basic science,” which means that we work towards answering fundamental questions about how our cells work, with the understanding that this knowledge can inform further translational research that is focused on treating or preventing human diseases. We will begin with a bit of background, then summarize some findings from one of our most recent studies, and end with an outlook of where we are heading and how it relates to ANE.


As you may already know, our DNA genome encodes for thousands of genes, and typically each gene contains the instructions to make a specific protein (e.g. the RanBP2 gene encodes the instructions to make the RanBP2 protein). Because our DNA genome serves as the sole source of these instructions, it must be protected from potential sources of damage. Thus, cells isolate the DNA genome from the rest of the cell by enclosing the DNA in its own compartment, called the nucleus. For the instructions to be communicated to the rest of the cell, they are copied into molecules called messenger ribonucleic acids (mRNAs), which are allowed to leave the nucleus.


To let mRNA molecules out of the nucleus without letting everything else in, cells have developed a specialized kind of portal called nuclear pore complexes (NPCs). Nuclear pore complexes are intricate molecular machines made up of about 34 different proteins, one of which is RanBP2. Despite decades of research, we are still just beginning to understand how each of these different proteins does its job. RanBP2 for example, is not only critical for holding the nuclear pore complexes together, but also helps mRNAs get out of the nucleus. There is still a great deal to learn about this process, as we only recently worked out how all the different components assemble into the whole machine.


The goal of the research in our lab is to understand at a basic chemical level how all the nuclear pore complex proteins do their respective jobs. An essential step towards this understanding is knowing what these proteins look like in atomic detail. This detailed knowledge of where every atom is located can also inform us about how mutations change the protein and lead to disease. In fact, many components of the nuclear pore complex have been linked to a large and diverse set of diseases, including ANE. Eventually the knowledge gained from this kind of research may reveal novel ways to treat or prevent these diseases. Last year, we published the results of a multi-year investigation of one of RanBP2’s neighbors in the nuclear pore complex, called Gle1. This study has changed the way we think about disease-causing mutations in all nuclear pore complex proteins.


Gle1, like RanBP2, helps mRNAs get out of the nucleus. Mutations in Gle1 have also been shown to cause a genetic disease called lethal congenital contractual syndrome (LCCS). As is typically the case, it is not known how mutations in Gle1 cause this disease, because we are still figuring out how normal Gle1 works in healthy cells. Thus, we first set out to thoroughly characterize normal Gle1 and how it interacts with other nuclear pore complex proteins. Through biochemical experiments, we found that the Gle1 protein is unusually fragile and has a tough time keeping its correct shape – what we call its atomic structure – on its own. In fact, we found that it relies on other nuclear pore complex proteins to stay intact. Now knowing that we could use other nuclear pore complex proteins to stabilize Gle1, we were able to do many previously impossible biochemical experiments with Gle1 for the first time. For example, we mapped out the details of how it assembles with several other nuclear pore complex proteins and measured how it controls the activity of enzymes that cooperate with Gle1 to enable nuclear export of mRNAs.


Perhaps most importantly, we were able to keep Gle1 stable for long enough to find out its exact shape using X-ray crystallography. X-ray crystallography is a powerful technique that essentially takes a three-dimensional snapshot of a biomolecule with such high resolution that one can map out where all of its thousands individual atoms are. Typically, it is a very tricky and time-consuming technique to perform, and often fails despite all best efforts. However, with the new knowledge of how to stabilize Gle1, we were finally able to use X-ray crystallography to visualize the thousands of atoms that make up Gle1. A major benefit of having this picture of Gle1 was that we could immediately see the location of the disease-associated Gle1 mutations and how they altered its atomic structure. We immediately realized that most of the disease-associated mutations actually make Gle1 even more unstable. One way that we demonstrated this experimentally was by comparing the stability of normal Gle1 or the disease-associated variants at multiple temperatures. Normally, a protein’s activity will speed up at higher temperatures – up until it gets too hot and the protein denatures. Denatured proteins have lost their shape and no longer work. This is what happens to egg whites, which are composed primarily of proteins and water, when they change from clear to opaque as they are cooked. The more fragile a protein is, the lower the temperature at which it denatures and stops working. Indeed, when we carefully measured the temperature at which normal Gle1 and the disease-associated Gle1 variants denatured, we found that the disease-associated variants denatured at much lower temperatures. This experiment indicated to us that mutations in Gle1 truly do make it more unstable.


This incremental path towards a discovery is common in basic biological research: an insight that Gle1 needs help from other nuclear pore complex proteins to maintain its shape enabled us to use X-ray crystallography. Using X-ray crystallography gave us a window into how mutations would affect the protein, which in turn led to the realization that these disease mutations further destabilize an already fragile protein. This insight is important to us because it was not a possibility we had previously considered when thinking about what mutations may be doing to nuclear pore complex proteins. An exciting outcome of our research is that some nucleoporin diseases may be treatable if we can design new molecular drugs that stabilize the affected proteins. We still have much more work to do to prove that destabilization of the Gle1 protein causes LCCS and that stabilizing Gle1 could help treat it. Also, it will be important to find out if similar things are happening with mutations in other nuclear pore complex proteins such as RanBP2. We have been eagerly pursuing similar studies on RanBP2, which has been an even more challenging protein to study, and we expect to have an atomic detail map of where ANE1-causing mutations are located soon.



Figure legend

Getting maps of nuclear pore complex proteins with atomic detail. Shown on the left is a simplified cross-sectional schematic of a nuclear pore complex. The nuclear pore complex creates a portal in the membrane that separates the nucleus from the cytoplasm (colored gray, labeled as the nuclear envelope). The approximate location of RanBP2 is indicated on the top face of the nuclear pore complex that is exposed to the cytoplasm. Different colored layers represent building blocks of the nuclear pore complex, with many proteins (also called nucleoporins) fitting together to form each building block. Transport cargoes such as mRNAs traverse the nuclear pore complex through the central transport channel, shown as a transparent layer in the center of the nuclear pore complex. The inset indicates the section of the nuclear pore complex where Gle1 is found. Shown on the right is a three-dimensional rendering of the atomic structure of the Gle1 protein. Names of the disease associated mutations in Gle1 are labeled in red, with the specific atoms affected shown as red spheres. Red sticks indicate chemical bonds between atoms.


Movie legend

The movie shows a rotation of a three-dimensional rendering of the atomic structure of the Gle1 protein. The atoms affected by mutations are shown as red spheres. Sticks between spheres indicate chemical bonds between the atoms.

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