Some of this has to be taken on faith. It simply surpasses our ability to visualize its realities. The same way we can understand billions of galaxies exploding away from us in the Cosmos, the same way we can understand a a hundred trillion human cells continually in motion, breeding, communicating, living and dying within our own bodies, so too can we understand the transformation of the mutated protein calreticulin. When the big ASH meeting in New Orleans ended a couple of months ago, the surprise MPN finish was the announced discovery of the CALR mutation. In a stroke, the discovery seemed to clear up the mystery of why we can have an MPN and not be JAK2 positive. It was interesting… in a general kind of way. To be honest, most of us hadn’t yet fully digested the 10 year old JAK2 mutation discovery and all that JAK-STAT business. Nor would we have to. There were pills available at a disgracefully hefty price to shut down the JAK2 V617F action for awhile and reduce our symptoms. So why bother about digesting yet another mutation and new vocabulary? CALR is different. For one thing, two big time labs arrived at the identical conclusion after seven or eight years of intensive work. So there would be no need to wait for replication of results. Mutations in the CALR gene on the short arm of chromosome 19 code for a mutated calreticulin protein that is mucking up our blood production system. This is real and measurable.
Mutations in the CALR gene on the short arm of chromosome 19 code for a mutated calreticulin protein that is mucking up our blood production system
The two scientists leading this work are among the very best in the MPN universe, with deep credits in discovery of the JAK2 mutations as well as CALR. Tony Green in London and Robert Kralovics in Vienna, live and work in a highly rarified world, cloning, measuring and manipulating infinitely small bits of molecular material, cutting DNA strips with enzymes, assembling proteins, calculating, speculating about a world roaring on far beneath our direct perceptions. This introduction to calreticulin is designed as a guide to follow their published findings in the New England Journal of Medicine and the interviews that follow in these pages. The CALR mutation is one of those fully understandable findings once you can accept the completely unbelievable size, scope and complexity of our biogenetic apparatus. As important as the JAK2 enzyme is in our bodily functions, calreticulin is equally pervasive and more tightly focused. Finally, and this is the bottom line that makes it worthwhile to dig into the findings: There is a potentially enormous payoff right around the corner. We already have CALR screening available at our local hematologist. Next step: drug targeting.
The CALR findings reported at the end of 2013 are very likely to bring about MPN solutions – like antibodies and vaccines – that will ultimately turn myeloproliferative neoplasm into a dimly remembered dull and ugly smear on the medical landscape. So take a breath and plunge on in to the molecular depths where calreticulin does its work.
It starts with the Cell. This grade school view of the cell holds up surprisingly well. But it doesn’t take us very far. For one thing, we have to dig into the nucleus to get a closer look at our double helix strand of DNA with its alphabet of life and death. We need to zoom in on that terrain if we want to see our 23 chromosomes surface during procreation because it’s on those wriggly paired structures that our genes reside…Including, on chromosome 19, the CALR gene that produces the code for the calreticulin protein. And then, there’s another drawback to our public school drawing. Cells aren’t flat and two dimensional. They’re living organisms that eat, eliminate waste, procreate, move around and follow orders with an unflinching obedience that would make fascist armies flush in embarrassment. Sort of like this: Courtesy, Wikipedia When messages arrive – via enzymes, hormones, cytokines, electrical signals, chemicals etc. – the cell has no choice but to respond. If the command is apoptosis, the cell will proceed to commit suicide. If the command is to produce reinforcements to fight infection, the cell will enter a reproductive frenzy Now look closely, because we’re zeroing in on calreticulin’s home territory. You see that yellow pimply membrane surrounding the nucleus? And around it there’s a red lobster-like membrane covering it. Together, that’s the endoplasmic reticulum — the (yellow) rough ER is studded with ribosomes, little protein factories, and the (red) smooth ER is the storage and gateway. Between the two ER structures is the ER lumen, a hive for tagging, modifying and storing new-born protein, the zip code and street address for calreticulin. To get an idea of the ER in action, here are electron microscope photographs of the ER both in its “resting” state around the nucleus and its active state as it houses the production of antibodies — a disease fighting protein — and spews them out to where they’re needed in the body.
The endoplastic reticulum – or ER – is the intermediate world between the cell nucleus and the greater organic world beyond the Sea of Cytosol. It’s a heaving, heavily populated, structure with traffic headed into and out of the nucleus. It’s home to the portable protein factories – the ribosomes – that turn DNA code into long strands of amino acids that, when folded into usable 3-D shape, form all the proteins in our body. And everyone of our 3.72 × 10(13) cells (except red blood cells that have traded their nucleus for a truckload of hemoglobin) have this complex endoplastic reticulum surrounding their cell nucleus. And it is home base for calreticulin. B.M. (before mutation) calreticulin has several functions, including helping to folding up those long strings of amino acids into three dimensional usable proteins and either escorting them out into the world or sending them back into the cell to be recycled if they fail. Calreticulin is also a scaffolding protein, one of those structures that joins other signaling elements together to send orders downstream. This is the Marek Michalak diagram of the CALR gene exons (expressed regions). Exon 9 is where the mutated action is. A.M. (after mutation) calreticulin does much the same thing…only not as well and adds a toxic signal to its activity. The CALR mutation usually produces one of two changes, the addition of 52 base pairs or deletion of 5 base pairs in the last section of the gene, exon 9. The effect in either case is to change the reading frame used to manufacture the calreticulin protein which results in a shift in the electrical charge. Instead of the negative charge needed to store calcium ions and release them where needed, the mutated protein has a weak positive charge. The full effect of this shift is still being explored but the loss of the so-called KDEL (a sequence of amino acids, Lysine, Aspartic acid, Glutamic acid, and Leucine) at the end of the protein that, among other functions, keeps calreticulin from leaving the ER doesn’t seem to effect the functioning of the protein. (Kralovics reattached the KDEL — see, you just have to take this stuff on faith. “Putting KDEL back on the mutant,” said Kralovics, ” but preserving the mutant peptide and just adding KDEL at the end, did not cancel the oncogenic signaling.”) “The oncogenic signaling.” The blood cancer upshot of the CALR mutation. What does matter in the mutated calreticulin protein is its new affinity for the thrombopoietin receptor.
That’s the punch line. The CALR mutation — whether deletion or insertion of base pairs in Exon 9 or some of the minor variations in between — affects a single cytokine (a cell signaling protein) called thrombopoietin receptor or TPO. The calreticulin mutated protein fuses to TPO and then acts as a regulator of blood lines, particularly megakaryoctyes and platelets.
To get some idea of the power of TPO — and now the fused calreticulin/TPO protein — the protein is coded by the Myeloproliferative Leukemia virus oncogene (MPL). This gene is capable of rendering hematopoietic cells immune to self-destruction signals, immortal. The activated TPO receptor signals through the JAK-STAT pathway. Finally, just to see how all these systems play together, here’s a two short animations that helps put things in perspective. And then do head over to meet Professor Tony Green and Robert Kralovics. It’s an encounter with history. http://youtu.be/fKEaTt9heNM Take me back to the Contents © MPNforum.com, 2014. Unauthorized use and/or duplication of this material without express and written permission is prohibited. Excerpts and links may be used, provided that full and clear credit is given to MPNforum.com with appropriate and specific direction to the original content.